Volume 6, Spring 2008 - Saddleback College

Saddleback 

Journal of Biology 

Zebra Tailed Lizard at Rainbow Basin California, Callisaurus draconoides 

Published by 

Saddleback College Biological Society 

Volume 6 Spring 2008 

Department of Biological Sciences 

Saddleback College 

Mission Viejo, CA 92692 

Editors, Tony Huntley and Steve Teh 

i 

Saddleback Journal of Biology 

Vol. 6, Spring 2008

TABLE OF CONTENTS 

Peer Reviewed Manuscripts from the Biology 3B Class 

Spring 2008 

Author(s) Title Page 

Merielle Ebol Growth Difference in the Goldfish, Carassius auratus, 1 

& 

Shanon Carney 

Exposed to Different Diets with Varying Levels of Proteins 

and Carbohydrates 

Kyle Lutz & The Effect of a Lactate Supplement on Maximal Cycling 4 

John I. Miller 

Performance in Man 

Stephanie Anstadt & The Effect of Silver Nitrate on the Inhibiting Growth of 7 

Teo Fernandez 

Escherichia coli 

Nathaly Leal- Arteaga & Effect of Tide Level on Nitrate and Phosphate Concentration 10 

Saori Shimamoto 

in Marine Water 

Lancelot Beier and Effect of Differing Color on the Timed Length of Aggressive 14 

Harrison Pham 

Response of Betta splenens 

Crystine Gill & Intestinal Candida albicans Overgrowth in Autistic Children 17 

Samantha Lopez 

with Food Allergies 

Ryan C. Clark & Comparison of Chlorophyll Content in Shade and Sun 20 

Josue J. Mandujano Leaves of the Lemonade Berry Plant (Rhus integrifolia) 

Thao Nguyen & The Effects of Ethinyl Estradiol on Aggressive Behavior in 22 

IxChel Cruz-Gonzalez 

Siamese Fighting Fish (Betta splendens) 

Kevin Murray & Antibiotics (Tobramycin and Polymyxin) Resistance in 26 

David Stapleton Staphylococcus aureus & Effectiveness of These Antibiotics 

Yuriko Kayama The Effects of pH on Escherichia coli Fermentation 29 

Kaung Ko & Antibiotics (Tobramycin and Polymyxin) Resistance in 33 

Spencer Roberts Staphylococcus aureus & Effectiveness of These Antibiotics 

Dorothy Chang & 

Effects of temperature on metabolic rate in 

37 

Grant T. Huttar 

Gromphadorhina portentosa 

Dayana Vera & The Effect of Creatine Monohydrate on the Run Time of 41 

Michael Moeller 

Sceloporus occidentalis 

Ryan G. White & The Metabolic Cost of Digestion in the Ball Python, 44 

Michael M. Hadley 

Python regius 

Nicole Baumgartner & Recreational Water Safety Following Rain at T-street Beach, 47 

Karl Neil 

San Clemente, California 

Takahiro Ueno & 

Arash Moghaddam 

The Effect of Salinity on the Photosynthetic Rate of 

Pickleweed, Salicornia virginica 

49 

Matt Apke & 

Zachary Beam 

The effects of various light sources on the fruiting bodies of 

Citrus limonium 

52 

ii 




Biology 20 Honors Manuscripts 

Fall 2007 & Spring 2007 


Michelle Huynh and Is MiracleGro the Best Fertilizer for Impatiens wallerana? 55 

Jiwon Park 

Cynthia Tran and 

Camille Barlow 

The Effects of Organic Fertilizer vs. Inorganic Fertilizer 

(Miracle Gro) on Growth of Tomato Plants 

57 


Peer Reviewed Manuscripts from the Biology 3B Class 

Fall 2007 


Lauren Ferris 

The Effects of fire on the Southern California Plant 60 

Succession and the Prevalence of Artichoke Thistle, Cynara 

cardunculus 

Gregory Nelson and The Effect of Mycorrhizae on the Growth and Development 63 

Elena Novak 

of Bush Beans Phaseolus vulgaris 

Thomas Caldwell Exhaustion due to Mental Stress and Metabolism of Sugar 66 

and Caffeine in Energy Drinks 

Heather Rufino and 

Alexa Milman 

Serial Lactate Levels Increase over Time under Anesthesia 

on Healthy Canine Patients Undergoing Elective, Minimally 

70 

Jasmine Mitchell and 

Tony Schofer 

Invasive, Lower Abdominal Surgery 

The Correlation Between the Vertical Jump Height and Calf 

Length in Athletes and Non-athletes 

74 

Chris Yang and 

Josue Mandujano 

Milad Danesh 

Catherine Pearson 

Shengchieh Chang 

Tarick Sheikh and Chris 

Glendinning 

Comparison of Chlorophyll Content of Leaves in a Green 

House and their normal environment of a Cyclamen Plant 

(Cyclamen Persicum) 

The Effects of Ginkgo Biloba on the Cognitive Thinking of 

Mus musculus 

Increased performance level due to carbohydrate vs. 

carbohydrate-protein composition in sports drinks 

Comparison of Drinks and Their Effects on Exercise 

Performance 

Correlation between attractiveness of male body odor and 

facial feature of Homo. sapiens 

77 

79 

83 

86 

90 

iii 



Biology 3A abstracts for papers presented at the 6 th Annual Biology 3A/3B 

Scientific Meeting (Spring 2008) 

The meeting organizers do not assume responsibility for any inconsistencies in quality or errors 

in abstract information. Abstracts are in numerical order according to the abstract number 

assigned to each presentation. Authorless abstracts appear at the end of all the abstracts, 

including non-submitted abstracts, if any. Abstracts begin on page 94. 


Biology 3A Abstracts 



Alexandra M. Franco THE EFFECT OF SODIUM CHLORIDE CONCENTRATION ON 94 

and 

THE GROWTH OF THE COMMON BEAN (Phaseolus vulgaris) 

Earl-Eugene E. Ringpis 

Chris J. LaCroix and PREFERENCE OF DOGS (Canis familiaris) FOR HIGH FAT DOG 

Jocelyn A. Finley 

FOOD VERSUS LOW FAT DOG FOOD 

94 

Raiff Josey and TESTING FOR INTELLIGENCE IN THE SLIME MOLD Physarum 

Christopher Luna 

polycephalum 

94 

Jason R. Riggio EFFECT OF pH ON RATES OF CLOSURE IN VENUS FLY TRAPS 

(Dionaea Muscipula) 

95 

Aubrey Michi and EFFECT OF TEMPERATURE ON PHOTOSYNTHETIC RATE OF 

Jeremy Ward 

Elodea canadensis 

95 

Anne Kathreane Ebol THE EFFECT OF GUAVA Psidium guajava AGAINST Escherichia 

and Hannah Rae Manuel 

coli 

95 

Anoush A. Garakani and THE EFFECT OF TEMPERATURE ON BACTERIAL COLONY 

Cassra Minai. 

FORMATION IN Escherichia coli 

96 

Brooke A. Hargis and CYNIPIDAE GALL DISTRIBUTION ON YOUNGER AND OLDER 

Candice B. Archer 

COAST LIVE OAK TREES, QUERCUS AGRIFOLIA 

96 

Claudia M. Shuldberg and 

Ximena M. Horne 

EXTRACTION OF IRON FROM BREAKFAST CEREALS 96 

Andrew J. Shires and OPERCULAR PUMPING RATES OF GOLDFISH (Carassius 

Christopher J. Walsh auratus) IN DIFFERENT CONCENTRATIONS OF GLUCOSE 

97 

Shayda Haghgoo and EFFECTS OF TABLE SUGAR AND STEVIA SWEETNER ON 

Erin Kang 

BLOOD GLUCOSE LEVELS IN HUMANS 

97 

Angel R. Vargas GROWTH INHIBITON OF HYDROGEN PEROXIDE ON THE 

PATHOGENIC BACTERIA, PSEUDOMONAS AERUGINOSA 

97 

Robert Powers COMPARISON OF LACTATE DEHYDROGENASE ACTIVITY 

BETWEEN THE THRESHER SHARK (Alopias vulpinus) AND THE 

98 

PIKE MACKEREL (Cololabis saira) 

Hanwool Park 

THE EFFECT OF A HAIR GEL ON THE GROWTH OF 

MALASSEZIA FURFUR 

98 

David Guzman 

THERE IS NO SIGNIFICANT OXYGEN PRODUCTION OF 

SPINACIA OLERACEA PLACED INTO CALCIUM CARBONATE 

98 

SOLUTION 

Yoko Kamei and THE EFFECT OF MODERN ROLLER COASTER RIDE ON HUMAN 

Diana Nguyen 

HEART RATE 

99 

Robert E. Maloney and THE AFFECT OF ROUNDUP ® ON OXYGEN PRODUCTION OF 

Bianca Christensen 

RED ALGAE (RHODOPHYTA) 

99 

Lindy A. Ackerman and 

Brittany N. Lincoln 

THE EFFECT OF SOIL pH ON THE GROWTH OF PHASEOLUS 

VULGARIS AND RAPHANUS SATIVUS 

99 

iv 







Nicholas Schmidt and IMPACT OF RED AND GREEN LIGHT ON GROWTH RATE OF 

Bobby Stangl 

SNAPDRAGONS (Antirrhinum majus) 

100 

Grady S. Counts and EFFECTS OF ENVIRONMENTAL TEMPERATURE ON THE 

Eric T. Rueda 

SPEED OF COMMON GARDEN SNAILS (Helix aspersa) 

100 

Hilda Gonzalez and THE EFFECT OF PH ON THE RESPIRATION RATE OF 

Natsumi Iwata 

GOLDFISH (Carassius auratus) 

100 

Tommy L. Roberts and 

Emmanuel Romero 

DETERRENT EFFECTS OF PEPPERMINT OIL IN MICE 101 

Samantha T. Dinh and THE EFFECT OF ROLLER COASTERS ON HEART RATE IN 

Cole P. Lyou 

RELATION TO GENDER 

101 

Harris M. Elhan and THE COMPARATIVE COST OF LOCAMOTION IN FEEDER MICE 

Tracy L. Kubas (Mus musculus) AND ROBOROVSKI HAMSTERS (Phodopus 

101 

roborovskii) 

Bobby Fujimoto EFFECT OF CHLORINE ON ALGAL GROWTH 102 

Grady S. Counts and EFFECTS OF ENVIRONMENTAL TEMPERATURE ON THE 

Eric T. Rueda 

SPEED OF COMMON GARDEN SNAILS (Helix aspersa) 

102 

Scott Mitchell and THE EFFECT OF TEMPERATURE ON THE METABOLIC RATE OF 

Amir Zand 

ANOLE LIZARDS (Anolis Carolinensis) 

102 

Roky Coria 

THE EFFICACY OF GARLIC (ALLIUM SATIVUM ) AS A 

BIOLOGICAL CONTROL OF MOSQUITO LARVA 

103 

John Lowd and 

EFFECTS OF AUDITORY AND CHEMICAL STIMULI IN 

Charles Steinfeld 

MAMMALIAN MEMORY (Mus musculus) 

103 

Sally A. Hutson A COMPARISON OF SUCROSE AND STEVIA EXTRACT AS A 

SUBSTRATE FOR YEAST METABOLISM 

104 

Natasha V. Polanski EFFECTS OF PESTICIDE ON OXYGEN PRODUCTION RATES OF 

RED ALGEA (Aharghiella tenera) 

104 

Anonymous POST FIRE PLANT FREQUENCY IN NORTH AND SOUTH SIDED 

HILLS IN SANTIAGO CANYON 

104 

Biology 3A abstracts for papers presented at the Annual Biology 3A 

Poster Presentation Scientific Meeting (Fall 2007) 

The meeting organizers do not assume responsibility for any inconsistencies in quality or errors 

in abstract information. Abstracts are in numerical order according to the abstract number 

assigned to each presentation. Authorless abstracts appear at the end of all the abstracts, 

including non-submitted abstracts, if any. Abstracts begin on page 105. 



Fall 2007 


Thao Nguyen and EFFECTS OF SPIRULINA ALGAE VERSUS REGULAR FISH 

Nelson Huang FLAKES ON METABOLISM IN BETTA FISH (Betta splendens) 

105 

Janelle Reed and 

Irina Alexandrova 

ASCORBIC ACID CONCENTRATION OF ORGANIC AND 

CONVENTIONALLY GROWN PRODUCE 

105 

v 





Fall 2007 


Kevin Murray and ANTIBACTERIAL EFFECTS OF RED WINE ON ORAL BACTERIA 

Arash Moghaddam 

105 

Izaak Miller and THE EFFECT OF PYRUVATE ON OXYGEN CONSUMPTION OF 

Lancelot Beier 

MUS MUSCULUS 

106 

Michael G. Moeller EFFECT OF LIGHT ON CHLOROPHYHLL CONCENTRATION IN 

ENGLISH IVY (Hedera helix) 

106 

Anne Merielle Ebol and SENSITIVITY OF PATHOGENIC BACTERIA STAPHYLOCOCCUS 

Shanon Carney 

AUREUS TO HONEY 

106 

Kaung Ko and THE EFFECT OF TEMPERATURE ON PHOTOSYNTHETIC RATE 

Spencer Roberts 

OF ELODEA CANADENSIS 

107 

Y Leho and 

THE EFFECT OF WEIGHT LOAD ON THE OXYGEN 

Christopher Triana CONSUMPTION OF PARAKEETS DURING TERRESTRIAL 

107 

LOCOMOTION 

Valerie Bowen and EFFECT OF SUCRALOSE AND SUGAR ON BLOOD GLUCOSE 

Samantha Lopez 

IN HOMO SAPIENS 

107 

Sherri Burnett and 

IxChel Cruz- Gonzalez 

Krystle Salazar and 

Matt Apke 

Saori Shimamoto and 

Yuriko Kayama 

Kyle Lutz and 

Yohsuke Kobayashi 

Deepa A. Thaker and 

Stanley Lin 

Nathaly Leal-Arteaga 

Aaron Echols and 

Crystine Gill 

Greg M. Fitzgerald and 

Michael B. Zilly 

Monica Mehran and 

Paris Aliyazdi 

Ryan G. White and 

Michael Hadley 

Micaela K. Dora 

Alvin P. Jogasuria and 

Takahiro Ueno 

John K. Davis and 

Jaclyn R. Kuluris 

Dorothy Chang and 

Grant T. Huttar 

COMPARISON OF LACTATE DEHYDROGENASE ACTIVITY IN 108 

SKELETAL MUSCLE OF BLUE ANCHOVY (ENCRASICHOLINA 

DEVISI) AND COW (BOS TAURUS) 

EFFECT OF PH ON GERMINATION RATE IN LIMA BEANS 108 

OXYGEN PRODUCTION OF BROWN ALGAE (Egregia laevigata) 108 

AND RED ALGAE (Gelidium robustum) IN DIFFERENT COLORS 

OF LIGHT 

THE EFFECT OF SODIUM BICARBONATE ON TIME TO 109 

EXHAUSTION IN THE WESTERN FENCE LIZARD (Sceloporus 

occidentalis) 

EFFECT OF CAFFEINE ON MEMORY IN MICE (Mus musculus) 109 

THE EFFECT OF PH ON THE GROWTH OF BACTERIA 

(Escherichia coli) 

THE EFFECTS OF OZONE ON ESCHERICHIA COLI ON SPINACH 

LEAVES 

THE EFFECT OF WAVELENGTH OF LIGHT ON THE 

DISCOLORATION OF WINE. 

THE EFFECT OF APPLES ON THE RIPENING OF VALENCIA 

ORANGES (Citrus aurantium) 

A COMPARISON OF THE WATER UPTAKE RESPONSES TO 

INTRAPERITONEAL BOLUS INJECTIONS OF ARGININE 

VASOTOCIN IN THE TOAD, BUFO AMERICANUS AND THE 

LEOPARD FROG, RANA PIPIENS 

EFFECT OF AMBIENT TEMPERATURE ON THE FORCE 

EXERTED BY CARIBBEAN HERMIT CRABS (Coenobita clypeatus) 

THE EFFECT OF HYDROGEN PEROXIDE ON THE 

GERMINATION RATE AND GROWTH OF STRING BEANS 

(Phaseolus vulgaris) 

EFFECT OF CAFFEINE ON BLOOD LACTATE IN EXERCISING 

HUMANS 

INHIBITORY EFFECTS OF CAM PLANT FLUID ON 

SATPHYLOCOCCUS AUREUS 

109 

110 

110 

110 

111 

111 

111 

112 

112 

vi 





Fall 2007 


Reza Ghassemi and DIABETES MELLITUS CONTRIBUTES TO THE WEIGHT 

Jonathan Willner 

DIFFERENCE IN FELIS DOMESTICUS 

112 

Madina Ali and 

THE EFFECTS OF VITAMINS A, C, AND E ON THE 

Rhonda Cheikh 

GERMINATION OF RADISH SEEDS 

113 

Nicole K. Baumgartner THE EFFECTS OF ACID RAIN ON O 2 PRODUCTION IN ELODEA 

and Karl M. Neil 

(Elodea camadensis) 

113 

Larry T. Lam EFFECT OF NORMAL SALINE AND CONTACT LENS SOLUTION 

ON THE EPITHELIUM OF THE CORNEA 

113 

Kasra Abolhosseini and 

Harrison Pham 

THE EFFECT OF JADE PLANT ON ESCHERICHIA COLI 113 

vii 



Fall 2007 Biology 3A Abstracts 

Growth Difference in the Goldfish, Carassius auratus, Exposed to Different Diets with 

Varying Levels of Proteins and Carbohydrates 

Anne Merielle Ebol and Shanon Carney 



Mission Viejo, California USA 

Good quality food is necessary to achieve adequate health and nutrition for all living 

organisms. Carbohydrates and proteins are two main components found in food, which 

are required for proper metabolism and growth. Several studies have been performed on 

goldfish to determine whether maximum growth based on weight and body size was 

achieved with a diet higher in protein or with a diet higher in carbohydrate. In the current 

study, fifteen goldfish were weighed before and after exposure to different diets consisting 

of various ratios of proteins and carbohydrates. While both food components play a role in 

growth, a difference in body mass was observed in the goldfish while exposed to the 

different food contents. Group A was given a diet higher in protein, Group B was given a 

diet higher in carbohydrate and Group C was given a diet containing both elements. The 

diets were administered for a period of one month and changes in body mass were 

evaluated every week. The average final weight measurements were: 1.76 ± 0.02 g (±se) for 

Group A, 1.61 ± 0.03 g (±se) for Group B, and 1.44 ± 0.03 g (±se) for Group C. The 

percentage weight gained for Group A was 15.03% from the high protein diet, Group B 

was 8.78% from the high carbohydrate diet, and Group C was 2.13% from the combo diet. 

Results indicated that there is a difference in growth in the goldfish, Carassius auratus, 

when given different diets with varying components (p


carbohydrates improved growth and feed utilization 

of the fish (Tan et al, 2006). 

Although both proteins and carbohydrates 

contribute to the development of the organism, the 

influence of each nutrient is not essentially equal to 

the other (Lovell, 1991). Since each component has 

various effects on the development of the fish, its 

effect on growth was evaluated. In the current study, 

growth of fifteen juvenile goldfish was observed 

before and after exposure to three different diets: a 

high protein, a high carbohydrate and a combination 

diet comprising of both elements. Growth was 

measured as an increase in body size, change in body 

condition and overall weight gain. As mentioned 

earlier, each nutrient has variable influence on 

growth; therefore, there should be a difference in 

growth or in the net weight gain of the goldfish 

between the different diets. 

Materials and Methods 

Experimental Diets 

Fifteen juvenile goldfish obtained from PetSmart 

at Mission Viejo, California were used in the study 

and were maintained in aerated, filtered water in 

three separate glass aquarium tanks. The goldfish 

were divided evenly into the three separate tanks and 

each tank was designated a different diet. Group A 

were given Micro Pellets (Kyorin Food Industry, 

Japan) with high protein content, Group B were given 

Baby Brine Shrimp Cubes (San Francisco Bay 

Manufacturing; Newark, California) with high 

carbohydrate content, and Group C were given 

TopFin Flakes (Pacific Coast Distributing, Phoenix), 

the control treatment, which contains both 

carbohydrates and proteins. 

When coupled with organic wastes discharged by 

the fish, excess feed in the tanks decreases dissolved 

oxygen in the water, which also decreases appetite 

and growth rate; hence, tanks were cleaned everyday 

before administering new sets of food, to ensure 

suitable environment for survival (Cacho et al, 1991). 

Weight Measurements and 

Calculations 

Each fish was weighed before the trial. Fish was 

removed from the tank using a net and was placed 

into a bucket containing tank water to carry out the 

measurements. Each fish was placed in a tared glass 

container of tank water on an electronic balance 

(Biology Department, Saddleback College) for body 

weight measurements, before being returned to their 

original tanks. 

Environmental changes, such as changes in 

temperature or pH levels, can cause stress to the 

normal physiology of the fish (Smith, 1966). In order 

to minimize stress, the groups were allowed to 

acclimatize to their new environment for one week 

prior to exposure to the different diets. After 

acclimatization for one week, each group was 

introduced to its new diet and was fed 1 gram of the 

food every 12 hours. Each group was given the same 

type of food for one week and subsequent weight 

measurements were made every week using an 

electronic balance. The study was conducted at room 

temperature (25–30 ºC) for four weeks in Mission 

Viejo, California starting February 24, 2008 to March 

12, 2008 and changes in growth were evaluated. 

Percentage of weight gained was obtained by using 

the nutritional index (Eq.1) (Bandyopadhyay et al, 

2005): 

Equation 1. 

Net Weight Gain = Final weight – Initial weight 

Weight Gain (%) = Net Weight Gain 

Food Analysis 

Initial weight 

X 100 

Percentage % 

Protein Carbos Fat Fiber Phosph 

orus 

(A) 49.0 20.0 4.0 0.8 2.0 

(B) 3.0 42.0 1.5 0.2 0.1 

(C) 39.0 43.0 9.0 2.0 1.0 

Table 1. Percent composition of diets administered 

to fish during trial. Percentage of vitamins and other 

minerals are not included in the overall composition 

of the food. Values acquired from each food 

container. 

Results 

Average initial weight measurements were 1.53 ± 

0.24 g for Group A, 1.48 ± 0.12 g for Group B and 

1.41 ± 0.07 g for Group C. Growth difference 

between the three experimental diet groups was 

demonstrated by the average final weight 

measurements obtained from the fish in comparison 

to its initial weight (Table 2). The results of the 

study indicate that there was a significant difference 

in growth observed between the groups of fish when 

9 


Spring 2008


given different types of diet, (p


Food preference and required nutrition 

change over the course of fish development and 

the types of food that are beneficial for the 

growing juvenile fish may not essentially be 

beneficial for the adult (Lovell, 1991). Aside 

from the purpose of determining the best food 

source, future experiments might include 

studying the difference in nutritional 

requirements of juvenile and adult goldfish to 

determine whether assimilation rates or 

absorption efficiencies vary between the two 

groups. 

Literature Cited 

Bandyopadhyay, P, Swain, S, and Mishra, S (2005). 

Growth and dietary utilization in goldfish fed 

diets formulated with various local agro-produces. 

BioResource Technology. 96: 731-740. 

Cacho, O, Kinnucan, H, and Hatch, U (1991). 

Optimal control of fish growth. American Journal 

of Agricultural Economics. 73(1): 174-183. 

Garling, D, and Wilson, R (1977). Optimum 

dietary protein to energy ratio for channel catfish 

fingerlings, Ictalurus punctatus. Journal of 

Nutrition. 106(9): 1368-1375. 

Kaiser, H, Endemann, F, and Paulet, T (2003). A 

comparison of artificial and natural foods and 

their combinations in the rearing of goldfish. 

Aquaculture Research. 34: 943-950. 

Lopez, R (2006). Exploring the assimilation rate of 

goldfish: a comparison of protein and 

carbohydrate diets. Biology Journal. 1: 273-282. 

Lovell, R (1991). Nutrition of aquaculture species. 

Journal of Animal Science. 69(10): 4193-4200. 

Lovell, T (1998). Nutrition and feeding of fish. 

Massachusetts, US: Kluwer Academic Publishers. 

266p. 

Priestley, S, Stevenson, A, and Alexander, A 

(2006). Growth rate and body condition in 

relation to group size in black widow Tetras 

(Gymnocorymbus ternetzi) and common Goldfish 

(Carrasius auratus). Journal of Nutrition. 136(7): 

2078S-2080S. 

Smith, M.W (1966). Influence of temperature 

acclimatization on sodium-glucose interactions in the 

goldfish intestine. Journal of Physiology. 182: 574- 

590. 

Tan, Q, Xie, S, Zhu, X, Lei, W and Yang, Y 

(2007). Effect of dietary carbohydrate-to-lipid 

ratios on growth and feed utilization in Chinese 

longsnout catfish (eiocassis longirostris). J. Appl. 

Ichthyol. 23: 605-610 

The Effect of a Lactate Supplement on Maximal Cycling Performance in Man 

Kyle Lutz and John I. Miller 




High lactate concentrations have traditionally been considered a causative factor in 

muscle fatigue during strenuous exercise in man. Recent research has challenged this view. 

A lactate supplement (SportLegs®) claims to increase time until fatigue in strenuous 

activity. Our study seeks to determine if supplementation with lactate before strenuous 

activity will increase time until fatigue in cyclists. Cycle power and time to failure were 

measured in two trials: the first unsupplemented and the second supplemented. Mean time 

to fatigue in trial 1 was 14.1 minutes and in trial 2 was 14.0 minutes. The supplement did 

however increase time to peak lactate production. Other studies have shown that high 

lactate concentrations have an insignificant effect on muscle fatigue and that other factors 

may be more important (Nielson 2003, Bangsbo 1992). We found that there was no 

significant difference in time to fatigue or maximum power output between cyclists taking 

the lactate supplement and those that did not. Our results strengthen the notion that 

lactate has a minimal effect on muscle fatigue. 

11 




Introduction 

It has widely been accepted that lactate build 

up contributes to fatigue during exercise. More 

recently, however, that notion has been increasingly 

challenged. During exercise, initially lactate 

concentrations rise rapidly. But with continued, 

submaximal exercise, there is a net usage of lactate. 

Lactate plays a role in gluconeogenesis and is 

shuttled from exercising and non-exercising muscles 

to splanchnic regions where it may be converted to 

glucose for use by the exercising muscles (Ahlborg, 

1982). 

Lactate is a by-product of glycolysis 

produced when oxygen levels are insufficient to 

metabolize pyruvate via the Citric Acid Cycle. 

Gluconeogenesis may consume lactate in splanchnic 

tissues to fuel active muscles during exercise. 

During prolonged (120 minutes), moderate intensity 

(30% and 58% maximal oxygen consumption, 

VO 2 max) exercise, a rise in splanchnic glucose 

output relative to work load has been associated with 

a 60 – 100% increase in the splanchnic uptake of 

gluconeogenic precursors, including lactate (Alhborg, 

1986; Ahlborg, 1982). In fact, after periods of 

moderate intensity exercise, previously active 

muscles become net consumers of lactate (Gladden, 

2004). 

During short bouts of intense activity (>60% 

VO 2 max) arterial lactate concentrations increase 

rapidly (Ahlborg, 1982). This results in an increased 

intramuscular lactate concentration ([La - ]) and net 

output of lactate into the blood. Whether this intense, 

short-term increase in [La - ] has a negative affect on 

muscle contractility or not is still a topic of debate. 

Research has shown that when [La - ] is kept at ~ 4 

mMolar by exogenous La - infusion, during moderate 

intensity exercise, lactate competes effectively with 

glucose as a carbohydrate fuel source, sparing 

glucose (Gladden, 2004). 

In addition to the effects of [La - ] on muscle 

performance, other factors such as concentration of 

hydronium ion ([H + ]) (pH), and potassium ion [K + ] 

have been proposed as potential mechanisms of 

fatigue. [K + ] at the onset of fatigue and ultimately 

muscle failure may be constant in trained and 

untrained human subjects. A delayed onset of this 

[K + ], and fatigue, has been associated with trained 

subjects (Nielsen, 2003). Studies have reported that 

lactic acidosis can protect against negative effects of 

altered [K + ] on muscle excitability and force 

(Gladden, 2004). 

While H + transport seems to be coupled with 

La - transport in inactive muscles (Bangsbo, 1995), H + 

transport (efflux out of the muscle cell) seems to 

exceed and be somewhat independent of La - transport 

in intensely active muscles (Bangsbo, 1993). Where 

some research has shown that high [H + ] may increase 

fatigue, other research illustrates that under normal 

physiological conditions the negative effects of 

elevated [H + ] are absent (Gladden, 2004). If a 

correlation exists between H + and La - co-transport, 

then a decreased [H+] by buffers in inactive muscle 

(Bangsbo, 1995) might impact [La - ], and thus impact 

energy production via gluconeogenesis. The 

mechanism for shuttling lactate between cells and 

tissues is still undetermined (Gladden, 2004). 

The SportLegs® supplement’s proposed 

mechanism states that by increasing lactate 

production prior to exercise, the body is stimulated to 

begin consuming lactate, therefore delaying the onset 

of fatigue from high [La - ]. While the net consumption 

of lactate by exercising muscle has been well 

documented with moderate intensity (


blood lactate was measured (see following 

paragraph) with the Lactate Scout using blood taken 

from the finger. They continued this until exhaustion. 

Blood lactate concentration was measured again 

every minute for the first five minutes during 

recovery. 

Each subject was required to draw blood 

using a lancet from one of the fingers. The Lactate 

Scout uses testing strips that require a 0.5 uL whole 

blood sample. The Lactate Scout automatically 

analyzes blood and displays a lactate concentration 

that is accurate between 0.5 mM and 25.0 mM [La - ] 

and gives results within 15 seconds. 

All readings for heart rate and power output 

are from the Saris Powertap and were obtained with 

the Saris Poweragent (Saris Company, Madison, 

Wisconsin) software. The Poweragent software 

converted data to comma-separated-values (*.csv) 

and all analyses were performed using Microsoft 

Excel (Microsoft Corporation, Redmond, 

Washington). A paired T-test was used to evaluate 

the differences between the two trials. 

Results 

Time until failure with and without the 

supplement had little variance, the largest change in 

an individual participant was an improved 

performance of 1.9 minutes. The mean time to failure 

without the supplement was 14.1 minutes and with 

the supplement was 14.0 minutes (Figure 1). Overall 

there was no statistically significant increase in time 

to failure between participants riding with the 

supplement and without the supplement. The p-value 

is 0.419 (One-tailed T-Test). 

Time Until Failure (m) 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

1 2 3 4 5 

Participant 

Without Supplement 

With Supplement 

Figure 1. Time to failure for each participant with 

and without the SportLegs lactate supplement. 

Use of the supplement increased the blood 

lactate levels in the participants. The mean lactate 

level without the supplement was 8.08 mM (Figure 2) 

and with the supplement was 9.89 mM (Figure 3). 

Time to peak lactate was significantly 

greater with supplementation (One-tailed Students T- 

test, p < 0.05). Exogenous lactate did delay muscle 

lactate saturation in the cyclists (Figure 2 and Figure 

3). 

16.0 

14.0 

12.0 

10.0 

8.0 

6.0 

4.0 

2.0 

0.0 

0 5 10 15 20 

Time (min) 

Figure 2. Blood lactate concentration 

during exercise in each cyclist without the lactate 

supplement. Mean lactate without the supplement 

was 8.07 mM. Cyclists in this group rode for a mean 

time of 14.1 minutes. A unique shape denotes each 

individual participant. 

30 

25 

20 

15 

10 

5 

0 

0 5 10 15 20 25 

Time (min) 

Figure 3. Blood lactate concentration in 

each cyclist during exercise with the lactate 

supplement. Mean lactate with the supplement was 

9.89 mM. Cyclists in this group rode for a mean time 

of 14.0 minutes. A unique shape denotes each 

individual participant. 

Discussion 

This study looked at the effects of 

exogenous lactate on muscle fatigue in cyclists. 

While supplementation (Trial 2) did increase arterial 

lactate concentration ([La - ]), there was no significant 

change in the time to failure during short bouts of 

near maximal intensity cycling between trials with 

supplementation (Trial 1) and without 

supplementation (Trial 2). 

Recent research indicates that during longterm, 

moderate intensity exercise lactate may be an 

important intermediate metabolite. Although short 

13 




bouts of exercise result in an accumulation of lactate, 

that accumulation has not been directly related to 

fatigue or performance. 

Traditionally, lactate was considered to be a 

waste product resulting in fatigue. Previous studies 

have shown that the detrimental effects of elevated 

[La - ] may be due more to [H + ] and [K + ]. Although 

[H + ]’s role in the onset of fatigue is highly debatable, 

the role of the [K + ] on fatigue seems to be more well 

documented. Nielsen, 2003, determined that muscle 

fatigue during exercise is caused, at least in part, by 

high interstitial potassium levels. Here again, [La - ] 

and lactic acid may help to maintain a proper [K + ] 

balance, reducing fatigue (Gladden, 2004). 

Although performance was not improved by 

La - supplementation, negative effects of a high [La - ] 

were also absent. During short-bouts of near maximal 

intensity exercise, blood [La - ] has no effect on 

performance. The findings in this research are 

consistent with others in that the contribution of [La-] 

to fatigue is insignificant (Alhborg, 1986; Ahlborg, 

1982; Gladden, 2004; Bangsbo, 1993). High [La-] 

associated with fatigue in other studies may have to 

do with other factors such as decreased ability of the 

body to undergo gluconeogenesis effectively. 

Ahlborg, G., Felig, P. (1982). Lactate and glucose 

exchange across the forearm, legs, and splanchnic 

bed during and after prolonged leg exercise. Journal 

of Clinical Investigation, 69, 45-54. 

Gladden, L. B. (2004). Lactate metabolism: A new 

paradigm for the third millennium. Journal of 

Physiology, 558.1, 5-30. 

Bangsbo, J., Johansen, L., Graham, T., Saltin, B. 

(1993). Lactate and H + effluxes from human skeletal 

muscles during intense, dynamic exercise. Journal of 

Physiology, 462, 115-133. 

Bangsbo, J., Aagaard, T., Olsen, M., Kiens, B., 

Turcotte, L. P., Richter, E. A. (1995). Lactate and H + 

uptake in inactive muscles during intense exercise in 

man. Journal of Physiology, 488.1, 219-229. 

Nielsen, J. J., Mohr, M., Klarkov, C., Kristensen, M., 

Krustrup, P., Juel, C., Bangsbo, J. (2003). Effects of 

high-intensity intermitten training on potassium 

kinetics and performance in human skeletal muscle. 

Journal of Physiology, 554.3, 857-870. 


Alhborg, G., Wahren, J., Felig, P. (1986). Splanchnic 

and peripheral glucose and lactate metabolism during 

and after prolonged arm exercise. Journal of Clinical 

Investigation, 77, 690-699. 

The Effect of Silver Nitrate on the Inhibiting Growth of Escherichia coli 

Stephanie Anstadt and Teo Fernandez 

Department of Biological Science 



During this experiment the effect of silver nitrate on the inhibition growth of 

Escherichia coli was studied. An agar solution was made and placed into a total of fifteen 

petri dishes. For this experiment a 0.5% silver nitrate solution was made from a 1.0% 

solution and were both used for this study. A controlled group with no silver nitrate 

solution was used. 0.5mL of E. coli were pipetted with a P1000 micro pipette and 

incorporated into the agar from which a lawn was made. The corresponding silver nitrate 

solution was incorporated into each correctly labeled petri dish by using chads. All dishes 

were placed into a 37°C incubator and results were read every 48 hour period. The 

hypothesis being tested in this experiment was supported by the results obtained during the 

second 48 hour period in which p= 0.008. 

Introduction By conducting this experiment the 

investigators were able to achieve a better 

14 




understanding of using silver nitrate (AgNO 3 ) to 

inhibit the growth of the bacteria Escherichia coli. 

An article published by Nataro and Kaper (1998) in 

the Clinical Microbiology Reviews, Escherichia coli 

is the predominant nonpathogenic facultative flora of 

the human intestine. Some E. coli strains, however, 

have developed the ability to cause disease of the 

gastrointestinal, urinary, or central nervous system in 

even the most robust human hosts. This makes quite a 

challenge in the inhibiting treatment of E. coli. 

According to Feng and Wu (2000) on the 

antibacterial effect of silver ions on E. coli and 

Staphylococcus aureus after the use of silver ions 

there were morphological changes in both bacteria. 

Silver is an agent known to have antibacterial 

properties and its inhibiting effect on E. coli would 

be a very cost effective way to treat outbreaks of this 

bacteria. Investigators Sondi and Sondi (2004) at the 

Center for Marine and Environmental Research 

found that nanosized silver particles damaged E. coli 

cells, showing formation of “pits” in the cell wall of 

the bacteria, while the silver nanoparticles were 

found to accumulate in the bacterial membrane. A 

membrane with such morphology exhibits a 

significant increase in permeability, resulting in the 

death of the cell. Schreurs and Rosenberg (1982) 

concluded that silver ions inhibit the respiratory chain 

of E. coli possibly at two sites of different 

sensitivities, and were also reported, in the journal of 

bacteriology, to exert an uncoupler-like action. It 

seems that silver will affect the E. coli in some way 

most often damaging the replication process and 

contributing to the death of the cell. Rosenkranz and 

Carr found that silver sulfadiazine blocked 

macromolecular synthesis in treated bacteria. Silver 

has been a known antibacterial agent against gram 

negative bacteria and could also have an effect on the 

gram positive bacteria E. coli. Gram positive bacteria 

is very resistant to antibiotic treatment. According to 

Jack and Tagg (1995) the journal of microbiology 

and molecular biology reviews published an article 

on the bacteriocins of gram positive bacteria and 

stated that the antibacterial action against a sensitive 

cell of a gram-positive strain is produced principally 

by destabilization of membrane functions. Silver 

nitrate is a chemical agent with properties that may 

demobilize the growth of E. coli. The hypothesis for 

this experiment is that the agar with the highest 

concentration of Silver Nitrate will have the greatest 

effects on inhibiting the growth of Escherichia coli. 

We anticipate the inability for the E. coli eukaryotic 

cells to replicate with the treatment of silver nitrate. 

department were used to prepare an agar solution 

within 500mL of water to determine the inhibition 

growth of Escherichia coli. The agar solution was 

introduced to fifteen petri dishes all correctly labeled 

to the amount of silver nitrate they were introduced to 

(0%, 0.5%, and 1.0%). After the agar solution 

hardened, 0.5mL of Escherichia coli were pipetted 

with a P1000 micro pipette onto each plate and 

spread with a sterile glass rod to form a lawn. The 

glass rod was dipped into ethanol, flamed, and cooled 

in between each plate. Following the incorporation of 

E. coli silver nitrate was placed in both the 0.5% and 

1.0% labeled plates. The 0% sample was used as a 

control group and left without silver nitrate. 

A 1.0% silver nitrate solution was obtained 

and used to make a 0.5% silver nitrate solution by 

diluting 1.0mL of it into 1.0mL of water. Chads were 

used to incorporate the corresponding silver nitrate 

solution into the correctly labeled petri dish. There 

were a total of four chads placed across from each 

other into each dish. The same procedure was 

followed for the remaining concentrations. Before 

placing a chad in each plate forceps were sterilized 

by dipping them into ethanol, placing them over a 

flame, and letting them cooled in between each plate. 

This process was strictly followed in order to avoid 

any possible contamination that could interfere with 

the case being studied. All petri dishes were placed 

into a 37°C incubator and results were interpreted 

within a 48 hour period. After each 48 hour period 

silver nitrate formed a perimeter of inhibition around 

each chad. The perimeter formed was measured in 

millimeters and used to determine the inhibition 

growth of E. coli. 

Results 


Ten grams of nutrient agar obtained from 

the Saddleback College Biological Science 

15 




Figure 1. Average inhibition growth of E. coli after the 

first 48 hour period. Error bars indicate SE. Average 

inhibited growth of E. coli in 1.0% AgNO3 was 0.52 ± 0.11 

(SE). The 0.5% AgNO3 sample obtained an average 

inhibition of 0.26 ± 0.02 (SE). A two tailed t- test was 

calculated to have a p value of 0.08. 

The inhition growth of E. coli under the two 

types of silver nitrate solutions appeared to increase 

within the 1.0% sample over a 96 hour period as 

shown in Figure 2, but started to remain constant 

within the 0.5% silver nitrate samples. Although the 

inhibition growth increased in both concentrations 

there was no net significance in the first trial, P value 

was more than 0.05 (p = 0.08). The experiment was 

run for 48 hours longer to further determine if there 

was a significance in inhibition. After the second trial 

p= 0.008, which indicated a statistical significance in 

the inhibition growth of E. coli under the 1.0% 

solution. The final results confirm our initial 

hypothesis and conclude that there is a statistical 

difference in the inhibition growth of E. coli under 

both silver nitrate solutions. 

concentration. According to an article by Wong and 

Jelacic (2000) in the New England Journal of 

Medicine, individuals that were treated with 

antibiotics for E. coli infection experienced 

symptoms of hemolytic uremic syndrome. Factors 

significantly associated with the hemolytic–uremic 

syndrome were a higher initial white-cell count 

(relative risk, 1.3; 95 percent confidence interval, 1.1 

to 1.5), evaluation with stool culture soon after the 

onset of illness (relative risk, 0.3; 95 percent 

confidence interval, 0.2 to 0.8), and treatment with 

antibiotics (relative risk, 14.3; 95 percent confidence 

interval, 2.9 to 70.7). Through trial and error 

investigators can uncover the most effective 

antibacterial agent to ward off E. coli. The 1.0% 

silver nitrate concentration did exhibit growth 

inhibition, as well as the 0.5% silver nitrate 

concentration and the results concluded that there is a 

significant difference with the use of silver nitrate. 

Previous investigators had similar findings in with 

the use of silver derivatives such as Feng and Wu, 

who concluded that morphological changes took 

place in bacteria treated with silver ions. Jack and 

Tagg also found that gram positive strains of bacteria, 

such as E. coli, can only by inhibited by 

destabilization of membrane functions. A function 

known of silver nitrate is to cause destruction of the 

cell according to Sondi and Sondi. This knowledge is 

a positive indication that silver nitrate is effective in 

the inhibition of E. coli, and if it could be derived in 

antibiotic form to treat infected humans, it would be a 

turning point in the field of medicine and bacterial 

control. 


Feng, Q.L. and Wu, J. (2000). A mechanic study of 

the antibacterial effect of silver ions on Escherichia 

coli and Staphylococcus aureus. Journal of 

Biomedical Materials Research. 52: 662-668. 

Figure 2. Average inhibition growth of E. coli after the 

second 48 hour period. Error bars indicate SE. Average 

inhibited growth of E. coli in 1.0% AgNO3 was 0.57 ± 0.06 

(SE). The 0.5% AgNO3 sample obtained an average 

inhibition of 0.26 ± 0.01 (SE). A two tailed t- test was 

calculated to have a p value of 0.008. 

Discussion 

Growth inhibition of E. coli and many other 

bacteria has been an issue at large in the medical field 

and investigators continue to pursue new research 

methods to improve the modes of treatment. The 

results of this trial concluded that there is a 

statistically significant difference in the inhibiting 

growth of E. coli between the 0.5% silver nitrate 

concentration and the 1.0% silver nitrate 

Jack, R.W. and Tagg J.R. (1995). Bacteriocins of 

gram positive bacteria. Microbiology and Molecular 

Biology Reviews. 171-200, Vol 59, No. 2 

Nataro, J and Kaper, J. (1998) Diarrheagenic 

Escherichia coli. Clinical Microbiology Reviews. p. 

142-201, Vol. 11, No. 1 

Schreurs, W.J. and Rosenberg, H. (1982) Effect of 

silver ions on transport and retention of phosphate by 

Escherichia coli. Journal of Bacteriology. 152: 7-13 

Sondi, I. and Sondi, B. (2004) Silver nanoparticles as 

antimicrobial agent: a case study on E. coli as a 

model for Gram-negative bacteria. Journal of Colloid 

and Interface Science. 275: 177-182 

16 




Wong, C. and Jelacic, S. (2000). The Risk of the 

Hemolytic–Uremic Syndrome after Antibiotic 

Treatment of Escherichia coli O157:H7 The New 

England Journal of Medicine. Volume 342:1930- 

1936 

Effect of Tide Level on Nitrate and Phosphate Concentration in Marine Water 

Nathaly Leal- Arteaga and Saori Shimamoto 




It is known that several factors such as tide level, temperature, water nutrient level, 

seasons and salinity affect phytoplankton activity. The objective of this study was to see 

the relationship between the tide level and the concentration of nitrogen gas and 

phosphorous ions, both of which affect the phytoplankton level in marine environments. 

The sea water was collected at Dana Point Harbor off the California coast on April 17, 

2008. A DR/850 Colorimeter was used to measure the concentration of two ions. Three 

10mL water sample were observed and the average value was analyzed. The nitrate ion 

concentration was 1.07 ± 0.3 ppm (± se) at low tide and 1.5 ± 0.1 ppm (± se) at high tide. 

The mean phosphate acid concentration resulted 0.28 ± 0.02 ppm (± se) (N=3) at low tide 

and 0.77 ± 0.09 ppm (± se) (N=3) at high tide. Both nitrate ions and phosphate acid levels 

increased as the sea level increased; however, there was no significant difference between 

low tide and high tide in the nitrate ion concentration (p>0.05). 

Introduction 

Combined inorganic nitrogen gas and 

phosphorus acid is the major limiting nutrient in 

many aquatic ecosystems (Small et al, 1989). 

Nitrogen fixation is the major way for blue-green 

algae and phytoplankton to precede the nitrogen 

metabolism by reducing the atmospheric nitrogen to 

ammonia. Nitrogen fixation is related to blue-green 

algal blooms, nitrogen compounds in lakes, and the 

role of the heterocyst (Horne et al, 1972). Heterocyst 

is the section where the cells in a filament carried out 

only by nitrogen fixation. Horne et al (1972) 

represented the role of the transparent heterocyst cell 

giving good measurement rates of nitrogen fixation 

by Anabaena; a freshwater algae that contaminate the 

water with a fishy odor and taste. 

Nitrogen gas ends up in the environment 

mainly through agricultural processes, and thereby 

also ends up in the ocean. The most widely applied 

nitrogen fertilizers is sodium nitrate; these fertilizers 

mainly contain nitrate, ammonia, urea, ammonium 

ions and amines are adding to the abundance of 

nitrogen compounds found in water masses, such as 

lakes, oceans and rivers. After fertilization, crops use 

a relatively small amount of added nitrogen 

compounds (Horne et al, 1972), therefore leaving the 

rest to run off into the water. 

Not only do the nutrients such as nitrogen gas 

and phosphorus gas control the phytoplankton 

population but other marine systems affect the water. 

May et al (2003) sustained observations and 

experimentations in South San Francisco Bay with 

numerical modeling analyses to search for general 

principles that define phytoplankton population 

responding to physical dynamics. Characteristics of 

shallow nutrient-rich coastal waters, tides, wind and 

the flow of water influence the phytoplankton 

concentrations. May et al (2003) indicated in their 

study that the sensitivity of estuarine phytoplankton 

dynamics to spatial and temporal variations in 

17 




turbidity depends on available light, rather than 

nutrients limiting phytoplankton growth. 

Large amounts of nitrate ions and phosphate 

gases may cause eutrophication, an excess of 

nutrients resulting in the abundance of photosynthetic 

algae and plankton causing a serious effect on the 

marine organisms’ life. Domoic acid (C 15 H 21 NO 6 ) is 

a naturally occurring amino acid phycotoxin 

produced by algae and plankton in all marine coasts 

(Pan et al, 2008). This extreme chemical 

proliferation is triggered by temperature in seasonal 

algae blooms during the months of March and June 

contaminating phytoplankton, shellfish and sardines 

which later poison lipid rich sea mammals. California 

sea lions are primarily affected by domoic acid; this 

neurotoxin attacks the hippocampus part of their 

brain causing memory loss, blindness and seizures 

that will lead to death. Borchelt (1997) has a handful 

of possible explanations for the increase of toxic 

tides: increase in the amount of nitrogen gas, 

phosphorus gas and other nutrients are disposed from 

land fertilizers and animal waste; sewage and effluent 

pollution in oceans. 

Temperature, salinity, wave conditions, sea 

levels caused by high tide and low tide influence 

phytoplankton activity- Nitrogen gas and Phosphate 

ion levels are well known to increase phytoplankton 

concentrations in ocean waters (Lehman, 2006). 

Therefore, in this study, nitrogen gas and phosphate 

ion levels are being tested to determine the 

relationship between tides and the abundance of 

phytoplankton which lead to severe toxin 

concentrations. 


This experiment was conducted over a period 

of three days in April of 2008. Water samples were 

collected behind the Ocean Institute at the Dana Point 

Harbor in Southern California and the analysis were 

performed in the laboratory at Saddleback College, 

Mission Viejo, CA. On Sunday April 6, 2008, three 

sterilized bottles were used to collect sea water 

during high tide. According to 2008 Dana Point 

Harbors Tide Calendar, high tide was at 9:30 am with 

a 4.7 MSL (mean sea level) which are recorded by 

stationed tide clocks and gauges. The bottles were 

then placed in a refrigerator to prevent bacterial 

growth. Later that day at 3:50 pm three other 

sterilized bottles were used to collect water samples 

during low tide, 0.6 MSL. The samples were also 

placed refrigerator to obtain accurate results. Samples 

were taken to laboratory to examine on April 7, 

2008. 

A DR/850 Colorimeter (Hach Company, CO. 

U.S.A) was used to measure the concentration of 

dissolved nitrate ions and phosphate ions in the water 

samples. This devise had several settings and packets 

that contain compounds that create a reaction; a blank 

test tube with sample water was always needed to 

calibrate the calorimeter between readings. Nitrogen 

was first tested, with a Nitra Ver 5 Nitrate packet 

mainly containing: Cadmium, Gentisic Acid, 

Magnesium Sulfate, Potassium Phosphate, 

Monobasic and Sulfanilic Acid. Using the low tide 

samples ten mL of sea water was placed into three 

special glass bottles with the Ver 5 Nitrate packets, 

they were shaken vigorously for one minute and were 

set aside for five minutes while the Colorimeter read 

the blank sample which was then zeroed out (0.0 

mg/L No3-N). The water samples were also tested for 

Phosphorus; a 3 Phosphate Reagent packet was used 

that contains: Ascorbic Acid, Potassium Pyrosulfate 

and Sodium Molybdate. Using low tide samples, 10 

mL of each bottle was placed in three special bottles 

that the colorimeter could read. Phos Ver 3 powder 

was added to each test tube, shaken for 15 seconds, 

were then left to stand for two minutes to allow the 

reaction occur. The blank tube was placed in the 

colorimeter to zero out (0.0 mg/L P04). Each water 

sample was placed in the device to be read. 

For both nitrogen gas and phosphorus gas, the 

average concentrations of the three measurements 

were recorded by summing all the three values and 

dividing by three. Those average values were 

demonstrated on bar graphs using Microsoft Excel 

2007 to compare the concentration differences at low 

tide and high tide. 

Results 

The nitrate ion concentration measurements were 

taken from the three water samples from low tide and 

were placed into the digital colorimeter that measures 

in gram per liter (mg/L) which is also known as parts 

per million (ppm). The samples displayed 1.6 mg/L, 

0.8mg/L and 0.8mg/L with an average concentration 

of 1.07 ± 0.3 ppm (± se) (N=3). For high tide, the 

nitrate concentration was measured to be 1.6mg/L, 

1.3 mg/L and 1.6mg/L with a mean of 1.5 ± 0.1 ppm 

(± se) (N=3). T-test was performed to see whether 

the difference is significant or not; resulting with a p- 

value of 0.1, the concentration of nitrate ions at low 

tide and high tide was not significantly different 

(Figure 1). 

Phosphate ion concentration was measured in 

the same manner for both low and high tide. The 

samples displayed 0.29 mg/L, 0.32 mg/L and 0.24 

mg/L and that average concentration was 0.28 ± 0.02 

ppm (± se). For high tide, the phosphate ion 

concentration sample was 0.57 mg/L, 0.69 mg/L, and 

0.94 mg/L. Due to the high concentration of 

phosphate ions between low and high tide, the water 

18 




turned to a bluish clear color with blue color 

precipitate floating in the water. 

Conc of N (ppm) 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

low 

high 

Figure 1. There was no difference in nitrate concentration 

between low tide (1.07 ± 0.3 ppm (± se) and high tide (1.5 

± 0.1 ppm (± se), (p = 0.1, one tailed t- test). Error bars 

show the standard error. 

1 

Conc of P (ppm) 

0.8 

0.6 

0.4 

0.2 

0 

low 

high 

Figure 2. Phosphate concentration was greater during 

high tide (0.77 ± 0.09 ppm (± se) than low tide ( 0.28 ± 

0.02 ppm (± se,) (p= 0.02, one-tailed t-test). Error bars 

indicate the standard error. 

Discussion 

For nitrate ion concentration was 1.07 ± 0.3 

ppm (± se) at low tide and 1.5 ± 0.1 ppm (± se) at 

high tide. Although hypothesis stated that nitrate ion 

concentration and the tide level were corresponding, 

the difference of nitrogen gas concentration at low 

tide and high tide were not significant. The 

concentration of nitrogen did not depend on the level 

of tide in this study. The result may suggest that 

dissolved nitrogen gas concentration varies 

depending on the season of the year or water 

conditions; for example salinity, temperature and 

tides. 

In this study, the phosphate concentration 

showed the relatively high value. For phosphate 

concentration, it was 0.3 ± 0.02 ppm (± se) at low 

tide and was 0.77 ± 0.09 ppm (± se) at high tide. 

Increased rate of phosphate supply led to the growth 

of two species of photosynthetic alga, O. agardhii 

and A. Formosa (Tilman et al, 1982). Tilman et al 

(1982) proved that nitrogen and phosphate limitation 

was the factor which led to a succession of algal 

growth. 

Horne et al, (1972) measured dissolved 

nutrients in the lake. In Oaks arm, nitrogen fixation 

was very low, but much higher in the two basins 

where high nitrogen fixation was occurring. This 

observation suggests that the possibility of a lasting 

nitrogen gas and phosphate gas limitation in Oaks 

arm following the peak of the bloom at the end of 

August. In contrast, organic dissolved nitrogen 

showed no significant variation. Horne et al (1972) 

provide a reasonable answer as to why low nitrogen 

concentration was found at certain times and only in 

non oligotrophic lakes. In oligotrophic lakes, these 

conditions of some combined inorganic nitrogen and 

sufficient dissolved organic nitrogen may never 

occur. In stratified non-oligotrophic lakes, the 

condition is normally only provided toward the end 

of a spring bloom and during autumn overturn. In 

non-stratified, non-oligotrophic lakes, the condition 

may occur at irregular intervals throughout the year 

were dependent on nitrogen turnover rates. Carbon, 

Nitrogen and Phosphorus are the chemical building 

blocks for living organism, therefore nitrogen 

fixation is a natural process that is needed in order for 

all organisms to survive, (Tzortziou, 2007 ) but the 

over use of nitrogen gas becomes a toxin that may 

also harm marine life. As water was collected and 

nitrogen gas and phosphorous gas levels were 

calculated with a DR/850 Calorimeter (Hach 

Company, CO. U.S.A) from Dana Point Harbor in 

Southern California at both low and high tide; Nitrate 

ions and Phosphorous gas levels did in fact increase 

as sea levels increased. This DR/850 Calorimeter 

devise has several settings and packets that contain 

compounds that create reactions obtaining accurate 

results. The mean of phosphate gas concentration was 

0.28 ± 0.02 ppm (± se) (N=3) at low tide and 0.77 ± 

0.09 ppm (± se) (N=3) at high tide. Nitrate ion 

concentrations were 1.07 ± 0.27 ppm (± se) at low 

tide and 1.5 ± 0.1 ppm (± se) at high tide; although 

there was no significant difference of nitrate ion 

levels between low and high tide. Nitrogen gas that 

ends up in the ocean waters by run offs of farmlands 

are contributing to the outbreak of Domoic acid (Pan 

et al, 1996) ; as the fish eat the polluted plankton, the 

toxins become more potent, then marine mammals 

consume this contaminated food source, affecting 

them tremendously. As a result their bodies can no 

longer digest the toxins and their brain’s 

hippocampus becomes paralyzed resulting in seizures 

19 




that make them too weak to defend themselves from 

other predators or they drown (Lefebvre et al, 1999). 

The ocean and the environment are being 

impacted by the amounts of chemicals and other 

pollutants around our neighborhoods carried via 

storm drains to surface waters which result in beach 

closings, polluted drinking water and endangered 

wildlife (Sixeas, 2000). The public is largely unaware 

of the problem because its effects are gradual and 

mostly hidden. Unlike most pollutants, nitrogen gas 

in reasonable levels is essential nutrient for marine 

life; too little nitrogen is a bad thing, but so is too 

much resulting in a decrease of biodiversity. Every 

individual can take small steps to decrease the use of 

toxic household chemicals and fertilizers. Septic 

systems should be inspected annually and the volume 

of wastewater can be reduced as well. The only way 

that there will be a great impact is if everyone 

educates themselves and those around them into 

taking care of our planet. Overall, this study supports 

the concept that Nitrogen levels and Phosphorous 

levels are affected by both low and high tides in 

Southern California. 


Borchelt, N. (1997). A Blooming Presence. 

Environment, 39 (10), 22-24. 

Horne, A.J, Dillard J.E, Fujita, D.K. and Goldman, 

C.R. (1972). Nitrogen Fixation in Clear Lake, 

California. Synoptic Studies on the Autumn 

Anabaena Bloom. The American Society of 

Limnology and Oceanography, 17 (5), 693-704. 

Lefebvre, K.A, Powell, C.L, Busman, M., Doucette, 

G. J., Moeller, P.D., Silver, J.B., Miller, P.E., 

Hughes, M.P., Singaram, S., Silver, M.W. and 

Tjeerdema, R.S. (1999). Detection of Domoic Acid in 

Northern Anchovies and California Sea Lions 

Associated with an Unusual Mortality Event. Natural 

Toxins, 7(3), 85-92. 

Lehman, P.W. (2006). The Influence of 

Phytoplankton Community Composition on Primary 

Productivity Along the Riverine to Freshwater Tidal 

Continuum in the San Joaquin River, California. 

Estuaries and Coasts, 30(1), 82-93 

May, C.L., Koseff, J. R., Lucas, L.V., Cloern, J. E. 

and Schoellhamer, D.H. (2003). Effects of Spatial 

and Temporal Variability of Turbidity on 

Phytoplankton Blooms. Marine Ecology Progress 

Series, 254, 111-128. 

Pan, Y., Rao, D.S., Mann, K. H., Brown, R.G., and 

Pocklington, R. (1996). Effect of Silicate Limitation 

on Production of Domoic Acid, A Neurotoxin, by a 

Diatom Pseudonitzschia multiseries. I. Batch Culture 

Studies. Marine Ecology Progress Series, 131, 225- 

233. 

Sixeas, V. (2000). Deteriorating Coasts. 

Environment, 42 (6), 6-7. 

Small, L.F, Landry, M.R, Eppley, R.W, Azam, F. and 

Carlucci, A. F. (1989). Role of Plankton in the 

Carbon and Nitrogen Budgets of Santa Monica 

Basin, California. Marine Ecology Progress Series, 

56, 57-74. 

Tilman, D., Kilham, S.S. and Kilham, P. (1982). 

Phytoplankton Community Ecology: The Role of 

Limiting Nutrients. Annual Review of Ecology and 

Systematics, 13, 349-372. 

Tzortziou, M., Neale, P.J., Osburn, C.L., Megonigal, 

J.P., Maie, N and Jaffe, R. (2008). Tidal Marshes as a 

Source of Optically and Chemically Distinctive 

Colored Dissolved Organiz Matter in the Chesapeake 

Bay. The American Society of Limnology and 

Oceanography, 53(1), 148-159. 

20 




Effect of Differing Color on the Timed Length of Aggressive Response of Betta splenens 

Lancelot Beier and Harrison Pham 




Siamese Fighting Fish are famous for their aggressive behavior towards other male 

fighting fish. It is known that fin size has a major effect on the evoked response of the 

opposing fish but few studies have been preformed to test whether or not color has an effect 

on the aggressive response of bettas. This study introduced betta fish to images of other 

male bettas in aggressive display that varied in colors. The aggressive response was 

measured by calculating the mean timed display length per aggressive show for each 

different color stimuli. The mean timed length of aggressive display for green, red, yellow 

and blue stimuli were 7.24 seconds (± 0.833 s.e.), 6.59 seconds (± 0.957 s.e.), 6.95 seconds 

(± 0.678 s.e.) and 7.69 seconds (± 1.306 s.e.) respectively. When compared, statistical 

analyses showed that there was no significant difference between the average lengths of 

aggressive displays when presented with differing colors of stimuli. 

Introduction 

Male Betta splendens, or Siamese Fighting 

Fish are well known for their aggressive behavior, 

especially towards other male betta fish. They typically 

exhibit an unlearned, or instinctual, aggressive display 

prior to attacking an opposing fish; this aggressive 

response consists of alternating displays (Thompson 

1963). The first method involves the displaying fish to 

be facing the opponent head-on with its opercular 

covers flared to increase its apparent size. The second 

method involves the displaying fish to be profile to the 

opponent fish while fanning out its dorsal, caudal and 

pelvic fins also to increase its apparent size (Bando 

2004). Betta fish respond to an opponent with an 

aggressive response that consists of numerous shows. 

They typically flare their fins and opercula for a short 

period of time per show. This will continue until the 

fish attack one another usually ending when one 

retreats or dies (Bronstein 1981a). 

Domesticated Betta fish, which can be easily 

found at almost all pet and aquarium supply stores, 

have been selectively bred to have larger fins and 

greater varieties of color. In addition to fin size and 

color, domesticated Betta fish are much more 

aggressive than their wildtype counterparts (Simpson 

1968). Fin size has already been shown to be a major 

contributor to the aggressive response seen in male to 

male Betta interaction (Allan and Nicoletto 1997), but 

this study looks to test whether or not color of fish 

plays an important role as well. It is hypothesized that 

the new variety of colors will evoke different degrees 

of aggressive response, which will be monitored as the 

length of display per show. 


Five male Betta fish were purchased from 

Petco pet stores (Capo Beach, Ca). The fish were kept 

in a quiet, sun-lit room at a residential home in Dana 

Point in individual containers that were visually 

separated from each other. In this way, they were in 

complete isolation for five days before testing was to 

begin, as to get them acclimated to being alone, and 

more importantly, not seeing other male Betta fish 

(they were kept in visual contact before being 

purchased and their aggressive response had been 

diminished due to over exposure to other males). After 

isolation and the return of their aggressive response the 

first of the trials was preformed in which the aggressive 

display was monitored in response to visual stimuli. 

This study looked to test whether or not color of visual 

stimuli affected the average time of aggressive display 

per show. 

Betta fish were in isolation when not in trial, 

kept in dechlorinated water that was changed twice a 

week, and fed once every three days. When trials began 

the fish were introduced into a larger container (8”x5”) 

with flat surfaces so that they could see outside the 

container clearly with no distortion for even slight 

distortion of a curved container could weaken the 

aggressive response of Bettas (Bronstein 1983). Three 

of the four sides were covered with white paper so as to 

keep any other outside stimuli from distracting the fish. 

21 




On the fourth side was placed the experimental visual 

stimuli. The visual stimuli consisted of a profile image 

of a betta fish in an aggressive show (Figure 1). Image 

stimuli has been shown to elicit an aggressive response 

comparable to live stimuli (Thompson 1966). The 

stimulus was kept at a constant distance roughly 4cm 

away from the container. This was done for it has been 

shown that the aggressive response of bettas is 

inversely proportional to the distance between the 

subject and stimulus (Bronstein 1981b). The image was 

edited using Adobe Photoshop to exhibit the four 

colors that were used in experimentation: blue, red, 

yellow and green. 

Figure 1. Profile view of Male Betta splendens in 

aggressive display as was used as the variant visual 

stimuli (edited to be Green, Red, yellow & blue). 

As each fish was introduced into the testing 

container they would be given five minutes to become 

accustomed to the new environment. Soon after, the 

stimulus was introduced to the fish by placing the 

image in visual contact with the fish from outside the 

container. Once the first aggressive display was 

observed a five minute timer was started. At the same 

time a stopwatch would be used to record the length of 

time the fish showed aggressive display out of the five 

minutes. In addition, a number counter would also 

count the number of times an aggressive show 

occurred. At the end of the five minutes all timing and 

counting would cease and the data recorded. (The trial 

length was chosen to be five minutes because during 

initial testing the fish showed that they would lose 

interest after this amount of time). More trials would 

follow in the same fashion using the remaining four 

fish. After each fish was introduced to the visual 

stimulus a thirty minute time period was allowed to 

pass before they were to be introduced to the next 

stimuli. This same process was repeated for each color 

of the visual stimuli for each fish. This was the 

conclusion of trial set no. 1. Five more days were 

allowed to pass before trial set no. 2 was to be 

preformed so as to give the fish time to be acclimated 

once more to being isolated from other fish and visual 

stimuli. As betta fish are exposed to other male betta 

for extensive amounts of time, their aggressive 

response diminishes. A recovery period is required in 

order to enable their full response (Clayton and Hinde 

1968). Trial set no. 2 was carried out in the same way 

as the first trial. 

Calculations 

The data collected consisted of total amount 

of time the fish showed aggressive display (t r ) out of 

the five minutes as well as number of times an 

aggressive show was observed during each trial (N f ). t r 

was then divided by N f allowing the average length of 

aggressive response per show to be obtained (t ave ). 

t r / N f = t ave 

Each color of visual stimuli had two sets (one 

for each trial set) of t ave for each of the five fish. This 

was then averaged together to give the final data points 

that were to be inserted into an Analysis of Variance 

(ANOVA) test between groups test to test for 

significance (Table 1). Significance was determined at 

P < 0.05. 

Results 

The mean length of display per show for the 

green stimuli was 7.24 seconds (± 0.833 s.e.), for the 

red stimuli it was 6.59 seconds (± 0.957 s.e.), for 

yellow it was 6.95 (± 0.678 s.e.) and for blue it was 

7.69 seconds (± 1.306 s.e.) Although there are 

differences in the mean lengths of displays per show 

there is not an apparent contrast between them (Figure 

2). 

Mean Length of Display per Show; 

tave (sec) 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

Green Red Yellow Blue 

Color of Stimuli 

Figure 2. Mean length of Display t ave per show for each color 

of visual Stimuli. Y-error bars indicate Standard Error 

An Analysis of Variance between Groups test 

confirms this observation and does not support this 

experiments hypothesis for there is not a significant 

difference of mean length of display per show between 

the four variant stimuli. (P = 0.878). 

22 




fish# Green Red Yellow Blue 

#1 5.19 5.48 5.92 3.69 

#2 7.28 6.6 8 7.87 

#3 6.29 10 4.77 11.5 

#4 10.2 6.6 8.12 6.38 

#5 7.26 4.25 7.91 8.96 

Ave. 7.24 5.59 6.94 7.68 

Table 1. Mean length of display (t ave ) for all 

five fish for each color in seconds. 

Discussion 

Color of stimuli had no significant effect on 

the mean length of aggressive display per show. This 

could be due to a number of factors. Betta fish are 

territorial animals that usually spend their time in the 

still waters of rice patties (Bronstein 1980). 

Throughout experimentation the fish were constantly 

being transferred from one container to another leaving 

little chance for the fish to ever claim their territory. 

Because the fish were introduced into a foreign 

container during the trials their aggressive response 

may have been limited on account that they were no 

longer protecting their territory, but invading that of 

another fish. Although they still evoked an aggressive 

display it may be more out of necessity so that it can 

try to intimidate the perceived threat, the stimuli 

(Bronstein 1983). 

Size of the opposing betta has been shown to 

be a major contributor to the aggressive response of the 

test betta (Allen and Nicoletto 1997). In this study the 

visual stimuli was a constant size but the test betta fish 

varied in sizes. Some fish were smaller than that of the 

stimuli and some were larger. Because of this it may be 

possible that the smaller fish would not produce such a 

long aggressive display due to intimidation. The larger 

fish would in turn not see the stimuli as a threat, thus 

be more inclined to show a longer display. The size of 

the stimulus remained the same throughout hoping that 

that the results would be consistent and therefore, still 

comparable. 

The resulting ANOVA test values showed that 

there was no significant difference between any of the 

four colors of stimuli. Furthermore, showing no 

indication that the fish would show an increased 

aggressive display towards a particular color. Color 

therefore, in the case of B. splendens, is not a visual 

trigger for aggressive displays. Color variation may 

exist in Betta fish not for male-male dominant behavior 

contact but instead for sexual selection. 


Allen, Joseph., Paul Nicoletto. (1997). Response of 

Betta splendens to Computer Animations of 

Males with Fins of Different Length. Copeia 

Vol. 1, 195-99 

Bando, Toshirhiro. (2004). Visual Perception of 

Texture in Aggressive Behavior of Betta 

splendens. Journal of Comparative Physiology 

A: Neuroethology, Sensory, Neural, and 

Behavioral Physiology. Vol. 169, 51-58 

Bronstein, Paul. (1980). Betta splendens: a Territorial 

Note. Bulletin of the Psychonomic Society. 

Vol. 16, 484-485 

Bronstein, Paul. (1981). Commitments to aggression 

and to nest sites in male Betta splendens. 

Journal of Comparative and Physiological 

Psychology. Vol. 95, 436-449 (a) 

Bronstein, Paul. (1981). Social Reinforcment in Betta 

splendens: A Reconsideration. Journal of 

Comparitive and physiological Physchology. 

Vol. 95, 943-950 (b) 

Bronstein, Paul. (1983). Agonistic Sequences and the 

Assessment of Opponents in Male Betta 

splendens. The American Journal of 

Psychology. Vol. 96:2, 163-173 

Clayton, Frances., Robert Hinde. (1968). The 

Habituation and Recovery of Aggressive 

Display in Betta splendens. Behaviour. Vol. 

30:1, 96-105 

Simpson, M.J.A. (1968). The Threat Display of the 

Siamese Fighting Fish, Betta splendens. 

Animal Behavior Vol. 1, 1-73 

Thompson, Travis I. (1963). Visual reinforcement in 

Siamese Fighting Fish. Science, New Series. 

Vol. 141:3575, 55-57 

Thompson, Travis I. (1966). Operant and Classically- 

Conditioned Aggressive Behavior in Siamese 

Fighting Fish. American Zoologist. Vol. 6:4 

,629-641 

23 




Intestinal Candida albicans Overgrowth in Autistic Children with Food Allergies 

Crystine Gill and Samantha Lopez 



Mission Viejo, Ca 92692 

Candida albicans overgrowth in the intestines has been proposed as a causative factor in 

a portion of autism cases. An overgrowth of C. albicans in the intestine can cause 

perforations in the intestinal walls that permit undigested substances to enter directly into 

the bloodstream, which can affect the central nervous system, and/or result in food 

sensitivities. Urine samples were collected from autistic and non-autistic children 

Homosapiens between the ages of 4-14 years old. Samples were plated on Bacto Candida 

BCG formula agar, for specific identification of Candida species. The plates were examined 

for fungal growth after 72 hours of incubation at 37° C. Variations in species of Candida 

were found, including Candida albicans, Candida stellatoidea, and/or Candida 

guilliermondii. Significant differences were not found in the frequency of Candida growth 

between specimens from autistic children and non-autistic children (p=0.234, one-tailed t- 

test). A correlation between children with food allergies and Candida growth was noted, 

but a significant difference was not supported (p=0.267, one tailed t-test). 

Introduction 

Autism is a condition, which can vary in degree of 

symptoms, characterized by impaired communication 

(delayed or lack of language), repetitive behaviors 

(preoccupations, motor mannerisms) and impaired 

social interaction (lack of eye gaze, peer relationships, 

sharing interests with others, reciprocity) (Vig and 

Jedrysek 1999). The diagnosis of Autism spectrum 

disorders has become an increasing syndrome among 

children within the United States. About one per 150 

children is diagnosed with Autism Spectrum Disorders 

(CDC). Presently, the specific etiology of autism is 

unknown. Genetics and environmental combinative 

factors are lead potential causes of the disorder 

(Szatmari 2000). 

A higher frequency of gastrointestinal dysfunction 

is found in among people diagnosed with autism and 

other developmental delays (Erickson and others 

2005). Studies have found evidence of immune 

response to intestinal fungus (Torrente and others 

2002, Edelson 2000). Further studies have addressed 

the alleviation of the overproduction of immune 

responses that manifest as GI dysfunctions (Torrente 

and others 2004). The presence of epithelial IgG and 

other immunoglobulins in foveolar and glandular 

epithelium suggests the body is responding to an 

infection within the digestive tract. 

The focus of this study is the causative factor of GI 

dysfunction in autistic children, proposed as Candida 

albicans. C. albicans is known to cause diseases 

varying from simple mucocutaneous infections to fatal 

candidiasis. An overgrowth of C. albicans can invade 

intestinal tissue causing Spontaneous Intestinal 

Perforation (Robertson and others 2003). Undigested 

proteins or substances may enter directly into the 

bloodstream, possibly causing toxic effects possibly 

attributing to characteristics of autism, or causing food 

sensitivities (Andrutis 2000). 

A symptom of a Candida can be food allergy or 

food sensitivity. Those diagnosed with autism show 

cognitive dysfunctions, which may be a secondary 

result of by the toxic effects to a fungal infection. 

The presence of C. albicans in urine is expected to 

be found at a greater frequency in children diagnosed 

with autism and having food allergies than in nonautistic 

children having food allergies 


Twenty-one children between the ages of four and 

fourteen years old participated in this study (mean age: 

7); ten males and eleven females. Twelve, of the 

twenty-one participants, are diagnosed with autism, 

ranging from severe to high functioning. Food allergies 

are reported in six of the autistic children and two of 

the non-autistic children. All participants reside within 

Orange County, California. 

Urine samples were collected in sterile specimen 

cups between April 1-9, 2008. Urine was plated, via 

aseptic techniques, within 48 hours from collection 

24 




onto Agar plates, prepared with bromcresol green (pH 

6.1 ± 0.1; at 25 °C) according to a Bacto Candida BCG 

agar formula (Difco, NJ). Urine not plated within 1 

hour was refrigerated at 10°C until plating. 

Inoculated plates were incubated at 37 °C. 

Examination for Candida albicans growth on the 

plates, was performed after 24 hours and after 72 hours 

of incubation. Difco Manual was used as a reference 

for morphology and pH indications to identify various 

Candida species. 

The morphology of the fungal colonies present was 

interpreted as Candida albicans, Candida stellatoidea 

and/or Candida guilliermondii (Difco reference 

manual). An unpaired, t-test and A-Nova test was 

performed in MS Excel (Microsoft Corporation, 

Sylmar, Ca) to assess the statistical differences 

between the quantity of specimens positive for 

Candida fungus in children diagnosed with Autism 

compared to children not diagnosed with Autism. 

Results 

After three days of incubation, inoculated agar plates 

were observed for growth and morphological 

identification. Twelve of the twenty-one plates had 

positive Candida growth (table 1). Fifty percent of the 

plates with specimen from children diagnosed with 

autism and sixty seven percent of the plates with 

specimen from non-autistic children were positive for 

Candida growth. 

Table 1. Results of urine samples plated on pH 

indicated agar, to indicate intestinal Candida growth in 

children with and without autism and food allergies. 

+Growth 

+ Food 

allergies 

-Growth 

+ Food 

allergies 

+ Growth 

-Food 

allergies 

-Growth 

-Food 

allergies 

Autistic 

children 4 2 2 4 

Non-autistic 

children 2 0 4 3 

Plates that were positive for Candida growth varied in 

morphology most commonly found configuration was 

round with raised margin, smooth margins, and 

convexed elevation. Few of the colonies showed 

variations in morphology including one concentric 

configuration, undulate or irregular margins, and 

elevations ranging from raised to drop-like. All 

growths were characteristically deep green to blue with 

margins fading to a pale yellow (figure 1). A decrease 

in pH was also observed as the blue in the agar change 

d to green or yellow around colonial growths. 

Figure 1. Candida growth showing characteristic 

morphology round configuration with smooth (some 

undulated in this figure), raised margin, and convexed 

elevation. Deep green to blue coloring, with 

diminishing color to pale yellow toward margins. 

Interpretation of identification was ambiguous, due 

to less than 100% agreement between defined 

morphological characteristics and morphology 

characteristics observed on the plates. However, each 

colony was deduced to be one of two types of Candida. 

Eight of twelve growths were identified as either 

Candida albicans or Candida stelloidea. Three were 

identified as Candida albicans or Candida 

guilliermondi. One was deduced to be Candida 

albicans. 

There was not a significant difference in the number 

of positive plates of Candida growth between autistic 

and non-autistic children (p= 0.234, one-tailed t-test; 

n=21). 

Sixty-seven percent of the plates from children 

having food allergies were positive for Candida 

growth. There was not a significant difference in the 

number of plates positive for Candida growth between 

children with and without food allergies (p= 0.267, 

one-tailed t-test; n=6). 

Discussion 

This study did not support a significant difference in 

Candida growth between autistic children and any 

other group including autistic children without food 

allergies, non-autistic children with and without food 

allergies. 

The small number of participants yielded a limited 

result with narrow statistics in this study. 

False positives may be a variable, as independent 

collection of specimens may have been exposed to 

non-aseptic environments. Additionally, females are 

25 




more prone to Candida contamination in their samples 

due to exposure to naturally occurring Candida of the 

external genitalia. 

One autistic subject having reported food allergies 

regularly takes an anti-fungal medication. Those results 

were negative for fungal growth; wherein he has had 

positive results in independent studies. 

Ambiguity in interpretation of morphology could be 

reduced in future studies with an alternative agar 

medium to further distinguish species of Candida. 

While the results of this test supported the null 

hypothesis, reports of GI dysfunction in autistic 

children remain prevalent. The presence of epithelial 

IgG and other immunoglobulins in foveolar and 

glandular epithelium in biopsies (Torrente and others 

2004), suggests the body is responding to an infection 

within the digestive tract. This study may not support 

the causative factor to be Candida overgrowth leading 

to undigested substances in the blood stream; further 

investigation is needed to determine the cause of 

elevation in the immune response system. 

A further occurrence of interest, with regard to 

intestinal Candida overgrowth in autistic children, is 

the perpetuation of Candida growth due to diet. Many 

autistic children are on gluten-free and casein-free 

diets. The fermentation of carbohydrates provides 

lactic acid, which inhibits the Candida from an 

overgrowth (Matsen 2004). Although, this reason gives 

cause to speculation, as GI dysfunction is frequent in 

those without special diets as well. 

There is not a current standard of measurement of 

dysfunction level of the gastrointestinal system 

(Yamaguchi N 2006). As such, a hypothesis was not 

made based on subjective data. It remains to be an area 

of further study to determine the correlation between 

GI dysfunction and autism. As further research 

methods are introduced and additional studies 

performed the cause of GI dysfunction can be 

determined and symptoms then alleviated. 


Andrutis K.A. 2000. Intestinal Lesion Associated 

with 

Disseminated Candidiasis in an Experimental 

Animal Model. Journal of Clinical Microbiology. 

38(6), 2317-233. 

Edelson S, Carter D. 2000. The neurotoxic 

etiology of 

the autistic spectrum disorders: a replication 

study. 2000. Toxicology & Industrial Health. 

16(6):239-247. 

Eichler, Evan E., Zimmerrman, Andrew W. (2008). A 

Hot Spot of Genetic Instability in Autism. The New 

England Journal of Medicine. 

Ericson C, Stigler K, Corkins M, Posey D, Fitzgerald J, 

McDougle C. 2005. Gasterointestinal Factors in 

Autistic Disorder: A Critical review. Journal of 

Autism and Developmental Disorders. 35 (6): 713- 

726. 

Matsen J. 2004. Intestinal Yeast and bacterial 

overgrowth. Better Nutrition. 66(9)29-31. 

Office of Communications and Public Liaison. (CDC) 

2006.Autism Fact Sheet. National Institute of 

Nerological Disorders and Stroke. 

Roberts N, Kuna J, Cox P, Lakhoo K. 2003. 

Spontaneous intestinal perforation and Candida 

peritonis presenting as extensive necrotizing 

enterocolitlis. Acta Paediatrica. 92(2): 258. 

Szatmari P. (2003) The causes of autism spectrum 

disorders. British Medical Journal. 326(1):732. 

Torrente F, Anthony A, Path M, Heuschkel R, 

Thomson M, Ashwood P, Murch S. 2004. Focal- 

Enhanced Gastritis in Regressive Autism with 

Features Distinct from Crohn's and Helicobacter 

Pylori Gastritis. The American Journal of 

Gastroenterology 99 (4): 598–605. 

Torrente F, Ashwood P, Day R, Machado N, Furlano 

R, Anthony A, Davies S, Wakefield A, Thomson 

M, Walker-Smith J, Murch S. 2002. Small 

intestinal enteropathy with epithelial IgG and 

complement deposition in children with regressive 

autism. Moleculary Psychiatry 7(4): 375. 

Vig, S, Jedrysek, E. 1999. Autistic features in young 

children with significant cognitive impairment: 

Autism or mental retardation? Journal of Autism 

& Developmental Disorders. 29(3):235-248. 

Yamaguchi N, Sugita R, Miki A, Takemura N, 

Kawabata J, Watanabe J, Sonoyama K. 2006. 

Gastrointestinal Candida colonisation promotes 

sensitisation against food antigens by affecting the 

mucosal barrier in mice. Gut 55:954-960 

26 




Comparison of Chlorophyll Content in Shade and Sun Leaves of the Lemonade 

Berry Plant (Rhus integrifolia). 

Ryan C. Clark and Josue J. Mandujano 




Chlorophyll is a green, photosynthetic pigment that absorbs sunlight, and 

uses this energy to produce ATP and NADPH. It is, therefore, the foundation for 

the life functions of all plants. Chlorophyll content varies with different plants but 

can vary in different leaf types of a certain plant. The amount of chlorophyll in a 

leaf depends on the quantity of sunlight the leaf in question receives. Given that 

photosynthesis occurs with more efficiency if it has more sunlight, it was predicted 

that the sun leaves, which receive more direct sunlight than the shade leaves would 

contain higher chlorophyll content than shade leaves. A spectrophotometer and 

chlorophyll extraction were used to determine whether sun or shade leaves, would 

contain more chlorophyll. Five mL of 80% concentrated acetone were mixed with 

two 6 mm leaf chads in scintillation vials. A 1 mL solution was inserted via styrene 

cuvette into the spectrophotometer for analysis. It was discovered that in the 

samples taken, half of the shade leaves of the lemonade berry contained more 

chlorophyll than the sun leaves and half of the sun leaves contained more 

chlorophyll than the shade leaves. However, the total combined average of the 

samples taken show that the sun leaves have a higher chlorophyll content than 

shade leaves. The results of the experiments therefore, supported the hypothesis 

that the sun leaves would contain a higher concentration of chlorophyll than the 

shade leaves. 

Introduction 

Pigments are chemical compounds which 

reflect only certain wavelengths of visible light (Speer, 

1995). Chlorophyll is a green pigment that contains a 

porphyrin ring and it is located in the thylakoids. It is 

the utilization of this porphyrin ring with its freemoving 

electrons that is the basic pathway by which 

chlorophyll captures sunlight’s energy. Of the several 

different kinds of chlorophyll, chlorophyll “a”, which 

is found in all plants and algae that photosynthesize, is 

the most important type of chlorophyll (Speer, 1995). 

Chlorophyll a is the type of chlorophyll that 

makes photosynthesis possible. It does this by passing 

on its energized electrons to molecules which will 

manufacture sugars (Speer, 1995). A second type of 

chlorophyll, chlorophyll b only occurs in plants and 

green algae that transfers energy to chlorophyll a. 

Photosynthesis is divided into two different and distinct 

stages – the Light Reaction, and the Calvin Cycle 

(Farabee, 2001). In the Light Reaction, which occurs 

continuously during the process, in the grana of the 

thylakoid membrane contained in the chloroplast in 

Photosystem II, photophosphorylation occurs. This is 

due to light energy causing the removal of an electron 

from P680 in Photosystem II (Campbell and Reece, 

2005). The P680 replaces the electron by taking it 

from a water molecule, which is split into its H + ions 

and O 2− ions. The O 2− ions then combine to form 

diatomic oxygen, which is released. The electron is 

captured by the primary electron acceptor and passed 

from Photosystem II to Photosystem I via an electron 

transport chain. While the electron moves through the 

electron transport chain to Photosystem I, it moves to a 

lower energy level, and it, along with other electrons 

moving along the chain, provides energy for the 

synthesis of ATP. Light energy excites an electron in 

P700 reaction center of Photosystem I, the electron is 

boosted to higher energy potential, and the electron is 

captured by Photosystem I’s primary electron acceptor. 

The electron that moved down the transport chain from 

27 




Photosystem II replaces the electron excited in 

Photosystem I, and as this excited electron is passed 

down a second electron transport chain through the 

protein ferrodoxin, the enzyme NADP + reductase 

transfers electrons from ferrodoxin to NADP + to form 

NADPH (Campbell and Reece, 2005). 

The Calvin Cycle of photosynthesis, which 

occurs in the stroma of the chloroplast, takes place 

three times (Farabee, 2001). Once every revolution of 

the cycle, a carbon dioxide molecule is attached to 

RuBP, which reacts with rubisco to form a product that 

immediately breaks down into two molecules of 3- 

phosphoglycerate. Each of the 3-phosphoglycerates 

obtains another phosphate group from ATP and 

becomes 1, 3-biphosphoglycerate. That is reduced by 

NADPH to from G3P. One G3P is released, the 

remaining molecules of G3P are turned into RuBP with 

the assistance of ATP, and the cycle repeats (Campbell 

and Reece, 2005). Chlorophyll content varies with 

different environmental factors. A plant that is 

healthier will have a higher amount of chlorophyll 

overall, not just in one type of leaf it possesses, but 

both sun and shade leaves. The amount of chlorophyll 

in a leaf is directly related to the amount of direct 

sunlight it receives. It is the main purpose of this 

experiment to determine which leaves, either sun or 

shade, will contain a higher chlorophyll content. It is 

predicted that the sun leaves will contain more 

chlorophyll than the shade leaves. 


Leaf samples were acquired for the analysis of 

chlorophyll content in shade and sun leaves of the 

lemonade berry plant (Rhus integrifolia). The samples 

were collected on 7 April 2008. The area from which 

the samples were obtained was near the entrance to the 

trail of Seaview Park in Laguna Niguel, CA. One 

hundred leaves were collected: fifty sun leaves, and 

fifty shade leaves. From each of 10 different mature 

lemonade berry plants, five sun leaves and five shade 

leaves were taken. The leaf samples were placed into 

labeled plastic bags which differentiated the sun from 

the shade leaves, and corresponded to the plant from 

which they had been chosen. 

The leaves were prepared for chlorophyll 

analysis on 8 April 2008 from 12:00 PM to 5:30 PM at 

Saddleback College. One hundred scintillation vials 

were filled with 5 mL of 80% acetone, measured using 

a Labnet International Inc. Labmax Dispenser. Two 

leaf chads, made with a standard hole-punch (diameter 

of 6 mm), were added to each vial, which were then 

labeled according to specimen number and condition, 

whether shade or sun. All vials were placed in a 4°C 

environment for a period of 48 hours. 

Chlorophyll measurements were taken on 10 

April 2008 using a Beckman DU 730 

spectrophotometer, calibrated for measurement of 

chlorophyll content in acetone at two wavelengths in 

nm. One milliliter of an 80% acetone solution was 

measured into a Plastibrand styrene cuvette, for the 

purpose of zeroing-out the spectrophotometer. Then, 

one milliliter of each of the sample mixtures was 

measured into a Plastibrand styrene cuvette and the 

readings of chlorophyll content were taken for each 

leaf. For each of the categorized groups of samples, 

the average amount of chlorophyll content was 

calculated in mg/L. 

Results 

The sun leaves of the lemonade berry plant 

contained more chlorophyll than the shade leaves. The 

combined averages of the groups of samples showed 

that half of sun-leaf groups had a higher chlorophyll 

concentration and half of the shade-leaf groups had a 

higher chlorophyll concentration. However, the total 

combined average of all sun leaf and shade leaf 

samples showed that sun leaves contain more 

chlorophyll (Figure 1). The total average measurement 

of milligrams of chlorophyll per liter of 80% 

concentrated acetone in the sun leaves of the lemonade 

berry plant were 4.03 mg/L (+ se, N=50). The total 

average measurement of milligrams of chlorophyll per 

liter of 80% concentrated acetone in the shade leaves of 

the lemonade berry plant were 3.73 mg/L (+ se, N=50). 

The difference between the average amounts of 

chlorophyll in sun leaves from the average amounts of 

chlorophyll in shade was not very significant (Figure 

1). A two-tailed, paired t-test revealed that the sun 

leaves contained more chlorophyll than the sun leaves 

(p= 1.52 ×10 -6 ). 

Avg. Chlorophyll Concentration 

(mg/L) 

5.00 

4.50 

4.00 

3.50 

3.00 

2.50 

2.00 

1.50 

1.00 

0.50 

0.00 

Shade Leaves 

Sun Leaves 

Lemonade Berry (Rhus integrifolia) 

Figure 1. Bar graph showing the mean + SE values 

for chlorophyll concentration. 

Discussion 

In measuring and comparing the chlorophyll 

content in sun and shade leaves of the lemonade berry 

plant, the results showed that the difference in amount 

of chlorophyll in shade leaves from sun leaves varies 

28 




from plant to plant. It was also demonstrated that same 

sample types can themselves vary significantly from 

one plant to another. 

The experiment attests that the major factors 

contributing to chlorophyll concentration in the sun and 

shade leaves is overall location of the plant, and how 

much direct sunlight the leaf type receives. In the 

experiment concerning the monitoring of chlorophyll 

in sugar maples tree leaves (Cate and Perkins, 2003), it 

was stated that angle of incidence and PAR irradiance 

affect chloroplast distribution and angle (Haupt 1982) 

and chlorophyll content index values are significantly 

affected by the incident irradiance, typically giving 

lower values at higher irradiances (Hoel and Solhaug, 

1998). In addition, in an experiment to determine the 

absorption of light by chlorophyll solutions (G. 

MacKinney, 1941), it was found that solvents, 

including 80% anhydrous acetone, have an affect on 

the absorption coefficients of chlorophyll a and b, 

altering them. 

Given that fact that the overall average of 

chlorophyll content was higher in sun leaves versus 

shade leaves, it would seem that sun leaves of the 

lemonade berry plants typically contain more 

chlorophyll than the shade leaves. This may be due to 

the fact that since the sun leaves receive much more 

sunlight to undergo photosynthesis than the shade 

leaves, they have more chlorophyll and, therefore, 

more effectively undergo the process that sustains the 

plant. Since the sun leaves have a larger amount of 

chlorophyll spread out over their surface area, it 

increases the leaves’ ability of going through 

photosynthesis more efficiently and producing the 

necessary amount of nutrients the plant needs to be and 

remain healthy. 


Campbell, N. A. and Reece, J. B. 2005. Biology: 

Seventh Edition. San Francisco, CA: Pearson 

Education, Inc. 1,231 p. 

Cate, T. M. and Perkins, T. D. 2003. Chlorophyll 

content monitoring in sugar maple (Acer saccharum). 

Tree Physiology. 23, 1077−1079 

Haupt, W. 1982. Light-mediated movement of 

chloroplasts. Plant Physiol. 33, 205–233. 

Hoel, B. O. and K. A. Solhaug. 1998. Effect of 

irradiance on chlorophyll estimation with the Minolta 

SPAD-502 leaf chlorophyll meter. Plant Science. 82, 

389–392. 

Farabee, M. J. 2001. Photosynthesis. 

. 

MacKinney, G. 1941. Absorption of Light By 

Chlorophyll Solutions. The Journal of Biological 

Chemistry. 132, 315-322 

Speer, B. R. 1995. Photosynthetic Pigments. 

. 

The Effects of Ethinyl Estradiol on Aggressive Behavior in 

Siamese Fighting Fish (Betta splendens) 

Thao Nguyen and IxChel Cruz-Gonzalez 




A natural and unfortunate effect of the unprecedented growth in the industrial and 

agricultural sector and the general expansion of human communities is that biological 

hormones and chemicals are increasingly making their way into the environment. Ethinyl 

estradiol, an estrogen derivative, is commonly included in many oral contraceptives taken 

by women and excreted in their urine. Due to inefficiency in water treatment facilities, 

industrial and municipal waste products such as ethinyl estradiol can be found in aquatic 

ecosystems in concentrations as high 64 μg/L. Basal aggression levels of six Siamese 

fighting fish, Betta splendens, were measured by timing their agonistic response triggered 

by their own reflection in a mirror. Ethinyl estradiol was then added to the water at a 

concentration of 60 μg/L. The Betta fish were allowed to acclimate themselves to the new 

29 




environmental conditions for one week. Then aggression levels were once again measured. 

There was not a significant difference in aggression responses between the pre- and postexposure 

to ethinyl estradiol. 

Introduction 

Pharmaceutical drugs in water supplies 

Pharmaceuticals, household chemicals, and 

biogenic hormones are released directly into the 

environment after passing through wastewater 

treatment processes, which are often not designed to 

completely remove these chemicals from the effluent. 

Many of these chemicals are in streams throughout the 

United States, including antibiotics, various 

prescription drugs, nonprescription drugs, steroids, 

reproductive hormones, personal care products, and 

other extensively used chemicals (Kolpin et al., 2002). 

Thus, the extent of pharmaceutical and personal care 

products (PPCPs) in the aquatic environment and their 

consequences are beginning to be monitored by some 

scientists (Potera, 2000). 

U.S. Geological Survey personnel tested a 

total of 139 streams; Table 1 lists the prescription drugs 

that were found and the respective concentrations 

(Kolpin et al., 2002). This list contains drugs that are 

used for a variety of different conditions in the human 

body. According to Christian Daughton, chief of the 

Environmental Chemistry Branch of the U.S. 

Environmental Protection Agency (EPA) 

Environmental Sciences Division in Las Vegas, 

Nevada, researchers worldwide have discovered more 

than sixty different PPCPs in water sources (Potera, 

2000). 

Table 1. List of some prescription drugs detected in 

streams across the United States (from Koplin et al., 

2002, pg 1204) 

Chemical N RL Feq (%) Max Median 

Albuterol 84 0.029 0 ND ND 

Cimetidine 84 0.007 9.5 0.58 0.074 

Codeine 84 0.1 10.4 1 0.2 

Digoxin 46 0.28 0 ND ND 

Dilitiazem 84 0.012 13.1 0.49 0.21 

Fluoxetine 84 0.018 1.2 0.012 0.012 

Gemfibrozil 84 0.015 3.6 0.79 0.048 

Metfibrozil 84 0.03 4.8 0.15 0.11 

Paroxetine 84 0.26 0 ND ND 

HCL 

Rantidine 84 0.01 1.2 0.01 0.01 

Warfarin 84 0.001 0 ND ND 

Environmental engineer Pierre Labadie and 

his students at the University of Sussex obtained water 

samples from the Ouse River at two sites: Ditchling 

(200 m downstream) and Lewes (500 m downstream). 

30 



Varying concentrations of levels of estrogen were 

detectable in both the depth profiles of Ditchling and 

Lewes. Average estrogen concentrations in the water 

column at the Ditchling site, during the month of 

December, were predicted to be among the highest, 

whereas, at Lewes, no subsurface peak was observed, 

and estrogenic activity declined with increasing 

sediment depth (Labadie et al., 2007). 

It should be noted that estrogen was targeted 

for this experiment due to the potential of interfering 

with normal reproduction and development in fish 

living downstream from sewage plants. Little 

information is known about the effects of 

environmental occurrence, transport, and ultimate fate 

of many pharmaceuticals that are designed to stimulate 

a physiological response in humans and animals 

(Kolpin et al., 2002). 

Effects of estrogen on humans 

Approximately 39% of women today are 

taking some kind of birth control method. Birth control 

pills, also called “the Pill”, are typically prescribed to 

patients to change the way the body works and prevent 

pregnancy. 

Hormones are chemical substances that 

control the functioning of the body’s organs. Most birth 

control pills, or "combination pills", contain a 

combination of the hormones estrogen and 

progesterone to prevent ovulation (the release of an egg 

during the monthly cycle). A woman avoids pregnancy 

by not ovulating. One hormone pill is taken orally 

each day, in the form of tablets, at about the same time 

for 21 days. 

In some cases, the effects of hormones on 

behavior depend on the environment. Estrogens can 

affect physiology and behavior through two general 

pathways. It can alter the activity of neural cells by 

genomic action. When estrogen binds to a classical 

estrogen receptor in the cytoplasm, it enters the nucleus 

and changes gene expression (which ultimately leads to 

changes in proteins). This is why it is often necessary 

to wait several days or weeks for effects of hormone 

manipulations to occur (Kolpin et al., 2002). 

As a sex hormone, estradiol is present in both 

females and males. It not only has a critical impact on 

reproductive and sexual functioning but also other 

organs, including bone structure. 

Effects of estrogen on aquatic animals


There have been very few studies that have 

tested the relationship between aggression in aquatic 

animals and estrogen. A study performed by Lai et al. 

(2002) looked at the effects of natural and synthetic 

steroid estrogens in relation to their environmental 

occurrence. It was found that fatal effects have only 

been observed in juvenile fish exposed to a high 

concentration of estrogens. The development of sexual 

and reproductive characteristics in vertebrates is 

sensitive to exogenous estrogens. If the alterations of 

sexual characteristics affect the reproductive 

performance of the organisms, either temporarily or 

permanently, the exposure to steroid estrogens may 

have an effect at the population level. Exposure to 

estrogens may cause other physiological effects in 

animals. It is apparent that exposure to steroid 

estrogens results in a diverse range of effects on a large 

number of species. 

Betta splendens, commonly referred to as 

Betta fish or the Siamese fighting fish, are a breed of 

fish that live in the waters around Southeast Asia and 

are from the family Anabantidae. Betta splendens are a 

labyrinth fish that gulp air to get oxygen. They have a 

special auxiliary breathing mechanism called the 

labyrinth that has a pair of irregular passages to provide 

supplemental oxygen to the blood. Hence, they can live 

in low oxygen level waters. The male Betta fish are 

renowned for their long tails and fins that they use to 

display aggression toward perceived threats. They 

exhibit a behavior of fixed action patterns that are 

sequences of movements determined genetically (Allen 

and Nicoletto, 1997). Researchers found that 

environmental stimuli promote these patterns. In the 

case of the Betta, the stimulus is most likely another 

male Betta. 

The male fighting fish is so aggressive, or 

agonistic, that in a community tank where other fish 

are present there can only be one male Betta. When 

fighting, males will nip at the other’s fins until one of 

them is exhausted. Betta fish will display their fins to a 

mirror since they do not recognize their reflections and 

think another male is infringing on their territory. The 

stretching of the fins and opening of the gills to display 

the membrane enables the male to look twice his 

resting size and is called flaring, or displaying. 

Betta splendens have historically been used in 

experiments because of their easily recognized and 

well documented social displays (Oiverira et al., 1998). 

This experiment exposed male Betta fish to high levels 

of ethinyl estradiol, and it was expected that they 

would behave less aggressively to stimuli. 

Methods and Materials 

Subjects and setup 

Six male Betta fish were purchased from Pet 

Palace (Glendora, CA) on April 2, 2008 and stored 

separately in 1 L Pyrex beakers: each containing 1 L of 

water. Azusa municipal tap water was dechlorinated 

by allowing it to sit for 24 hours in an open mouthed 

container. For the duration of the experiment, the 

beakers housing the Betta fish were wrapped in white 

paper in order to minimize external stimulation. All 

the Betta fishes were approximately the same size, 

appeared to be in good health, and maintained in 

similar conditions. They were fed four Aqua Culture 

Betta Pellet (HBH Pet Products, Springville, UT) twice 

daily. 

Testing procedure 

After a week of living under “normal” 

conditions, basal levels of aggression were measured. 

One at a time, the Betta fish were transferred into a 

paper wrapped 2.5 L Aqueon Mini-tank (Central 

Garden & Pet Company, Walnut Creek, CA) filled 

with dechlorinated water and allowed to acclimate to 

the new environment for an hour. A portion of the 

white paper was then removed and replaced with a 3 x 

4.5-inch reflective mirror. The time it took the fish to 

respond to the mirror was measured in seconds using a 

stop watch. Aggression was determined by the time it 

took to begin fin flaring which includes the pelvic fin, 

the ventral fin, the caudal fin, the dorsal fin, the gill 

operculum, and the 90° twist of the body. 

In order to simulate estrogen pollution, thirty 

Yasmin 28 (Berlex, Nomtville, NJ) birth control pills 

each containing 0.03 mg ethinyl estradiol were ground 

using a ceramic mortar and pestle. Since ethinyl 

estradiol has low solubility in water, the resultant 

powder was first dissolved in 4 mL of ethanol to make 

a stock solution of 7.6 mM. Now able to dissolve in 

water, 220 μL of the stock solution was then added to 

10 mL of dechlorinated water. Each fish received 1 ml 

of this diluted solution in their 1 L beakers for a final 

concentration of approximately 60 μg/L. The dosed 

Betta fish were allowed to acclimate to the new 

environmental conditions for a week. Once again, the 

Betta fish were transferred to the 2.5 L aquarium now 

containing estradiol treated water, and their agonistic 

behavior was recorded in manner described above. 

Data analysis was completed using Microsoft 

Excel (Microsoft Corporation, Sylmar, CA). A paired 

t-test was performed to assess whether there was a 

significant difference in the time to start the fixed 

action pattern of agonistic aggressive pre and post 

ethinyl estradiol exposure. Differences were considered 

significant at P


Results 

The mean body mass of the Betta fish was 

1.55± 0.03g (±SE). Overall there was not a statistically 

significant difference in initial aggression response and 

ethinyl estradiol acclimated aggression response. 

Although the actual approach of the Betta fish to its 

image is not specific to the agonistic display, it was 

included because it signaled the start of Betta 

aggression. The average time it took for the Betta to 

notice, then approach its mirrored image was not 

affected by exposure to ethinyl estradiol (Table 2). 

The average time it took for the initial flaring of the 

Betta’s pelvic fin, ventral fin, caudal fin, gill 

operculum, and twist its body 90° were longer in the 

treated fish, but statistically the averages were not 

significantly different (Table 2). 

Type of 

Fin Flaring 

Mean ± SE 

pre-exposure 

(seconds, N=6) 

Mean ± SE 

post-exposure 

(seconds, N=6) 

paire 

d t- 

test 

(two 

tailed 

) 

Approach 39.91 ± 9.34 46.56 ± 54.16 0.657 

Pelvic 55.95 ± 17.29 74.135 ± 84.04 0.415 

Ventral 92.77 ± 29.00 102.08 ± 89.12 0.484 

Caudal 127.00 ± 27.01 125.77 ± 84.71 0.979 

Dorsal 92.01 ± 29.47 105.36 ± 56.76 0.503 

Gill 

Operculum 

90°Body 

twist 

127.52 ± 25.60 148.03 ± 76.83 0.649 

117.78 ± 105.8 159.71 ± 91.17 0.517 

Table 2. Type of flare, Mean times ± Standard error 

pre- and post-ethinyl estradiol exposure, and paired t- 

test of the means. 

Discussion 

Unfortunately ethinyl estradiol did not have a 

statistically significant effect on the agonistic 

aggressive behavior of male Betta fish. The slight 

increases in mean times observed could be attributed to 

differences in physiological condition of each Betta 

fish (Karino and Someya, 2002). The results of this 

experiment were negative, but other experiments have 

shown that estrogen affects fish behavior including 

aggression. Therefore, future experiments should 

increase exposure and/or concentration, or expose 

immature fish (Clotfelter and Rodriquez, 2006). If 

aggression is not changed, other behaviors like nest 

building and mating rituals can be studied. 

Possible sources of error can come from the 

fact that a pill of Yasmin 28 contains other ingredients 

besides ethinyl estradiol. It is possible the observed 

changes in behavior are actually caused by those other 

ingredients. Also, at times, it was difficult to 

differentiate the exact time of the fin flaring. If this 

experiment were to be done again, the researchers 

suggest that a video recorder be used so that the results 

can be reviewed and the exact movements and timing 

can be determined. 

It should be known that the concentration of 

ethinyl estradiol used during this experiment mimics 

exceptionally polluted waters and is much higher than 

concentrations normally found in natural water systems 

(Quintana et al., 2003; Lai et al., 2002). In the future, 

it would behoove researchers to repeat the experiment 

using varied concentrations of estrogen to determine 

exactly where behavior begins to diverge from the 

norm. Although even then, the results would not 

necessarily be an indication of what actually occurs in 

nature, because estrogen degrades and reacts with other 

naturally occurring chemicals in ways that are not 

simply or easily predicted (Lai et al., 2002). 

In conclusion, ethinyl estradiol is a specific 

type of estrogen, but as a class, other estrogen derivates 

are known to have affected dramatic changes in 

population size, reduction in immune responses, have 

serious detrimental effects on maturing embryos and 

other immature aquatic vertebrates (Clotfelter and 

Rodriquez, 2006; Quintana et al., 2004). Although it 

may seem that estrogen pollution of water can only 

affect aquatic species, it is also able to migrate through 

river bed sediments and potentially contaminate the 

ground water that humans use (Labadie et al., 2007). 

As the human population continues to expand, the 

public needs to become aware of the effect that our 

agricultural and industry is having on our environment 

and our water sources. 


Allen, J.M. and Nicoletto, P.F. (1997). Response of 

Betta splendens to Computer Animations of Males with 

Fins of Different Length. Copeia, 1997 (1): 195-199. 

Clotfelter, E.D. and Rodriquez, A.C. (2006). 

Behavioral Changes in Fish Exposed to 

Phytoestrogens. Environmental Pollution, 144, 833- 

839. 

Karino, K. and Someya, C. (2002). The Influence of 

Sex, Line, and Fight Experience on Aggressiveness of 

the Siamese Fighting Fish in Intrasexual Competition. 

Neurotoxicology and Teratology, 24, 29-36. 

Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, 

E.M., Zaugg, S.D., Barber, L.B., and Buxton, H.T. 

(2002). Pharmaceuticals, Hormones, and Other 

Organic Wastewater Contaminants in the U.S. Streams, 

1999-2000: A National Reconnaissance. 

32 




Environmental Science & Technology, 36 (6): 1202- 

1211. 

Labadie, P., Cundy, A.B., Stone, K., Andrews, M., 

Valbonesi, S., and Hill, E.M. (2007). Evidence for the 

Migration of Steroidal Estrogens through River Bed 

Sediments. Environmental Science Technology, 41, 

4299-4304. 

Lai, K.M., Scrimshaw, M.D. and Lester, J.N. (2002). 

The Effects of Natural and Synthetic Steroid Estrogens 

in Relation to their Environmental Occurrence. Critical 

Reviews in Toxicology, 32 (2): 113-132. 

Oiverira, R.F., McGregor, P.K., Latruffe, C. (1998). 

Know Thine Enemy: Fighting Fish Gather Information 

from Observing Conspecific Interactions. Proceedings: 

Biological Science, 265 (1401): 1045-1049. 

Potera, C. (2000). Drugged Drinking Water. 

Environmental Health Perspectives, 180 (10): 108- 

110. 

Quintana, J.B., Carpinteiro, J., Rodríguez, I., Lorenzo, 

R.A., Carro, A.M., and Cela, R. (2004). Determination 

of Natural and Synthetic Estrogens in Water by Gas 

Chromatography with Mass Spectrometric Detection. 

Journal of Chromatography A, 1024, 177-185. 

Effects of Increased Levels of Lactate on Cognitive Ability 

Kevin Murray and David Stapleton 




This study investigates the effect of lactate on cognitive ability, in the form of memory 

recall. Sports and competition often hinge on the ability of a participant to make good 

decisions. This is especially true near the end of a game or competition when the game is 

on the line. Often near the end of a physical competition the participants become fatigued 

and have elevated levels of lactate, and this fatigue can lead to poor decision making. This 

study reproduced fatigue by inducing higher levels of lactate, due to strenuous physical 

exercise on a stationary bike. Higher levels of lactate were expected to create a lesser 

cognitive ability; in this case the cognitive ability was the outcome of a memory recall test. 

Although there were some differences in cognitive abilities at different lactate levels, this 

study found there was no significant difference in the average cognitive ability between 

baseline and elevated levels of lactate for the ten participating subjects. 

Introduction 

Competitive sports, team and individual, often 

hinge on the decision making of the participants. Early 

on in the competition individuals are at peak levels 

physically and mentally; however, this may not hold 

true for the later phases as individuals tire (Fleury and 

Bard, 1987). The physical effects of extended exercise 

can be readily seen and measured, but differentiating 

the decision making capability remains somewhat 

nebulous. This decision making ability at later stages of 

a competition can be critical, making the difference 

between winning or losing. 

Extended exercise pushes individuals into the 

oxygen starved levels of anaerobic activity, generating 

increasing levels of lactate and depleting levels of 

available glucose. While the body can generate 

supplies of glucose for the brain through 

gluconeogenesis, disposing of some of the excess 

lactate, there are still increased levels in the blood 

stream. This excess lactate flows across the blood 

brain barrier (Nemoto and Severinghaus, 1974) and 

may have an affect on the decision making capability 

of individuals. There is also evidence of extracellular 

lactate in the brain in studies done on rats (De Bruin et 

al., 1990). This lactate was the result of stress, 

primarily stress brought on by exercise. Further 

evidence supports the presence of increased lactate in 

the brain during cognitive stimulation (Urrila et al., 

2003). 

There is some evidence to point to an adverse 

effect from lactate in the brain (Bakker, et al., 

2004)(Kaufmann, et al. 2004)(Weiskopf, et al., 2002), 

as well as evidence that shows that the presence of 

33 



34 




lactic acid may challenge certain memory inhibition 

(Gibbs, et al., 2007). 

This study hopes to resolve some of the 

confusion by providing a measure of the memory 

capabilities at varied levels of lactate, across a small 

sample population. Our hypothesis for this experiment 

is: there is a significant difference in the memory 

capability between resting and elevated levels of 

lactate. 


Subjects: 

Ten test subjects (N = 10) were selected from 

male and female individuals of ages 17 to 50 years in 

age, and levels of fitness from semi-sedentary to 

athletic. Minimum standards were observed in that all 

subjects were adults and capable of 30 minutes of 

sustained exercise, on a stationary bike, at moderate 

levels. 

Design of the study: 

The measure of memory capability across 

increasing levels of lactate involved a small sample 

population of 10 individual adults with clean bills of 

health. Lactate was generated by exercise at a heart rate 

in the anaerobic range appropriate for the individual’s 

age and relative fitness as following formula: 

Max Heart Rate (Male) = 210 - Age * 0.8 

Max Heart Rate (Female) = 205 - Age * 0.7 

Target Heart Rate = (Max Heart Rate - Resting 

Heart Rate) * Anaerobic Threshold % 

Memory capability was measured by recall of a 

number of items from a computer image viewed by the 

test subject for two seconds. Measurement at multiple 

levels of lactate including resting levels provided 

adequate measures to differentiate memory tests. The 

full test session consisted of the following: 

Take resting lactate and heart rate 

measurements 

Test memory 

10 minute warm-up, 

Increase effort to reach target heart rate for 

anaerobic exercise (based upon age, sex and 

resting heart rate, see formulae above) 

1 minute at target heart rate, 

Take lactate measurement and record heart 

rate 

Test memory 

5 minute recovery period at very low heart 

rate 

We do realize that there were many different 

people being tested with many different fitness levels 

so intensity of exercise was defined according to 

Lactate Threshold (LT) to normalize any stress to 

subjects (Farina et al., 2004). 

Measurement of anaerobic activity: 

Subjects wore an Xplorer GLX PasPort PS 

2002 with Exercise Heart Rate sensor PS 2129 Polar 

T31 wireless chest band heart rate monitor to measure 

the subject’s heart rate over the test interval. 

Measurement of lactate in blood: 

Subjects’ lactate was measured through a 

blood sample tested using a Lactate Scout portable 

lactate measuring device. The samples were taken 

using antiseptic technique, cleaning and sterilizing the 

area to be lanced. 

Measurement of cognitive ability: 

Cognitive ability was determined using two 

pictures, one each for the before and after test. The 

pictures contain a variety of items, 20 items in each 

picture, and were shown to the subject for two seconds 

and then removed. Subjects then were asked to name 

the items from the picture that they remember. 

Data analysis: 

The data collected was comprised of a lactate 

measurement paired with a cognitive value for the 

number of items remembered from the picture. The 

paired values for resting and exercising states will be 

compared to assess the impact of lactate on cognitive 

ability. 

Statistical analysis: 

The statistical software in Excel (Microsoft® 

version 2003) was used for all statistical analyses. 

Comparisons between responses were made using 

Student t-Test. Statistical significance was accepted at 

5%. Results are presented as means ± standard error 

(SE), unless stated otherwise. 

Results 

As can be seen in Table One, five subjects 

scored slightly better on memory test one (low lactate) 

than on memory test two (elevated lactate). Four 

subjects scored the same on tests one and two. One 

subject scored better on memory test two than on 

memory test one. 

Figure One shows the average scores, out of 

twenty, for the two memory tests. Memory test one 

yielded a mean score of 5.2 ± 0.47 (S.E.M.). Memory 

test two yielded a mean score of 4.7 ± 0.45 (S.E.M.). 

There is a small difference in the average scores on the 

two memory tests. A one-tailed t-Test assuming 

unequal variances was run, p = 0.22, p>0.05 therefore 

there is no significant difference in the cognitive 

abilities at baseline or elevated lactate levels. 

Baseline lactate levels ranged from 2.0 

mmol/L to 5.3 mmol/L. The elevated lactate levels 

ranged from 8.1 mmol/L to 22.6 mmol/L.

35 




Mean Recall 

6 

5 

4 

3 

2 

types of variables were difficult to control for and may 

have led to the undesirable results. 

With a better designed cognitive ability test 

and perhaps more subjects a different result could 

possibly have been obtained. However, in the case of 

this study, the original thought that lactate levels have a 

significant effect on memory capabilities is rejected, 

and the findings suggest there is not a significant 

effect. 

1 

0 

Memory Test 1 Memory Test 2 

Figure 1. Mean for memory test one, performed at baseline 

lactate levels, was 5.2 ± 0.47(s.e.m.). Mean for memory test 

two, performed at elevated lactate levels, was 4.7 ± 

0.45(s.e.m.). One-tailed t-Test assuming unequal variances P 

= 0.22. Error bars indicate standard error of the mean. 

Discussion 

According to this study lactate level has no 

significant effect on cognitive abilities. There was a 

slight difference between the memory recall for lower 

lactate level and higher lactate levels, however, the 

results were not consistent. Five subjects score higher 

on the first cognitive test when their lactate levels were 

rather low. Four subjects scored the same regardless of 

their lactate level. Finally, one subject scored better on 

the cognitive test when their lactate levels were 

elevated. The latter result was interesting because this 

subject looked visually to be the most fatigued. This is 

in direct contrast with the findings by Fleury and Bard. 

However, this result seems to be in concordance with a 

finding by Urrila et al., which found that higher lactate 

levels were associated with cognitive functioning. 

As there was no difference in the cognitive 

abilities one may assume the levels of lactate crossing 

the blood brain barrier were not sufficient during short 

term bouts of activity above the anaerobic threshold to 

impair cognitive ability. 

A possible source of error in this study was 

the design of the memory test. Roediger found that in 

the case of pictures verses words on a memory tests, 

subjects were able to use priming, to help them 

remember, when pictures were used in the memory 

test. So if a subject sees two items that are related to 

each other in some way the subject may remember both 

easier because they associate one with the other. 

Another reason for insignificant results could 

have included the small number of subjects. It was 

also difficult to control the subjects’ environment and 

activity prior to testing. One subject became physically 

ill after testing, and the researchers later learned the 

subject had not eaten for a long period of time. These 

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36 




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The Effects of pH on Escherichia coli Fermentation 

YURIKO KAYAMA 

Department of Biological Sciences, Saddleback College, 28000 Marguerite Parkway, Mission Viejo, 

CA 92692, USA 

The studies of Escherichia coli are closely related to human lives because they live in 

mammals’ gastrointestinal and sometimes cause diseases after changing to pathogens. In this 

study, the fermentation of E. coli was examined in different pH conditions (pH 1, 3, 5, 7, and 9) at 

36 ºC for 24 and 48 hours by measuring gas production. The hypothesis being tested is that gas 

production during E. coli fermentation under different pH conditions will be significantly different. 

As a result, the gas production in pH 1 was 0.14 ± 0.02 (±se) mls: 0.02 ± 0.02 (±se) mls in pH 3, 0.69 

± 0.12 (±se) mls in pH 5, 0.56 ± 0.13 (±se) mls in pH 7, and 0.64 ± 0.13 (±se) mls in pH9. Gas 

productions was significantly different between different pH groups (p=0.0002

37 




efficiently with the energy by using mixed acid 

fermentation. According to the studies of Bragramyan 

and Trchounian (2003), oxidation of glucose in 

bacteria involves a chain of sequential chemical 

reactions with some enzymes and H+ and CO 2 in 

mixed-acid fermentation of E. coli. 

In this study, Escherichia coli will be used 

because it is closely related to our lives and has many 

advantageous characteristics as a production host, such 

as rapid growth under aerobic and anaerobic conditions 

and simple nutritional requirements (Chang et al., 

1999). E. coli live in mammals’ gastrointestinal under 

anaerobic condition and also have to be able to survive 

in the low of the gastric stomach to colonize the 

intestinal tract of humans if they are injected from 

mouth (Diez-Gonzalez et al., 1998). Thus, there are 

the important relationships between pH and E. coli 

fermentation. 

The previous study shows that increasing the 

pH at the interval of 0.5 resulted in an increase in 

cumulative volume of gas production up to pH 6 and 

thereafter the production dropped (Chittibabu et. al., 

2006). In the same study, the poor hydrogen 

production at low pH, lower than 5.5 could be due to 

the increased formation of acidic metabolites, which 

destroys the cell's ability to maintain internal pH 

(Chittibabu et. al., 2006). However, this study was 

demonstrated from pH 5 to pH 7, and it does not show 

the effects of various pH values on the gas production 

of E. coli. Therefore, the effects of various pH groups 

on E. coli fermentation will be tested (pH 1, 3, 5, 7, and 

9) in this study, and the volume of gas produced in 

fermentation tubes will be measured. The hypothesis 

being tested is that gas production during E. coli 

fermentation under different pH conditions will be 

significantly different. 


Materials and medium 

Escherichia coli used in this study were 

provided from Biological Department at Saddleback 

College. For the medium for Escherichia coli, 1% of 

glucose solution was prepared by using five 100-mL 

volumetric flasks. Glucose was used for the important 

sugar source for the fermentation of E. coli (Xu et al., 

1999). Each glucose solution was adjusted to be 

appropriate pH value (pH 1, 3, 5, 7, 9) with 1.0 M HCl 

or 1 % NaOH. The pH values were checked with pHindicator 

strips (range 0-14 and 0-6) (Gibbstown, NJ, 

USA). 

Fermentation conditions and measurement 

Five fermentation tubes were used for each pH, 

and 25 fermentation tubes were filled with 14.5 mls of 

glucose solution and 0.5 mls of E. coli solution without 

any bubbles inside. Anaerobic condition was made by 

using fermentation tubes. Aseptic technique was 

performed in each time of transfer of E. coli to 

fermentation tubes. For the fermentation tubes, a 10- 

mL graduated cylinder was used while 0.5 mls of E. 

coli solution were measured by using a micropipette. 

The fermentation tubes were put in the incubator at 36 

ºC for 48 hours. The volume of gas production in the 

fermentation was measured after 24 and 48 hours. The 

amount of gas produced in each tube was indicated by 

a scale in milliliter. 

Statistical analysis 

Data were analyzed, using Microsoft Excel 

2003 (Microsoft, Redmond, WA) with ANOVA to 

determine if there were significant differences between 

pH groups. The difference was considered significant 

at P < 0.05 for Bonferroni Correction. All data 

reported were means ± SE unless noted otherwise. 

Results 

Gas Production 

Gas production in the fermentation of E. coli 

significantly differs between different pH groups 

(p=0.0002

38 




Gas Production (ml) 

0.9 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

pH1 pH3 pH5 pH7 pH9 

Figure 1. Mean gas production is 0.14 ± 0.02 mls (±se) 

in pH1: 0.02 ± 0.02 mls in pH3, 0.69 ± 0.12 mls in 

pH5, 0.56 ± 0.13 mls in pH7, and 0.64 ± 0.13 mls in 

pH9 (±se). Maximum production of gases was 

observed in pH5, 0.69 ± 0.12 mls (±se), and minimum 

production was in pH3, 0.02 ± 0.02 mls (±se). Gas 

production in the fermentation of E. coli significantly 

differs between different pH groups (p=0.0002, 

ANOVA). Error bars show the standard errors. 

Decrease of the volume of gas production 

For four tubes in pH 3 and one tube in pH 1, 

the decrease in volume of gas production between 24 

hours and 48 hours after the initiation of the 

fermentation in E. coli was observed. Also, mean gas 

production after 48 hours in pH 3 is smaller than after 

24 hours while the increases of gas production were 

observed for other pH groups (Table. 1). Mean gas 

production of 24 hours after the incubation was 0.11 ± 

0.02 mls (±se), and mean of 48 hours after was 0.02 ± 

0.02 mls (±se). 

Table 1. Mean gas production in milliliters after 24 

hours and 48 hours of Escherichia coli fermentation in 

different pH groups (pH 1, 3, 5, 7, and 9). N=5 for 

each pH. The incubation was carried out at 36 ºC. The 

values were expressed as means ± se. 

Gas Production (ml) 

Time pH 1 pH 3 pH 5 pH 7 pH 9 

Also, gas production in higher pH (pH 5-9) was 

greater than in lower pH (pH 1-3) (Fig. 1). Although 

the maximum production was observed in pH 5, 0.69 ± 

0.12 mls (±se), there were much greater gas production 

in pH 7 and 9 than pH 1 and 3. From these results 

show that higher pH (5-9) allows E. coli to produce 

larger amount of H 2 and CO 2 , and, thus, optimum pH 

in this study. These pH values can include the result of 

Chitteibabu, et al. (2006) that optimum pH is 6.0 for 

maximum yield of hydrogen in Escherichia coli BL- 

21. In another study, optimum pH is 6.2 with glucose 

and a mixed culture (Oh et al., 2003). Furthermore, 

Glass et al. (1992) concluded that E. coli grew well at 

all alkaline pH values (to pH 9.0). Thus, E. coli has 

wide range of optimum pH for the fermentation, pH 5- 

9, and does not carry out the fermentation effectively in 

pH 1 and 3. The results in this study are reliable 

because they match previous studies. 

In this study, pH 5 showed the maximum 

production, 0.69 ± 0.12 mls (±se) although lower pH 

than pH 5 showed a less amount of gas production, 

0.14 ± 0.02 mls (±se) in pH 1 and 0.02 ± 0.02 mls in 

pH 3. The gas production rapidly decreased from pH 5 

to pH 3 (Fig. 1). These results show that there is a 

border line between pH 3 and 5 and suggest that E. coli 

cannot carry out the fermentation efficiently at the 

lower pH than that border line. Jordan et al. (1999) 

also showed that poorer pH homeostasis was most 

evident below pH 5. However, the minimum gas 

production was not in pH 1. The minimum gas 

production was observed in pH 3 in this study as it is 

between 4.0 and 4.5 in the study of Glass et al. (1992). 

Thus, the gas production in E. coli fermentation is not 

proportional to pH values. The fermentation of E. coli 

is regulated by many factors such as a fermentating 

substrate, pH, redox potential, and temperature 

(Bragramyan and Trchounian, 2003). Also, E. coli (K- 

12 and O157:H7) produce many organic compounds, 

converting glucose to acetate, formate, and ethanol 

(Diez-Gonzalez and Russell, 1997). The combination 

of these factors and processes gives the different results 

in each time. However, further analysis is required for 

the details of the combination of them. 

24 hours 0.12±0.02 0.11±0.02 0.56±0.08 0.43±0.12 0.51±0.13 

Minimum pH for growth and Internal pH 

48 hours 0.14±0.02 0.02±0.02 0.69±0.12 0.56±0.13 0.64±0.13 

There are many enzymes that play a role in 

mixed-acid fermentation of E. coli. Enzyme repression 

Discussion 

and inhibition of their activity by oxygen regulate the 

pH dependence and optimum pH 

gateway of mixed-acid fermentation because these 

enzyme function only under aerobic or anaerobic 

Gas production in the fermentation of E. coli condition (Bragramyan and Trchounian, 2003). From 

showed that there are significant differences between results, the gas production in the fermentation of E.coli 

different pH groups (Fig. 1; p=0.0002, ANOVA). This was greater in higher pH (Fig. 1). Some enzymes in a 

result shows that the activity in the fermentation of E. process of the fermentation require H + source 

coli depends on pH values. 

(Bragramyan and Trchounian, 2003). However, the

39 




induction of enzyme was observed in weakly alkaline 

medium although acidification of the medium also 

promoted induction of the enzyme (Bragramyan and 

Trchounian, 2003). In fact, the greater gas production 

was observed in higher pH (pH 5-9). Thus, if we focus 

on just enzyme activity, some of the activities of the 

enzymes are encouraged in acidic condition, but the 

entire process of fermentation requires high pH 

condition. 

E. coli need to maintain their internal pH to survive 

when extracellular pH decreases. In E. coli 

fermentation, acetate, a product of fermentation, 

inhibits the growth of E. coli as the accumulation of 

acetate ions increases intracellular potassium that 

causes the acidification of cytoplasm (Diez-Gonzalez 

and Russell, 1997). According to Chitibabu et al. 

(2006), the poor hydrogen production at low pH, lower 

than 5.5 could be due to the increased formation of 

acidic metabolites, which destroys the cell's ability to 

maintain internal pH. Also, protein confirmation 

depends on the protein’s environment, and protein is 

denatured if the pH or/and other factors are altered 

(Campbell and Reece, 2005). Protein of E. coli that do 

not have tolerance for acidity can be inactivated by low 

pH. In the study of Diez-Gonzalez and Russell (1997), 

the decline in cell protein was observed when acetic 

concentration is higher. As a result, the fermentation of 

E. coli rapidly decreased because they cannot maintain 

their internal pH. Thus, the lower pH inhibits the gas 

production in E. coli fermentation below pH 5. In 

higher pH, greater gas production was yield because 

there is no stress of acidity. The border line between 

pH 3 and 5 indicates the minimum pH for growth of E. 

coli. Thus, the amounts of gas production above pH 5 

are close to each other and much greater than below pH 

5. Also, E. coli produced much less gas in lower pH, 

and the amounts of gas are close. 

Acknowledgments 

I would like to thank the Saddleback College 

Biology Department and Professor Teh for providing 

the materials (including Escherichia coli) and for 

assisting with the development of the experiment 

protocol. 


Bagramyan, K. and A. Trchounian. (2003). Structural 

and functional features of formate hydrogen lyase, and 

enzyme of mixed-acid fermentation from Escherichia 

coli. Biochemistry: 68(11), 1159-1170. 

Campbell, N. A. and J. B. Reece. (2005). Biology: 

Seventh edition. San Francisco, Benjamin 

Cummings. 

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Homofermentative production of D - or L - 

lactate in metabolically engineered Escherichia 

coli RR1. Applied and Environmental 

Microbiology, 65(4), 1384-1389. 

Chittibabu, G., K. Nath, and D. Das. (2006). Feasibility 

studies on the fermentative hydrogen 

production by recombinant Escherichia coli 

BL-21. Process Biochemistry, 41, 682-688. 

Diez-Gonzalez, F., and J. B. Russell. (1997). The 

ability of Escherichia coli O157:H7 to decrease its 

intracellular pH and resist the toxicity of acetic 

acid. Microbiology, 143, 1175-1180. 

Diez-Gonzalez, F., T. R. Callaway, M. G. Kizoulis, and 

J. B. Russell. (1998). Grain feeding and the 

dissemination of acid-resistant Escherichia coli 

from cattle. Science, 281, 1666-1668. 

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Doyle. Fate of Escherichia coli O157:H7 as 

affected by pH or Sodium Chloride and in 

fermented, dry sausage. Applied and Environmental 

Microbiology, 58(8), 2513-2516. 

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L. R. Booth, and S. F. Park. (1999). 

Augmentation of killing of Escherichia coli O157 

by combinations of lactate, ethanol, and low-pH 

conditions. Applied and Environmental 

Microbiology, 65(3), 1308-1311. 

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Fermentative biohydrogen production by a new 

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Kakizono, and N. Nishio. (1997). Enhanced 

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processes with Escherichia coli. Appl 

Microbiol Biotechnol, 51, 564-571.


Antibiotics (Tobramycin and Polymyxin) Resistance in Staphylococcus aureus & 

Effectiveness of These Antibiotics 

Kaung Ko and Spencer Roberts 

Department of Biochemistry and Biological Sciences 



Staphylococcus aureus is a major problem; causing a number of illnesses and diseases. It is 

regarded as one of the primary infections in post-surgery and is known to grow resistance 

against antibiotics. In this experiment, antibiotic effectiveness of Tobramycin Opthalmic 

solution (0.3%), and Polymyxin B Sulfate (with Trimeththoprim) were tested; both are 

common antibiotics for treating staph infections in the eyes. It was hypothesized that S. 

aureus will grow more resistance to Polymyxin, based on the previous studies of how these 

two drugs interact with the bacterium. Three generations of S. aureus were treated with the 

antibiotics. The antibiotics were diluted and then placed on the S. aureus; each antibiotic 

was placed in to separate Petri dishes and grown with S. aureus. The antibiotic 

concentration(s) given to the S. aureus were, 20%, 40% and 50% respectively for the three 

consecutive generations. In the first generation, the mean radius of the zone of inhibitions 

observed for Tobramycin and Polymyxin 20% diluted solution was 0.26 cm ±0.07 and 0.38 

cm ±0.07 respectively. However, the mean radius of the zone of inhibitions observed in the 

second generation was 0.69 cm ±0.04 and 0.20 cm ±0.09 and was 1.2 cm ±0.1 and 0.18 cm 

±0.06 respectively in the third generation. ANOVA multiple variance comparison tests give 

p


Figure. 1. Molecular structure of Tobramycin 

Polymyxin (Firgure 2) is also an antibiotic, 

whose structure has a cyclic peptide with a long 

hydrophobic tail. It works by interacting with the 

bacterial membrane’s phospholipids and disrupting the 

membrane. Polymyxin is produced from a Grampositive 

bacterium Bacillus polymyxa, and is very 

toxic for the Gram-negative bacteria, disrupting the 

lipopolysaccharide layer in the outer membrane. It can 

potentially be effective in killing Staph a. (Nakamura T 

et al., 2003) 

Figure. 2. Molecular structure of Polymyxin 

Since both Tobramycin and Polymxcin are 

toxic for the Gram-negative bacteria, they should easily 

be able to kill staph, a simple Gram-positive infection. 

Antibiotic resistance is the ability of virus, bacteria or 

microorganisms to endure and survive (resist) 

antibiotic effects. It naturally evolves through natural 

selection and mutation by applying evolutionary stress 

on the population (Cirz et al., 2005). In order to 

research upon the antibiotic with the longest effect on 

the bacterium before the evolution favors it to become 

resistant, several generations of Tobramycin and 

Polymyxin resistance S. aureus colonies will be grown, 

and effect will be determined by the size of zone of 

inhibition. It is predicted that the antibiotic with the 

longest effect will have a larger zone of inhibition, and 

the opposite for the other. 


On 27 March 2008, one liter of nutrient agar 

was plated on 34 sterile Petri dishes. 

On March 31 st , 2008, nutrient broth containing 

S. aureus were obtained from the Microbiology stock 

room. Tobramycin and Polymyxin ophthalmic 

solutions were obtained from Longs Drug store in 

Laguna Hills, California. To decrease the concentration 

of Tobramycin and Polymyxin, both were diluted: one 

mL of antibiotic solution to every four mL of sterilized 

water, giving 20% strength. First, one mL of S. aureus 

solution was spread onto four of the prepared agar 

plates using P1000 pippetteman. The sterilized glass 

rod, dipped into ethanol and flamed before spreading, 

was used for bacterial lawn preparation. Containers and 

materials were exposed slightly to the flame to keep 

sterilized every time before using. Five sterilized discs 

were dipped in the diluted Tobramycin solution and 

other five in Polymyxin solution. The discs were 

placed on the Staphylococcus spread nutrient plates 

and the plates were incubated overnight for 24 hours at 

38 o C. 

The procedure began with 20% dilution of the 

antibiotic and bacteria colonies allowed to grow for 24 

hours. The radii of zone of inhibitions were measured. 

Colonies in the zone of inhibition were pulled using a 

sterilized loop and cultured in nutrient broth for 24 

hours. They were plated as before but with different 

antibiotic concentration on the chads; this time 40% 

and allowed to grow for 24 hours. Radii of the zone of 

inhibition were measured for that generation. The 

procedure was repeated with the final antibiotic 

concentration of 50%. 

For the data analysis the sizes of the zone of 

inhibitions produced by Tobramycin and Polymyxin at 

each generation were compared and mean radius of 

zone of inhibition were calculated. The multiple 

analysis of variances between the data sets were also 

calculated utilizing Microsoft Excel software and 

ANOVA software. 

Results 

The radiuses of zone of inhibition (cm) of 

Tobramycin, for all five zones after the 1st generation, 

were 0.4 cm, 0.4 cm, 0.1 cm, 0.1 cm, and 0.3 cm. The 

radiuses of zone of inhibition (cm) of Polymyxin, for 

all five zones after the 1st generation, were 0.6 cm, 0.5 

cm, 0.3 cm, 0.3 cm, and 0.2 cm. Polymyxin is more 

effective in killing S. aureus in the 1 st generation with 

mean radius of the zone, 0.38 cm ±0.07 compared to 

the mean radius of 0.26 cm ±0.07 by Tobramycin 

(Table 1.). 

The radiuses of zone of inhibition (cm) of 

Tobramycin, for all eight zones after the 2nd 

generation, were 0.7 cm, 0.6 cm, 0.7 cm, 0.6 cm, 0.7 

cm, 0.6 cm, 0.9 cm, and 0.7 cm. The radii of zone of 

inhibition (cm) of Polymyxin, for all eight zones after 

the 2nd generation, were 0.5 cm, 0.4 cm, 0.0 cm, 0.0 

41 




cm, 0.0 cm, 0.0 cm, 0.1 cm, and 0.6 cm. Tobramycin 

is more effective in killing S. aureus in the 2 nd 

generation with mean radius of the zone, 0.69 cm 

±0.04 compared to the radius of 0.20 cm ±0.09 by 

Polymyxin (Table 2.). There is higher resistance with 

Polymyxin. 

The radii of zone of inhibition (cm) of 

Tobramycin, for all eight zones after the 3rd generation 

were 1.1 cm, 0.9 cm, 1.5 cm, 1.6 cm, 0.5 cm, 1.5 cm, 

1.5 cm, and 1.0 cm. The radiuses of zone of inhibition 

(cm) of Polymyxin, for all eight zones after the 3rd 

generation were 0.1 cm, 0.35 cm, 0.15 cm, 0.0 cm, 0.5 

cm, 0.2 cm, 0.1 cm, and 0.0 cm. The 3 rd generation 

Tobramycin is more effective in killing S. aureus with 

mean radius of the zone, 1.2 cm ±0.1 compared to the 

radius of 0.18 cm ±0.06 by Polymyxin (Table 3.). 

There is higher resistance with Polymyxin. 

There is a significant difference between the 

radiuses of the zone of inhibitions produced by 

Tobramycin and Polymyxin at each generation; P value 

is less than 0.05 (Table 4.), and it can be concluded 

from Figure 3 that as next generation was entered, S. 

aureus became more resistance to Polymyxin and 

much less to Tobramycin. 

Polymyxin 

2nd Gen 

Polymyxin 

2nd Gen 

0.5 0.4 0 0 0 

0 0.1 0.6 

Table 3. Radius of the zone of inhibitions observed for 

Tobramycin and Polymyxin 50% diluted solution of 

the 3 rd generation. Mean radius for Tobramycin is 1.2 

cm ±0.1 and for Polymyxin is 0.18 cm ±0.06. 

Tobramycin 

3rd Gen 

Tobramycin 

3rd Gen 

Radius of Zone of Inhibition 

(cm) 

1.1 0.9 1.5 1.6 0.5 

1.5 1.5 1.0 



the 1 st generation. Mean radius for Tobramycin is 0.26 


Radius of Zone of Inhibition (cm) 

Polymyxin 

3rd Gen 

Polymyxin 

3rd Gen 

0.1 0.35 0.15 0 0.5 

0.2 0.1 0 

Tobramycin 

1 st Gen 

Polymyxin 

1 st Gen 

0.4 0.4 0.1 0.1 0.3 

0.6 0.5 0.3 0.3 0.2 

Table 4. Statistical significance calculated based on the 

ANOVA results and the table produced by the 

software. T represents the Tobramycin and P represents 

the Polymyxin followed by the generation number. In 

all generations, there is a significant difference. 



the 2 nd generation. Mean radius for Tobramycin is 0.69 


Radius of Zone of Inhibition (cm) 

Comparison Significant? (P


Mean Radius of Zone of Inhibition 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

-0.2 

1st Generation 2nd Generation 3rd Generation 

Generation 

Tobramycin 

Polymyxin 

Figure 3. Mean radius of zone of inhibitions produced 

by Tobramycin and Polymyxin at each generation. 

Error bars representing ±se. 

Discussions 

The results showed that there were significant 

resistance levels due to effectiveness of Tobramycin, 

and Polymxin B Sulfate in treating S. aureus. The first 

generation of Staphylococcus, treated with the 20% 

antibiotic solution(s) of Tobramycin and Polymyxin 

showed an average radius of 0.26cm ±0.07 and 0.28cm 

±0.07 zones of inhibition respectively. Second 

generation of S. aureus (treated with 40% antibiotic 

solution) showed an average radius of 0.69cm ±0.04 

and 0.20cm ±0.09 zones of inhibition respectively. 

The third generation of S. aureus (treated with 50% 

antibiotic solution showed an average radius of 1.20cm 

±0.1 and 0.18cm ±0.06 zones of inhibition 

respectively. After statistical analysis, it was 

concluded that S. aureus did not develop resistance to 

Tobramycin. Polymyxin B Sulfate grew progressively 

weaker to the bacteria with each new generation 

grown. This drug is concluded to be the least effective 

towards the bacterium. Further analysis of these results 

indicates that Tobramycin is a very effective drug to 

treat bacteria infections such as S. aureus. (Lucas et 

al.). Although the Tobramycin used in the first 

generation had smaller zones of inhibition than the 

Polymyxin, the difference can be accounted for the fact 

that Tobramycin did not spread as rapidly as the 

Polymyxin, therefore did not kill as much bacterium. It 

can be concluded that Tobramycin is much more 

effective at inhibiting bacterium reproduction. Its 

inhibition of translation of mRNA into a protein is 

much more effective, as opposed, to Polymyxin 

disrupting the outer polysaccharide layer of the 

bacterium. 

The experiment demonstrates the speed at 

which evolutionary adaptation bacterium possesses 

when put under evolutionary stress as Cirz et al. had 

explained and demonstrated (Cirz et al., 2005). It 

should also be noted the speed at which the mutations 

occurred at on an evolutionary scale. According to 

Maree et al., the resistance did occur. This happened 

within three generations of the S. aureus. It is not 

known whether the S. aureus was already resistant to 

the Polymyxin or if it mutated in the small amount of 

time that the bacterium were grown. 

Acknowledgements 

The authors appreciatively acknowledge 

Professor Steve Teh and Dr. Tony Huntley for their 

guidance and Saddleback College Biology Lab for the 

contribution of S. auereus, measuring devices and other 

required testing materials. 


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Inoue, T., Shoji, H., Hara, M., Shimada, N., Koide, H 

(2003). Hemoperfusion with Polymyxin B- 

Immobilized Fiber in Septic Patients with Methicillin- 

Resistant Staphylococcus aureus-Associated 

Glomerulonephritis. Nephron Clin Pract 2003;94:33- 

c39 

Reddy, S.L, Grayson, A.D, Smith, G., Warwick, R., 

and Chalmers, J.A (2007). Methicillin 

resistant Staphylococcus aureus infections 

following cardiac surgery: incidence, impact 

and identifying adverse outcome traits. 

European Journal of Cardio-Thoracic 

SurgeryVolume 32, Issue 1, July 2007, Pages 

113-117 

Effects of temperature on metabolic rate in Gromphadorhina portentosa 

Dorothy Chang and Grant T. Huttar 




Concentrations of carbon dioxide production of hissing cockroaches 

(Gromphadorhina portentosa) were measured in sealed containers; acclimation to 

environments occurred for 10 minutes and roaches were left to metabolize the air, CO 2 

production recording every second for 15 minutes. Eight roaches were exposed to 

temperatures of 10.0, 23.0, and 38.0C under constant conditions and with multiple 

replications. The mean body mass of the roaches were 6.966 grams (g). The mean metabolic 

rate (MR) of roaches was greatest in elevated temperature of 6.755 10 -5 9.388 10 -6 

(standard error) milliliters per gram of body mass per second (mL/g/sec) followed by room 

temperature with 3.770 10 -5 mL/g/sec 4.791 10 -6 (se) and cold temperature with 2.497 

10 -5 mL/g/sec 8.154 10 -6 (se). A p-value of 2.068 10 -3 was acquired through an ANOVA 

simple factor test, but a significant effect of temperature on MR was not seen due to the 

fact that the probability was a combination of the three variables’, therefore lowering the 

actual p-value. 

Introduction 

Metabolism is the process by which materials 

and energy are used in an organism and exchanged 

between the organism and its surroundings. The 

functional capacity of a life form can be observed 

through the rate of O 2 inhaled and CO 2 expelled. 

Metabolic rate refers to the cost of energy for an 

organism to function at a given condition—such as 

variable-induced environments—opposed to a natural 

one. Basal metabolic rate represents the amount of 

energy expended when the subject is at ease in a 

thermoneutral zone in a post-absorptive state (Hails, 

1983). An organism’s metabolic rate increases 

exponentially with temperature, affecting metabolism 

by impacting the rates of bodily reactions (Gillooly, 

2001). 

Metabolic rates of insects have been studied to 

expand knowledge regarding energy costs of various 

types of locomotion or basal metabolic rates. The 

demands of active metabolism of flight make it an 

extremely expensive form of movement (Dudley, 

2000) when compared to the rates of oxygen 

consumption when at rest. Contrary to more popular 

studies, the metabolic rates of uncommon and larger 

44 




species of insects remain relatively unknown. The 

amount of oxygen input or carbon dioxide output in a 

resting state may differ substantially than a flying form 

of locomotion, contributed by factors such as dietary 

energy content or temperature, even among species of 

similar body masses. However, there is a direct 

relationship between metabolic rate and body mass: 

metabolic rate per gram of body mass decreases with 

mass by a power less than 1.0 (Mueller & Diamond, 

2001), therefore the total metabolic rate of an animal 

increases proportionally with mass. 

Gromphadorhina portentosa is a species of 

large hissing cockroaches originating from 

Madagascar. No published previous experiments have 

been conducted on metabolic rates of this species. 

Gathering information about furtive, hissing roaches 

could offer insight for the comparison of energy costs 

of a more diverse range of insects. 


Procedure A. Cockroach metabolism at ambient, 

elevated, and cold temperatures 

Eight Madagascan hissing cockroaches were 

individually placed on an analytical balance; weights 

recorded (g) and labeled 1-8 for identification. Each 

roach was placed in a plastic container and sealed by a 

stopper that encircled a Pasco Temperature Sensor 

attached to a Pasco Xplorer GLX device used to 

measure carbon dioxide (CO 2 ) production in parts per 

million (ppm) with readings every second (sec) for 15 

minutes. Food consumption was restricted from the 

roaches for at least one hour prior to testing to 

eliminate increased metabolic rate from postabsorption. 

MRs of the roaches were to be observed at 

three variables: room temperature (23.0C), cold 

(10.0C), and an elevated temperature (39.0C). They 

were labeled according to weight and left to acclimate 

to their ambient temperature and surroundings for 10 

minutes. Roaches were handled carefully to keep them 

in a calm state; sudden movements may lead to an error 

in analyzing the results. The concentration of CO 2 in 

the closed system of the container was recorded onto 

the Xplorer GLX every second for 15 minutes. 

Between each test, the roaches were released to their 

normal surroundings to stabilize metabolic rates so 

there would not be extreme changes in CO 2 production. 

This same procedure was repeated with the 

warm temperature, except the system was left in a 

heating chamber adjusted to 39.0C. A light was 

installed to replicate the room temperature environment 

and to encourage the roaches to stay in an aroused, but 

resting, state. The procedure was again repeated for 

cold temperature. A thermoregulated refrigerator set at 

10.0C was used with a light set inside to maintain 

constant environmental factors. 

Once complete, the data was transferred from 

the Xplorer to a flash drive device and the table of 

information (ppm/sec) was transferred as a .glx file to 

be analyzed using Pasco’s Data Studio, and exported as 

a text file (.txt) that could be read using Microsoft 

Excel. 

Procedure B. Calculating the rate of oxygen 

consumption (STP) 

To determine the metabolic rate of G. 

portentosa, the data from all the roaches were graphed 

as a scatter plot and a linear trendline was added. The 

slope of this line was the rate of CO 2 production 

(ppm/sec) and had to be set as a ratio to the amount of 

moles (mol) of air in the container. This was calculated 

by obtaining the volume of the container the, minus the 

volume of the CO 2 sensor probe and the individual 

roaches. Volumes were measured by filling the 

container to the brim with water and pouring all the 

liquid into a graduated cylinder, the final volume was 

the volume of the container. The volume of the 

cockroaches (V total ) was determined by submerging 

them into a 250mL graduated cylinder of a known 

initial volume (V i ) of liquid and subtracting that from 

the final volume (V f ): 

V total = V f – V i 

The CO 2 sensor probe was found in a similar fashion 

but with an 800mL beaker. 

The volume of air in the container was 

converted to standard temperature and pressure (STP) 

using Boyle’s Gas Law: 

P 1 V 1 = P 2 V 2 

T 1 T 2 

P 1 = atmospheric pressure (atm) 

V 1 = volume of air in container (L) 

T 1 = temperature variables (C) 

P 2 = 1.00 atm 

V 2 = volume of air in container at STP 

T 2 = 273.15K 

Atmospheric pressure (P 1 ) was found using a 

barometer in an adjacent room. The temperature, T 1 , 

was the environment in which the roaches were set in. 

The adjusted volume was then converted to moles of 

air in the container by the Ideal Gas Law: 

PV = nRT n = RT 

PV 

P = pressure (atm) 

V = volume (L) 

n = moles of air in container 

R = gas constant (0.08206 Latm/molK) 

T = temperature (K) 

Using the graph of the CO 2 concentrations 

versus time, a linear fit trendline was added in addition 

45 




with the equation of the line and the R 2 value (Fig 2). 

The slope of the line represented the metabolic rate in 

different units. The slope (ppm/sec) was multiplied by 

the total time the roaches remained in the container to 

make the value a measurement only of CO 2 per million 

molecules of air: 

Slope ppm/sec Time sec = [CO 2 ] ppm 

To determine the number of moles of CO 2 

within the volume of air in the container, a ratio was 

set between the ppm of CO 2 per million molecules of 

air: 

Parts of CO 2 = Moles of CO 2 

Parts of Air Moles of Air 

The moles of CO 2 were applied to the Ideal Gas Law to 

calculate the volume (L) of CO 2 : 

PV = nRT V = nRT 

P 

The volume of CO 2 produced was converted 

to milliliters (mL), divided by the body mass (g) of 

each roach, and divided by the duration of the trial 

(sec), ultimately yielding the metabolic rate (mL/g/sec) 

or (mLg -1 sec -1 ). Calculations were applied to all 

roaches and their averages for the three temperatures. 

Results 

A one way ANOVA test with replication 

(ANOVA simple factor) was conducted since there 

were three groups to account for. There were 

differences between the MR means for the three 

temperatures (Figure 1). Standard deviation represents 

the probability of the subjects’ results within a group 

skewing from a projected mean value (Figure 2). The 

combined p-value 2.068 10 -3 is less than the critical 

value ( = 0.05). A post hoc test was performed by 

GraphPad Software to determine if there was a 

considerable difference and between which groups. 

Paired p-values—room temperature vs. hot, room 

temperature vs. cold, hot vs. cold—are greater than = 

0.05, therefore the null hypothesis was not rejected and 

the conclusion cannot be drawn stating there is an 

association between temperature and metabolic rate. 

Mean Metabolic Rate (m L/g/sec) 

CO2 Concentration (p p m ) 

0.00009 

0.00008 

0.00007 

0.00006 

0.00005 

0.00004 

0.00003 

0.00002 3500 

0.00001 

3000 

0 

2500 

2000 

1500 

1000 

500 

Figure 2 Mean metabolic rates with corresponding 

standard deviation values: room temperature was 

3.770 10 -5 ml/g/sec 1.355 10 -5 ; cold 

temperature was 2.497 10 -5 ml/g/sec 2.157 10 - 

5 ; hot temperature was 6.755 10 -5 ml/g/sec 

2.484 10 -5 . 

y = 1.2545x + 850.1 

R 2 = 0.9984 

y = 0.7764x + 916.18 

Temperature (C) 

R 2 = 0.9848 

Room Hot Cold 

y = 0.3476x + 1482.4 

R 2 = 0.8836 

0 

400 600 800 1000 1200 1400 1600 1800 2000 

Time (sec) 

23C 39C 10C Linear (39C) Linear (23C) Linear (10C) 

Figure 1 Comparison of average metabolic rates of 

roaches from three variables. Elevated temperature 

was the highest with 6.755 10 -5 mL/g/sec 9.39 

10 -6 (se); room temperature was 3.770 10 -5 

mL/g/sec 4.79 10 -6 (se), elevated; and cold 

46 



temperature was 2.497 10 -5 mL/g/sec 8.15 10 -6 

(se). 

Figure 3 Comparison of averaged data from three 

temperatures showing slopes and correlation values. 

Slopes values converted to mL/g/sec from ppm/sec 

represent metabolic rates. 

Discussion 

G. portentosa rely mainly on carbohydrate 

synthesis (Weins & Gilbert, 1995) to provide energy 

for metabolism. The measurements of the volume of 

carbon dioxide (VCO 2 ) during cockroaches’ calm 

states and graphed slopes (ppm/sec) could be converted 

into metabolic rate in relation to body masses and the 

amount of time they respired (ml/g/sec). The volume of 

CO 2 is equal to the volume of oxygen (VO 2 ) because


VCO 2 /VO 2 = 1.0 during the oxidation of carbohydrates 

(Suarez, Lighton, Joos, Roberts, & Harrison, 1996) as 

illustrated from the balanced chemical equation: 

C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O 

Masses of eight roaches were taken before 

each test proceeded to make body mass comparable to 

metabolic rate for three variables: room temperature, 

heat, and cold-induced environments. Conditions were 

kept similar under each test, such as leaving the 

roaches exposed to light to deter them from changing 

basal to resting metabolism, where the MR would be 

lower in the latter. 

The average metabolic rates from each of the 

separate temperature trials were found to have a 

combined p-value that is lower than each of the pairs. 

There are no significant differences (p-values > 0.05) 

between any of the three variables. A large p-value 

indicates the difference in results probably happened 

by chance and that the hypothesis is not accepted. The 

null hypothesis stating there is no association of 

temperature on metabolic rate is considered valid, and 

the alternative hypothesis stating there is a significant 

difference between the MRs of the three variables is 

rejected. Therefore, temperature and MR of G. 

portentosa are independent. 

A possible reasoning for this result is due to the fact 

that cockroaches are homeothermic ectotherms, 

allowing the roaches to change their body temperature 

and better adjust to the environment that they are 

placed in. Their ability to adapt to a diversity of 

conditions may be attributed to the millions of years of 

evolution and being selected to new environments. 

Though there is no significant association 

between temperature and MR, R 2 values of the graphs 

display a relatively strong correlation (average 

0.9556) between the concentration of CO 2 and time, 

demonstrating a pattern where CO 2 and time is directly 

related: over time, CO 2 production increases within the 

trials of all three temperatures though MRs remains 

constant (Figure 3). 

Possible sources of error may include short 

time frames of data collection, a small sample size, and 

faulty equipment setup. The sample size tested was 

only 8 cockroaches while a larger sample size would 

have given a better understanding of the species as a 

whole. The test was conducted for a 15-25 minute time 

frame; the short breadth of this trail period may not 

have given the subjects enough time to acclimate to the 

surroundings and thus give inaccurate readings. During 

the high temperature trial, two separate incubators were 

used; the second had a less precise way of controlling 

the temperature which could have lead to inconsistent 

temperature conditions. Also one of the Xplorer GLX 

CO 2 detectors went into power-saving mode after a 

short period and shut off before the completion of 

timed trials, making the sample size of CO 2 production 

smaller than it should have been. An undersized 

amount of data collection could have lead to a less 

precise mean MR for the entire group for that trial. 

Another possibility is that during testing, a CO 2 sensor 

probe may not have been sealed completely to the 

container, remaining slightly ajar as to allow a higher 

concentration of CO 2 produced by a roach in the 

chamber to diffuse out into the surroundings. 

Studying insect metabolism gives a stronger 

understanding of the Kingdom Animalia and more 

specifically the subphylum Insecta. This information 

can be used in future experiments if the subject chosen 

is cockroaches, but tested under a separate variable 

instead of temperature. In addition, it could give 

examples of small organisms’ metabolic rates and to 

see if their results are within reasonable parameters or 

not. 


Dudley, R. (2000). The biomechanics of insect flight: 

form, function, evolution. Princeton University Press. 

Gillooly, J.F. (2001). Effects of size and temperature 

on metabolic rate. Science 293. 2248. 

Hails, C.J. (1983). The metabolic rate of tropical birds. 

Condor 85. 61-65. 

Mueller, P. & Diamond, J. (2001). Physiology. 

Metabolic rate and environmental productivity: Wellprovisioned 

animals evolved to run and idle fast. 

Proceedings of the National Academy of Sciences of 

the United State of America, Vol. 98, No. 22. 12550- 

12554. 

Suarez, R.K., Lighton, J.R.B., Joos, B., Roberts, S.P., 

& Harrison, J.F. (1996). Physiology. Energy 

metabolism, enzymatic flux capacities, and metabolic 

flux rates in flying honeybees. Proc. Natl. Acad. Sci. 

USA, Vol. 93. 12616-12620. 

Weins, A. & Gilbert, L. (1995). Biological sciences. 

Regulation of cockroach fat body metabolism by the 

Corpus Cardiacum in vitro. Science 29, Vol. 150, No. 

3969. 614-615. 

The Effect of Creatine Monohydrate on the Run Time of Sceloporus occidentalis 

Dayana Vera and Michael Moeller 

47 







Creatine is a natural occurring amino acid that is found in muscle and nervous 

tissue in all vertebrates. Along with its associated enzyme, creatine kinase, creatine and 

phosphocreatine create a new energy transport chain that is twelve times faster than 

oxidative phosphorylation. In this experiment the run time until exhaustion was measured 

of the Western Fence lizard (Sceloporus occidentalis) prior to consumption of creatine and 

after the consumption creatine. Runs were done using a table treadmill kept at a constant 

speed until the lizards reached exhaustion. The results showed that the hypothesis was 

confirmed and that there was a significant difference between the two groups. This shows 

that creatine plays a role in the formation of energy in the lizards and allowed them to 

utilize it more readily. 

Introduction 

Creatine and phosphocreatine are present in 

all vertebrates along with the enzyme creatine kinase. 

Creatine can keep the ATP to ADP ratio high while 

acting as a buffer with the reaction [ATP + Creatine ↔ 

Phosphocreatine + ADP] through the creatine kinase 

enzyme (Lee Fillers and Lyengar, 1988). Creatine 

kinase also plays a key role in energy metabolism in 

cells catalyzing the reversible transfer of phosphoryl 

group from phosphocreatine to MgADP to form 

creatine and MgATP (Woods and Guan, 1988) 

(Andres, Ducray, Schlattner, and Wallimann 2008). 

High energy phosphate buffers in the form of 

phosphocreatine are also known as phosphagens 

(Bessman and Geiger, 1981). In the form of 

phosphocreatine, a common phosphagen, creatine, can 

act as an intracellular energy pathway to help deliver 

ATP. Further studies performed by, Rae, Digney, 

McEwan, and Bates (2003), have showed that creatine 

supplementation has a significant positive effect on 

working memory and processing speed, this effect was 

found along with ATP which can be re-synthesized 

from phosphocreatine 12 times faster than via oxidative 

phosphorylation. This is because ATP synthesis is 

more complex than a direct phosphoryl transfer 

reaction catalyzed by the enzyme creatine kinase 

(Powers and Riordan, 1975). Phosphocreatine is an 

important source of chemical energy in the heart, brain, 

skeletal muscle, and macrophages; although no muscle 

cells or macrophages can synthesize creatine. Cells 

must take in creatine via the plasma by means of up hill 

reaction (Loike, Azalutsky, Kaback, Miranda and 

Silverstein, 1988). Creatine kinase has been identified 

as a key enzyme in the homeostasis of maintaining 

energy in vertebrates (Feng, Xu and Yan 2008). 

Creatine is available in almost every nutrition 

center and has many different forms in which can be 

found as. Creatine is commonly taken by weight lifters 

to have more energy in their muscles during exercise. 

Some studies have shown that creatine loading during 

exercise generates muscle mass (Gallo, MacLean and 

Tyreman, 2008). Other cases added that creatine can 

generate more energy in a system (Young, Bertram, 

Theil, Petersen, Poulsen, Rasmussen, Malmendal, 

Neilsen and Vestergaard 2007). For this experiment we 

will use creatine monohydrate as our source of 

creatine. By over loading Western Fence lizards with 

creatine monohydrate the reaction involving 

phosphocreatine will shift and enable the use of the 

creatine pathway to help synthesize and re-synthesize 

ATP from phosphocreatine. We hypothesize that if we 

give creatine monohydrate to Western Fence lizards 

(Sceloporus occidentalis) they will then be able to run 

for a much longer period of time demonstrating greater 

endurance. 


Western Fence lizards (S. occidentalis) were 

collected from Serrano Creek Park in Lake Forest, 

California. The lizards (N=10) were housed in a glass 

aquarium, which contained sand suitable for their 

environment. Temperature was kept well regulated 

between 75º and 85º Fahrenheit with a heat lamp and 

Ultra Violet Lamp lighting the aquarium from above. 

For an entire period of one month, the lizards were fed 

three times a week and water was provided 

accordingly. 

Prior to obtaining the lizards, 500 pinhead 

crickets were purchased from Pet Smart in Rancho 

Santa Margarita, Ca, and 600 grams of Creatine 

Monohydrate pure powder was purchased from the 

Now Sport Nutrition website. Furthermore, for the first 

two consecutive weeks of the experiment the lizards 

were fed 40 pinhead crickets three times a week. 

Before running the lizards, to obtain the control run 

48 




time, all ten lizards were submitted to an individual 

training run time on a motor driven treadmill provided 

by the Biological Science Research Lab at Saddleback 

College. Each lizard was placed on the treadmill with a 

small plexiglas box over their body in order to sustain 

them on the treadmill. Treadmill speed was 

continuously adjusted to complement the lizards’ 

sprinting speed. 

Once already trained, by the end of the second 

week the lizards’ run times were tested for one final 

control run and a simple minute stop watch was used to 

determine the lizards’ run time in seconds until 

exhaustion was reached. Exhaustion appeared obvious 

when each lizard flipped over on their back without 

moving for a certain time. After time had been 

determined, the lizards were not subjected to another 

run for the next ten days. Moreover, in the following 

two weeks the diet of the lizards was rapidly changed. 

Instead of only feeding them 40 crickets three times a 

week, a new supplement, creatine monohydrate pure 

powder, had been introduced to their diet along with 

the crickets. 

During this experimentation session of two 

weeks long, 40 crickets were placed in a freezer size 

Ziploc plastic bag to dust them in 100 grams of 

creatine powder. Once the crickets had been dusted, 

they were evenly distributed among all ten lizards, 

which had already been separated within the room. 

After consuming the food, they were returned back to 

their short term home, the aquarium. This process 

occurred three times a week for two consecutive 

weeks, in order to test for a higher endurance in the 

lizards’ run times. 

According to previous studies, all lizards 

needed to be trained to run on the motor driven 

treadmill one more time before the final 

experimentation session, otherwise a greater standard 

error could perhaps be obtained. Therefore two days 

before the actual finale, all ten lizards were trained 

exactly as the previous time. On the last day of the two 

weeks, all lizards consumed crickets dusted in 100 

grams of creatine 30 minutes to 1 hour before exercise. 

Prior to experimentation, each lizard was removed 

from the carry on bin into an isolated plastic bucket. 

Since temperature in the research lab stated 68ºF, warm 

water was left running in the sink were the bucket had 

been placed to maintain a satisfying temperature for the 

lizards. Five minutes before run time, each lizard then 

was placed one by one in a blue, plastic bin to reach 

normal respiration. After five minutes, the lizard from 

the blue bin was placed onto the treadmill and tested 

for the run time until complete exhaustion under the 

consumption of creatine monohydrate pure powder. 

Process was critically repeated for all ten lizards’ run 

times in the same manner and time was recorded in 

seconds same as the control variable. 

Results between both groups were later 

compared, by using a t-test of two variables and 

average means of the total run times. 

Equation: Average Mean = (n 1 +…+ n 10 ) 

Total 

Results 

Run time in Western fence lizards (S. 

occidentalis) could be increased with a relatively small 

amount of consumption of creatine monohydrate pure 

powder. The endurance and run times of the lizards 

were determined among ten lizards which underwent a 

normal run and a run under the consumption of creatine 

powder, an amino acid which generates energy. Times 

were recorded between both control and experiment 

variables (Table 1), displaying the effect of the 

supplement in the run times of the lizards. 

Endurance in the lizards was greatly affected 

by the amount of creatine provided to them, 

demonstrating a great significant difference in both 

groups through the mean averages of their run times 

(Figure 1). Lizards appeared thoroughly fatigued in the 

first two weeks of the experiment and rather fast and 

energetic by the end of the month period due to the 

ATP generated from the dietary supplement. 

Run Time in Seconds 

250 

200 

150 

100 

50 

0 

No Creatine Creatine Consumed 

Figure 1. A statistical significance between the No creatine 

group with a mean average of 174.5 seconds (S.E. ± 3.7) and 

Creatine consumed group with a much greater average of 

202.3 seconds (S.E. 4.3) both with a value P < 0.05. 

Discussion 

Creatine kinase plays an important role in 

energy metabolism catalyzing the reversed transfer of a 

phosphyl group to creatine and MgATP (Wood and 

Guan, 1988). In addition, creatine by means of 

phosphagen can become an intracellular energy 

delivering massive amounts of ATP causing greater 

49 




energy therefore greater endurance in the body 

(Bessman and Geiger, 1981). In this study, both 

groups, the Western Fence lizards with no creatine 

consumed and the same lizards later with creatine 

monohydrate in their systems were carried out and 

exercised throughout the study in the same conduct. 

However, all ten lizards under the consumption of 

creatine demonstrated a hyper active behavior which 

later had an effect in their running times. 

Results were determined by obtaining the run 

times of all ten lizards and calculating for the mean 

average of both groups. By carrying out the same 

experiment with both groups, the small amount of 

creatine monohydrate pure powder introduced to the 

lizards’ diet showed a great impact on their energy 

metabolism. In previous studies, muscle cells and 

macrophages demonstrated that it is impossible for 

cells to synthesize creatine, therefore, the cells must 

take creatine in by plasma in an uphill reaction (Loike, 

Azalutsky, Kaback, Miranda, and Silverstein, 1988), 

which has been done so by running the lizards to 

exhaustion and increasing their sprinting time. 

This experiment indicates that severe activity 

by Western Fence lizards and many similar vertebrates, 

which undergo aerobic exercise, may require enough 

ATP energy to sustain the rigorous conditions 

presented to them. By consuming dietary supplements, 

which can stimulate the metabolism to release energy 

faster, will result in the accumulation of energy causing 

a greater potential for longer endurance. In this study, 

the creatine monohydrate passed on to the lizards has 

triggered the body to release large amounts of energy. 

Following that, we hypothesized that by 

giving creatine monohydrate to Western Fence lizards 

(S. occidentalis), the reaction involving 

phosphocreatine would enable the creatine pathways to 

synthesize and re-synthesize ATP from 

phosphocreatine developing a greater endurance and a 

longer run time. In conclusion, the results have proven 

such hypothesis to be accurate, leaving us with clear 

certainty that in fact creatine monohydrate has an 

increasing significant effect in the vertebrate’s aerobic 

conditions. 


Andres, R., Ducray, A., Schlattner, U. and Wallimann, 

T. (2008). Functions and effects of creatine in the 

central nervous system. Laboratory of Fundamental 

and Applied Bioenergentics. Cedex 9 

Bessman, S. P, and Geiger, P.J (1981). Transport 

Of Energy in Muscle: The Phosphorylcreatine Shuttle. 

Science 211.4481: 448-52. 

Feng, S., Xu, Z., and Yan, Y. (2008). Blocking creatine 

kinase refolding by trace amounts of copper ions. 

Journal of Inorganic Biochemistry 102.4: 928-35 

Gallo, M., MacLean, I., and Tyreman, N. (2008). 

Adaptive responses to creatine loading and exercise in 

fast-twitch rat skeletal muscle. American Journal of 

Physiology-Regulatory Integrative and Comparative 

Physiology 295.4: 1319-28 

Lee, H. J, Fillers W. S, and Lyengar M. R (1988). 

Phosphocreatine, and Intracellular High-Energy 

Compound, is Found in the Extracellular Fluid of 

the Seminal Vesicles in Mice and Rats. Proceedings of 

the National Academy of Sciences of the United States 

of America 85.19: 7265-69. 

Loike, J. D, Zalutsky D. L, Kaback, E., and Silverstein 

S. C (1988). Extracellular Creatine Regulates Creatine 

Transport in Rat and Human Muscle Cells. 

Proceedings of the National Academy of Sciences of 

the United States of America 85.3: 807-11. 

Powers, S. and Riordan, J. (1975). Functional Arginyl 

Residues as ATP Binding Sites of 

Glutamine Synthetase and Carbamyl Phosphate 

Synthetase. Proceedings of the National Academy of 

Sciences of the United States of America 72.7: 2616- 

2620 

Rae, C, Digney A, McEwan S. R, and Bates T. C 

(2003). Oral Creatine Monohydrate Supplementation 

Improves Brain Performance: A Double-Blind, 

Placebo-Controlled, Cross-over Trial. Proceedings: 

Biological Sciences 270.1529: 2147-50. 

Wood, T. and Guan, Z. (1988). Creatine Kinase: 

Essential Arginine Residues at the Nucleotide Binding 

Site Identified by Chemical Modification and High- 

Resolution Tandem Mass Spectrometry. Proceedings 

of the National Academy of Sciences of the United 

States of America 95.7: 3362-3365 

Young, J., Bertram, H., Theil, P., Petersen, A., Poulsen, 

K., Rasmussen, M., Malmendal, A., Nielsen, N., 

Vestergaard, M. and Oksbjerg, N. (2007). In vitro and 

in vivo studies of creatine monohydrate 

supplementation to Duroc and Landrace pigs. Meat 

Science. 76.2: 342-51 

The Metabolic Cost of Digestion in the Ball Python, Python regius 

50 




Ryan G. White and Michael M. Hadley 



Mission Viejo, California 92692 

The Ball python, Python regius, is considered an intermittent feeder, like 

many reptiles, and fasts for long periods of time as a “sit-and-wait forager” prior to 

feeding. As an intermittent feeder, P. regius has a relatively low metabolic rate 

while fasting, yet with feeding, hypertrophy of digestive organs and increased 

digestive functions causes a rapid postprandial response known as Specific Dynamic 

Action (SDA). Specimens of the Ball python P. regius were subject to carbon 

dioxide production analysis prior to feeding and during digestion. During 

experimentation, subjects were held within a sealed container to prevent gas for 

leaking and carbon dioxide production was determined for a single thirty-minute 

interval once a day for two days prior to ingestion. On the third day, subjects were 

fed and carbon dioxide was determined in the same fashion for an additional four 

days. Mean carbon dioxide production value for all specimens (N=4) was 

0•065990•0003 (mean and s.e.). Evaluation of average carbon dioxide production 

values by means of specific T-tests indicated that there was a significant difference 

(P


(P


CO2 exhalation (m L/hr/g) 

0.160 

0.140 

0.120 

0.100 

0.080 

0.060 

0.040 

0.020 

0.000 

0 1 2 3 4 5 6 7 8 

Time from feeding (days) 

Figure 1. Mean carbon dioxide production of 

specimens prior to digestion and during digestion 

(N=4) of Ball python Python regius. Ingestion of 

prey took place at day 3. 

Discussion 

The carbon dioxide production and the 

specific dynamic action (SDA) of pre and post 

ingestion were observed within this experiment. 

Using an Explorer GLX Carbon Dioxide Analyzer, 

carbon dioxide production over thirty-minute 

intervals, once per day for two weeks were 

obtained. Values were adjusted to specific 

temperature and pressure. Total carbon dioxide 

production was calculated into milliliter of carbon 

dioxide production per gram of body weight per 

one hour. Mean carbon dioxide production value 

for all specimens (N=4) was 0•065990•0003 

(mean and s.e.). Evaluation of said figures by 

specific T-tests indicated that there was a 

significant difference between carbon dioxide 

production prior to ingestion of prey and post 

ingestion of prey (P


Beach, San Clemente, California 

Nicole Baumgartner and Karl Neil 



Mission Viejo, CA, 92692 

Seven, one-hundred milliliter samples of beach water collected in front of a sewage 

run-off in San Clemente, California, were analyzed using multi-tube fermentation to 

determine if three days is a sufficient amount of wait time before people can re-enter 

recreational waters following a rainstorm. Three portions of 10, 1, and 0.1 milliliters of 

samples collected were added to nine lactose fermentation tubes, three into triple strength 

brother, and 6 into single strength broth, respectively. Measuring total coliform bacteria 

and using an index of Most Probable Numbers, confidence intervals for each sample were 

obtained, determined by the positive presumptive test of acid production in the tubes. Gas 

production was also monitored as a second indicator of bacterial presence. Days that 

rained were found to have the largest amount of tubes showing positive presumptive 

results. Days without rain were found to be negative for acid production. However, these 

results were inconclusive due to a small data set. Due to the inconclusiveness of these 

results, this study accepts the current three days suggested wait time, as it cannot conclude 

from the data that less time will suffice. 

Introduction 

Water pollution is caused by many factors 

and cannot be attributed to just one person or one 

type of pollution. Increased urbanization minimizes 

natural surroundings that biologically filter pollution 

before it reaches the ocean and increases the number 

of suitable surfaces where pathogens can grow and 

multiply. Despite California’s history of battling 

water pollution, beach closures due to heightened 

levels of bacteria occur frequently up and down the 

coast every year. A number of ocean-goers report 

illnesses including ear and eye infections, 

gastrointestinal problems, and rashes from excessive 

levels of coliform bacteria (Gaffield et al, 2003.). 

Thus, each rain washes potentially harmful levels of 

bacteria into our recreational waters. These high 

levels, given a subsequent amount of time, will die 

down, due to “salt water, sun, or age, predation by 

other organisms and dilution” (Ocean Water 

Protection Program). However, the Surf Riders 

Foundation as well as the state of California 

recommends that all beach-goers wait a minimum of 

seventy-two hours before returning to the water in the 

hopes of minimizing time spent in contact with 

polluted water while giving the bacteria enough time 

to die off or be diluted to levels no longer considered 

harmful by state and government regulations. 

Almost universally, microbes have been 

recommended as a measure of water quality for 

recreational waters. Bacteria naturally occur in 

water, and while most are not harmful, others, 

especially in large quantities, can cause problems. 

Indicator bacteria (total coliform, fecal coliform, and 

enterococcus) are measured to determine whether the 

waters are safe for recreation. Indicator bacteria are 

relatively easy to test for and fecal coliform bacteria 

outlive most other bacteria. This means that if an 

absence of fecal indicator bacteria is found, one can 

safely assume that there is an absence of other 

potentially dangerous bacteria as well. They are also 

good indicators of the “presence of harmful viruses, 

bacteria, or protozoa” (Ocean Water Protection 

Program). Dwight et al. (2004) found that surfers of 

Northern Orange County beaches reported almost 

twice as many symptoms of illnesses due to exposure 

of polluted recreational water than did surfers of the 

less urbanized Santa Cruz area, indicating that 

urbanized runoff contains larger quantities of 

pathogens. Another study of Avalon Bay, Catalina, 

California found that fecal indicator bacteria levels 

decreased during the day and hypothesized that the 

sunlight may have induced a die-off of the bacteria 

(Boehm et al., 2003). Lastly, another study found 

that fecal indicator bacteria are evenly distributed 

throughout the surf zone because of the wind that 

drives the waves up the coast, however large 

variability was found between samples (Kim et al., 

2004): meaning that samples collected in the surf 

54 




zone will represent a majority of pathogens present 

throughout the beach. This study hypothesizes that 

three days is an insufficient amount of time following 

a rainstorm for beach goers to return to the water due 

to elevated bacteria levels at the beach. 


Seven sterilized one hundred milliliter 

bottles were used to obtain samples of water 

collected from a storm drain run-off site located in 

San Clemente, California on January 28 through 

February 3 2008. The samples of water were 

collected in the surf zone directly in front of the runoff. 

The samples were then kept in the refrigerator 

until multi-tube fermentation tests could be 

performed three weeks later. 

Nine lactose fermentation tubes, 3 triple 

strength and six single strength, were arranged for 

each of the seven samples collected. 10 milliliters, 

one milliliter, and 0.1 milliliters of sample were 

transferred into each of the three lactose tubes, 

respectively, using sterile techniques (flaming the 

inoculation loop before transferring the sample). 

After a period of 24 hours of incubation at 37 o C, the 

tubes were observed for gas and acid production. 

Acid production in each tube is recorded as a positive 

presumptive test when the lactose color changed from 

green to yellow. Gas production, if present, was seen 

as a small amount of gas captured in the Durham 

tubes at the bottom of the large test tubes. 

Finally, the most probable number (MPN) 

was determined using an MPN index for various 

combinations of positive and negative results for a 

change in acid. From the MPN index, 95% 

confidence intervals were also determined for the 

sample. 

Results 

Days with a large amount of rainfall 

corresponded with positive results for acid 

production (Figure 1). The day with the most 

significant amount of rainfall had the highest MPN 

number and most acid production. This day also had 

Rainfall (in.) 

0.45 

0.4 

0.35 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0 

1/27/2008* 

1/28/2008 






2/3/2008* 

Figure 1. Total rainfall Date (in.) (D/M/Y) for each of seven days 

samples were collected. Days with asterisk (*) 

correspond to days showing positive presumptive tests 

55 

for acid production. 



the highest variability determined by the size of the 

95% confidence interval. 

Gas production and acid production did not 

correspond with the presence or absence of one or the 

other in a single tube (Table 1). Additionally, gas 

and acid production were not prevalent in one 

strength tube versus another. 

Date 10 mL 

3X 

lactose 

1 mL 

1X 

lactose 

0.1 mL 

1X 

lactose 

MPN 

1/27/2008 +/+ -/- -/-


the inconclusiveness of the results, waiting three days 

before using recreational water should result in 

decreased amount of illness from contaminated 

waters. 


Boehm, Alexandria B., Jed A. Furhman, Robert D. 

Merse, and Stanley B. Grant. "Tiered Approach for 

Identification of a Human Fecal Pollution Source At 

a Recreational Beach: Case Study At Avalon Bay, 

Catalina Island, California." Environmental Science 

and Technology 37.4 (2003): 673-680. 

Dwight, Ryan H., Dean B. Baker, Jan C. Semenza, 

and Betty H. Olson. "Health Effects Associated with 

Recreational Costal Water Use: Urban Versus Rural 

California." American Journal of Public Health 94.4 

(2004): 565-567. 

"Frequently Asked Questions." Ocean Water 

Protection Program. Orange County Healthcare 

Agency. 6 Apr. 

2008. 

Gaffield, Stephen J., Robert L. Goo, Lynn A. 

Richards, and Richard J. Jackson. "Public Health 

Effects of Inadequately Managed Stormwater 

Runoff." American Journal of Public Health 93 

(2003): 1527-1533. 

Kim, Joon H., Stanley B. Grant, Charles D.McGee, 

Brett F. Sanders, and John L. Largier. "Locating 

Sources of Surf Zone Pollution." Environmental 

Science and Technology 38 (2004): 2626-2636. 

"San Clemente History." Weather Undergroud. 24 

Apr. 2008 . 

Standard Methods for the Examination of Water and 

Wastewater, 13 th Edition New York. American 

Public Health Association, 1971. 

Standard Methods for the Examination of Water and 

Wastewater, 12 th Edition New York. American 

Public Health Association, 1967, p.608. 

The Effect of Salinity on the Photosynthetic Rate of Pickleweed, Salicornia virginica 

Takahiro Ueno and Arash Moghaddam 




Abstract 

Halophytes have developed methods to survive in environments of high salinity. 

Salicornia virginica a C 3 plant, relies on specialized mechanisms for storing and excreting 

salt from its cells. The plants were exposed to three different solutions of various salinities: 

0%, 3.5%, and 6.0%. Photosynthetic rates were determined by measuring the oxygen 

production rates (mg/L) over a period of one hour. The average oxygen production levels 

were 0.52 ± 0.020 mg/L in the distilled water, 0.69 ± 0.059 mg/L in the 3.5% solution, and 

0.28 ± 0.053 mg/L in the 6.0% solution. Results showed that the null hypothesis was 

rejected and a significant difference (ANOVA test) was found among the three groups. 

Introduction 

Throughout many of the various coasts along 

the United States, pickleweed (Salicornia virginica) 

flourishes as one of the most common forms of 

vegetation in intertidal marshes. These halophytic 

shrubs inhabit the higher marshes where water levels 

fluctuate throughout the day. As a result, this stemsucculent 

C 3 species must tolerate salinity levels higher 

than that of average seawater. Organisms like 

Salicornia virginica must rely on specialized 

mechanisms that efficiently excrete or filter out salt 

from their cells. The method of resistance can involve 

either salt tolerance or salt avoidance. 

Previous studies have examined changes in 

the growth patterns of these types of organisms, due to 

seasonal salinity changes (Satoh et al., 1983). Among 

terrestrial plants, the primary effect of changes in 

salinity was on the photosynthetic capacity within the 

56 




mesophylls (Pearcy & Ustin, 1984). As salt 

concentrations increase, the uptake of water by the 

plant is inhibited, thus greatly affecting processes 

involving respiration and photosynthesis. In some 

terrestrial species, transpiration is affected by increased 

salinity as the stomata’s water vapor conductance is 

decreased (Biber, 2006). The leaves of several 

organisms have even shown signs of damage with the 

slightest changes in salinity (Flowers et al., 1985). 

Based on these similar previous studies, it is perceived 

that increased salt concentrations interfere with the 

plant’s primary biological processes. 

The objective of this research was to observe 

the effects that salinity has on the photosynthetic rates 

of S. virginica. The hypothesis tested was whether the 

plant would have a maximum photosynthetic rate in a 

freshwater solution, and if a high salinity level would 

damage the plant. Photosynthetic rates were 

determined by measuring the rate of oxygen 

production. The plants were introduced to three 

different salinity levels: 0%, 3.5%, and 6.0%. All test 

groups were studied under the same ambient 

conditions. By subjecting the plants to two different 

saline solutions, we were able to experiment with the 

plant’s natural seawater environment and also one in 

which the salt level was significantly higher. 


S. virginica were obtained from the marshes in 

Newport, California. The plants were cut at the stems, 

leaving the lateral branches intact. Each plant was 

weighed out to 10 g to ensure uniform mass for all 

samples. 

Each of the saline solutions was prepared in a 

beaker using ordinary table salt (NaCl) and distilled 

water. The concentrations prepared were 0%, 3.5%, 

and 6.0%. Prior to the experiment, the plants were 

immersed in their respective solutions and left to 

acclimate for a period of thirty minutes. One hundred 

watt lamps provided a light source for the plants. The 

lamps were placed forty inches away from the beakers. 

To measure the photosynthetic rates, data 

loggers equipped with oxygen meters (Pasco TM PS- 

2002) were used. After the plants had been given time 

to adjust to their environments, the oxygen probes were 

inserted into the beakers and data collecting began. 

Plants were then left in the controlled setting for a onehour 

period. Oxygen production rates were measured 

in mg/L by the data logger device. At the conclusion of 

each test period, the maximum, minimum, and average 

oxygen production levels were recorded. A total of four 

tests were performed for each solution group. 

Results 

Oxygen production by S. virginica was 

observed with all groups throughout the experimental 

Oxygen Production (mg/L) 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

57 



period. The highest oxygen production levels were 

collected from the plants in the 3.5% saline groups, 

with the 0% groups having the next highest production 

levels (Fig. 1). The average oxygen production levels 

were 0.52 ± 0.020 mg/L in the 0% solution, 0.69 ± 

0.059 mg/L in the 3.5% solution, and 0.28 ± 0.053 

mg/L in the 6.0% solution (Fig. 2). 

The photosynthetic rates for the plants in the 3.5% 

and the 0% saline solutions increased at a higher rate 

throughout the one-hour period than the 6.0% saline 

group (Tab. 1). Although the 6.0% saline groups had 

lower oxygen production than other groups, none of the 

plants showed evidence of decreased photosynthetic 

rates or any signs of damage. 

0 

Oxygen Production (mg/L) 

in 

the 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

Salinity 

\when exposed to 

various saline 

solutions. 

0% 

Oxygen 

production 

3.50% 

6.00% 

Figure 1. Oxygen production (mg/L) of S. virginica when 

exposed to various saline solutions. Squares represent 

line for groups in 3.5% salinity; triangles represent 

groups in 0% salinity; and circles represent groups in 

6.0% salinity. 

levels were 0.52 ± 0.020 mg/L in 0% 

salinity, 

0.69 ± 

0.059 mg/L in the 3.5% 

solution, and 0.28 ± 0.053 mg/L 

0 5 10 15 20 25 30 35 40 45 50 55 60 

Time, min. 

Figure 2. Average oxygen production (mg/L) ±S.E.M. of 

four test groups of S. virginica when exposed to various 

saline solutions. Oxygen production levels were 0.52 ± 

0.020 mg/L in 0% salinity, 0.69 ± 0.059 mg/L in the 

3.5% salinity, and 0.28 ± 0.053 mg/L in 6.0% salinity.


Salinity Group 1 Group 2 Group 3 Group 4 Mean 

0% 0.55 0.561 0.482 0.49 0.521 ± 

3.5% 0.828 0.656 0.588 0.698 0.693 

6.0% 0.178 0.202 0.348 0.392 0.280 

6.0% solution. 

Table 1. Oxygen production levels (mg/L) of four test 

groups under 0%, 3.5%, and 6.0% salinity. 

Discussion 

All of the data shown indicate that regardless 

of the salinity, all of the plants produced some levels of 

oxygen within their respective solutions. The initial 

hypothesis predicted that the greatest oxygen 

production rates would be observed within the 0% 

salinity. The results demonstrated that the highest 

oxygen production rates occurred in the 3.5% solution. 

These results come to no surprise, as these plants are 

regularly exposed to seawater, which on average has a 

salinity of 3.5%. Therefore, these findings indicate that 

S. virginica and their cells have adapted to functioning 

most productively in similar salinities. The plants 

exposed to 0% salinity showed the second highest 

photosynthetic rates, and the 6.0% saline groups 

showed the least productive photosynthetic rates. A 

significant difference (ANOVA statistical test) was 

established between all of the groups, which implies 

that salinity has a great effect on the photosynthetic 

rates of S. virginica. 

These halophytes have demonstrated their 

ability to tolerate the constant salinity changes in their 

environment. In addition to the daily fluctuations in the 

water levels of their environment, some of these plants 

inhabit ecosystems that experience very low annual 

precipitation levels. These unique adaptations were 

apparent when the plants did not display any signs of 

damage or decreases in photosynthetic rates, even at a 

salinity level nearly twice that of seawater. 

Along with the experimental results and those 

from previous studies, it seems appropriate to assume 

that these plants could undergo photosynthesis at even 

higher levels of salinity than 6.0%. A further study on 

the salt tolerance of these plants would yield greater 

information on just how these organisms manage to 

survive in these extreme environments. Replicating this 

experiment and examining the plant at cellular levels 

would show how the cells maintain ideal conditions 

through homeostatic responses. As a result, 

comparisons could be examined between S. virginica 

and species that are intolerant to salinity changes. 


Biber, P.D. (2006). Measuring the Effects of Salinity 

Stress in the Red Mangrove, Rhizophora mangle L. 

African Journal of Agricultural Research, 1, 1-4. 

Flowers, T.J., Duque, E., Hajibagheri, M.A., 

McGonigle, T.P. & Yeo, A.R. (1985). The Effect of 

Salinity on Leaf Ultrastructure and Net Photosynthesis 

of two varieties of Rice: Further Evidence for a 

Cellular Component of Salt Resistance. New 

Phytologist, 1,3 37-43. 

Pearcy, R.W. & Ustin, S.L. (1984). Effects of Salinity 

on Growth and Photosynthesis of Three California 

Tidal Marsh Species. Oecologia, 62, 68-73. 

Satoh, K., Smith, C.M. & Forke, D.C. (1983). Effects 

of Salinity on Primary Processes of Photosynthesis in 

the Red Alga Porphyra perforata. Plant Physiol, 73, 

643-647. 

58 




The effects of various light sources on the fruiting bodies of Citrus limonium 

Matt Apke and Zachary Beam 




Photosynthesis is the main source of power among all plants. Without it, 

plants would be unable to create sugars and other nutrients essential in 

reproduction. In this experiment, several lemon trees (Citrus limonium) were placed 

under various light sources (n=50) including the colors of green, blue, and red (n=10 

for each group) for eleven weeks. A control was conducted with a group of plant 

placements under a white light to simulate a typical light source. A quantitative and 

qualitative analysis of the fruiting bodies of all the plants was conducted and 

recorded. Distinct differences were found among the groups showing evidence that 

the lemon trees much preferred wavelengths between 400 and 500 (blue to green on 

the electromagnetic spectrum). The average number of passing fruiting bodies was 

four under the white light, three under blue and green lighting, and two under the 

red lighting. 

Introduction 

Plants need light to grow and more 

importantly to grow fruit. Plants use light to 

undergo photosynthesis which in turn provides 

energy and nutrients to the plant, enabling it to 

produce and maintain some sort of fruit. A fruit 

producing plant usually has some sort of bud that a 

pollinator has to pollinate in order for the bud to 

become a fruit. The size, shape, and taste of the 

fruit depend on the quality of plant and the type of 

environment that the plants need to be in order to 

grow. For example, tropical fruits like bananas 

and coconuts need a location in which light is 

abundant (Shipley, 1998). Moderate fruits, like 

apples and strawberries, can be in an environment 

where a light source is not available at all times. 

Plant growth depends on the intensity of light. For 

example, the same plant that grows with light 

probably will not grow the same, or at all, with the 

absence of light. In the color spectrum most plants 

absorb all light colors, but reflect green, (Sunal, 

2000) which is why they appear green to our eyes. 

If the plants were another color, for example red, 

the difference in that light might affect how the 

plant produces its fruit. We hypothesize that the 

color source of light closet to the actual color of 

the plant (green) on the color spectrum, with grow 

the least efficiently because they will reflect the all 

of the available light. There is not enough research 

to determine if another light besides white light is 

needed in order to make plants produce fruit more 

often or maybe make a fruit sweeter, harder, or 

juicier. This experiment aims to discover if 

different color light sources have an effect on the 

quality and quantity of fruiting bodies on Citrus 

limonium (see Figure 1). The result from this 

experiment could determine whether white light or 

colored light is better for fruit production. 

Figure 1. Typical lemon tree, Citrus limonium, 

illustrated with fruiting lemons. 


Lemon trees (Citrus limonium) are 

generally known for quick growing rates so they 

have become the plant of study. The lemon trees 

were purchased from a Home Depot in Laguna 

Niguel, and came pre-pollinated by honey bees 

(Apis mellifera). The fifty plants were taken into a 

completely dark garage (in a home in Ladera 

59 




Ranch, California). The plants were then separated 

into four groups (each group n=10): red, blue, 

green, and white light. The light sources consisted 

of a flashlight covered by colored saran wrap. 

While the flashlights contained incandescent 

lighting, which is poor lighting for plant growth 

(Pratt, Bonner, 2005), it was the only affordable 

light source. All plants were watered uniformly 

daily (approximately 150mL) at the same time 

each day. All plants were in identical pots with a 

diameter of 13 inches and used the same brand of 

soil (Miracle Gro Fertilizer). At the conclusion of 

eleven weeks, the fruiting bodies were examined 

according to shape, color, size, and quantity. When 

and what each plant first produced their fruit was 

recorded and compared against each other in order 

to find any differences among the groups. 

Results 

The colors in which had the most fruiting 

bodies were not met with the most known effective 

light in driving photosynthesis; these being violetblue 

and red (Campbell, 2005). The plants under 

the following color lights went from most to least 

number of fruiting bodies: white, green, blue, and 

red (see Figure 2). White, being the control, had 

the most fruiting bodies as expected but green 

theoretically should have had the least number of 

products produced as green light (550nm) is the 

wavelength reflected. 

Although as the green light group 

produced the most fruit numerically, the quality of 

the products was lackluster. Blue and red produced 

fewer fruiting bodies but the quality of the lemons 

was somewhat better quality. This is far from 

surprising since blue and red were the light 

Figure 2. Average height of each group 

placed under different light sources over an 

11 week period. 

wavelengths most preferred by photosynthesis. A 

better rate of photosynthesis means more energy 

(ATP) available for reproduction and in 

consequence, better quality fruit. 

The white lit group produced four noteworth 

fruiting bodies and the overall plant grew 

2.1 inches; blue had three fruiting bodies with a 

net plant growth of 1.7; green grew three as well 

with a growth of 2 inches; and red had 2 fruits 

with 1.6 inches of growth. Growth of the plants is 

shown for each week in Table 1. No significant 

difference was found between the groups (P>0.05). 

red green blue white 

15.4 16 15.8 16.4 

15.6 16.2 16 16.8 

15.7 16.3 16.3 17 

15.9 16.4 16.4 17.2 

16 16.7 16.4 17.5 

16.2 16.8 16.7 17.6 

16.3 17 16.8 17.8 

16.4 17.3 17 18 

16.6 17.7 17.2 18.2 

16.7 17.9 17.3 18.4 

17 18 17.5 18.5 

Table 1. Average height (in inches) of the plants 

in each group over an 11 week period. 

Discussion 

As a typical light ray enters the 

chloroplasts and into the granum of a plant, there 

is a result in a separation of the spectrum and 

absorption of all colors but green. The white light 

of the experiment was no surprise to have created 

the most (quantitatively) and best (qualitatively) 

fruit. This is due to the fact that with white light 

has a greater range of availability of the specific 

light wavelengths photosynthesis prefers (Yoshida, 

2008). When specific colors are only exposed to 

the plants, it does not have those specific 

wavelengths and thus creates a lower quality of 

fruit. The results having green light source 

producing the most fruiting bodies shows evidence 

against our hypothesis since green is the actual 

wavelength of light we hypothesized would create 

the least amount of fruiting bodies. 

A possible reason for this occurrence 

could be that a saran wrap covering a white light 

may not completely block the wavelengths of 

lights preferred by photosynthesis. Perhaps the tint 

level of the differently colored saran wraps was the 

biggest variable in availability of light from the 

source. 

60 





Campbell, Reece. Biology Seventh Editioin. San 

Francisco: Pearson Education Inc., 2005. 

Pratt, H. Bonner, B (2008). Growing light, Plant 

Light, Artificial Light Plant Sunlight. Horticulture 

Growing 

Techniques. 

http://www.pfolighting.com/Horticulture- 

Grow-Info.aspx 

Shipley, Jennifer A. (2005). The Mysteries of 

Plants and Light: The Effects of Wavelengths of 

Light on Plant Food Production. 2005 Project 

Summary. 

ww.usc.edu/CSSF/History/2005/Projects/J1636.pd 

f 

Sunal, Dennis W. Sunlight and Plants. Sample 

Lesson from The Universtiy of Alabama. 2000 

Sample 

Lesson. 

astlc.ua.edu/lessonplans/LCPlants.htm 

Yoshida, T, Anderson, T. (2008). American 

Journal of Botany: Vol. 13,Biological steps of the 

effect of light and wave lengths on the growth of 

plants. No. 10 pp.706-736. 

61 




Is MiracleGro the Best Fertilizer for Impatiens wallerana? 

Michelle Huynh and Jiwon Park 

Department of Psychology and Foreign Language 



Fertilizers are used to enhance the growth and development of a plant. Fertilizers 

may contain nitrogen, potassium, and soluble potash, which provide nutrients for growth. 

Well-known fertilizers, such as MiracleGro, are considered to make plants healthy. The 

hypothesis suggests that when the group of Impatiens wallerana is given MiracleGro All- 

Purpose Liquid Fertilizer, the plants will grow at a faster and healthier rate than the 

groups that are given other or no fertilizer. In this study, the plants were organized into 

three groups: MiracleGro, Liquinox, and control. The groups were given the same amount 

of fertilizer and water for five weeks. Each plant was measured once each week and at the 

end of five weeks, the chlorophyll from the leaves was extracted and measured at two 

different wavelengths. The Liquinox group had the highest chlorophyll concentration 

(p=0.009, unpaired t-test); however, there was no difference in the stem length change 

among the three groups. The results show that MiracleGro is the most harmful fertilizer if 

given a large amount due to its high nitrogen content compared to Liquinox. 

Introduction 

Fertilizers are compounds that are used to help 

plants grow and develop. Plants can uptake fertilizers 

by the roots or through the leaves. Fertilizers can be 

organic, composed of naturally occurring elements, or 

inorganic, manmade chemicals. The three major plant 

nutrients are nitrogen, phosphorus and potassium, 

which are the components provided by fertilizers. 

Typically, the presence of nitrogen compounds in the 

soil is essential to plant growth. Thus, nitrogen plays 

important roles in providing proteins, hormones, 

chlorophyll, vitamins and enzymes. Phosphate (P 2 O 5 ) 

is integral for photosynthesis, protein formation and 

flowering. An absence of phosphate can lead to 

delayed growth and poor flower production. Soluble 

potash (K 2 O), or potassium, facilitates in the 

production of sugars, starches, carbohydrates, in 

protein synthesis and cell division in plants. It aids in 

the absorption and conservation of water, turgidity of 

plants and the enhancement of flower color. A lack of 

potassium will result in unhealthy leaves (Milius, 

2007). 

Chlorophyll is a pigment that gives plants its 

green color. The more chlorophyll content in a plant, 

the higher the rate of photosynthesis will be and the 

darker the color pigment will be (Milius, 2007). 

Based upon many advertisements and 

commercials, one can conclude that MiracleGro is the 

most efficient fertilizer compared to generic brands, 

such as Liquinox. Therefore, MiracleGro is the best 

fertilizer for plants compared to Liquinox and no 

fertilizer at all. With the addition of fertilizer, plants 

should have more chlorophyll, and thus higher growth 

rates. 


Eighteen plants of Impatiens lipstick, one of 

the garden cultivars of Impatiens wallerana, purchased 

at Home Depot in Mission Viejo, California on 

October 7, 2007, were planted with Premier Pro-mix 

general purpose growing medium potting soil and 

separated into three groups: Control, MiracleGro, and 

Liquinox. They were planted in Science and Math 

greenhouse at Saddleback College on October 8, 2007. 

Each pot was watered 60 mL. Every week, each 

individual plant was watered 200 mL of water twice a 

week for three weeks. 

Every Friday, each plant of the MiracleGro 

group received 3 mL of the MiracleGro All-Purpose 

Liquid Fertilizer plus 200mL of water. Each plant of 

the Liquinox group received 3 mL of Liquinox Fish 

Emulsion Plant Fertilizer plus 200 mL of water. Each 

plant of the Control group only received 200 mL of 

water, with no fertilizer. The longest stem of each plant 

from all three groups, which was marked with a string 

loosely tied around on the first day, and measured at 

the end of each week to chronicle the growth of the 

plants. In addition, a photograph was taken of each 

group of plants in order to record how the plants were 

developing and changing, in regards to the fertilizer, or 

lack thereof. 

62 




After five weeks of watering and feeding the 

plants with fertilizer, the chlorophyll concentration of 

each group of plants was determined using a Beckman 

DU 720 Spectrophotometer. From one leaf of each 

plant, two 5 mm diameter chads were cut using a 

standard hole punch and soaked in an 80% acetone 

solution for 48 hours in the refrigerator at 40°C. Then 

the absorption of acetone extracted chlorophyll was 

measured at wavelengths of 645 and 663 nm as 

described by MacKinney’s (1941). 

Results 

The control group started with 2 flowers, and 

after five weeks, had 57 flowers. The Liquinox group 

had no flower initially, and after five weeks, had 17 

flowers. The MiracleGro group 1 flower and no flower 

survived after five weeks. 

The stems of the Control group had an overall 

average growth of 3.02 cm, which was the highest 

exponential growth from all three groups. The 

MiracleGro group grew the least and after Week 4, 

there was no growth of the stems. The stems of the 

Liquinox group had an overall average growth of 2.74 

cm. 

After five weeks of growth (Figure 1), the 

control group had a 3.0 ± 0.7 cm change in length. The 

MiracleGro had a 1.5 ± 0.5 cm change in growth, while 

the Liquinox group had a 2.7 ± 0.4 cm change in 

length. There was no statistical difference between the 

three groups (p=0.15, ANOVA). 

Average Change in Stem Lengths 

(cm ) 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

Control MiracleGro Liquinox 

Groups 

Figure 1. There was no difference in the change in 

stem lengths among the three groups (p=0.15, 

ANOVA). 

Figure 2. Mean chlorophyll concentration of the 

control group was 47% less than the mean chlorophyll 

concentration of the Liquinox group (p=0.009, 

unpaired t-test). 

Discussion 

There was no difference in the change in stem 

lengths due to a short duration of time and a small 

sample size. The plants may have been still in the 

process of development. Long-term experiments can 

identify correlative relationships between two variables 

(Reed and Martiny, 2007). Therefore, short-term 

experiments are unable to demonstrate a change. 

The MiracleGro plants’ failure to mature was 

partially due the 11% nitrogen content of the 

MiracleGro fertilizer. The amount of nitrogen in 

MiracleGro was twice as much as the amount of 

nitrogen in Liquinox, which limited the MiracleGro 

group’s plant growth, like in Vitousek’s et al. (1993) 

study where the results showed that the contents of 

nitrogen inhibited plant growth. This does not support 

the hypothesis that MiracleGro fertilizer would work 

best for plants because the ratio of the three groups’ 

length was equal to each other. 

Liquinox facilitated an increase in the 

chlorophyll concentration in the leaves of the plants, 

which allowed for more photosynthetic processes to 

occur which enables more plant growth. The Liquinox 

chlorophyll concentration was 47% more than the 

chlorophyll concentration of the control group. 

Based upon our findings, we would choose the 

generic brand of Liquinox fertilizer for our house 

plants because it results in a higher concentration of 

chlorophyll content, which will allow for more 

photosynthesis to take place. 

Acknowledgements 

We would like to thank the Saddleback 

College Department of Biological Sciences for 

providing the potting soil, pots, and fertilizers to 

conduct our study in the Math and Science 

63 




Greenhouse. We would also like to thank Professor 

Steve Teh for his profound knowledge and relentless 

dedication in assisting us with this study. 


MacKinney, G. 1941. The absorption of light by 

chlorophyll solutions. J. Biol. Chem. 140: 315-322. 

Milius, S. 2004. Rewriting the nitrogen story: 

plant cycles nutrient forward and 

backward. Science News 166.1:5(2). 

Reed, H., Martiny, J., 2007. Testing the functional 

significance of microbial composition in natural 

communities. FEMS Microbiology Ecology 62:161- 

170. 

Vitousek, P., Walker, L., Whiteaker, L., Matson, P., 

1993. Nutrient limitations to plant growth during 

primary succession in Hawaii Volcanoes National 

Park. Biogeochemistry 23:197-215. 

The Effects of Organic Fertilizer vs. Inorganic Fertilizer (Miracle Gro) on Growth of 

Tomato Plants 

Cynthia Tran and Camille Barlow 




The effects of organic and inorganic (chemical) fertilizers were studied on the growth of 

tomato plants within a four week period. One group of eight tomato plants were given an 

organic fertilizer and the other eight a chemical (Miracle Gro) fertilizer. Plants grown in a 

chemical fertilizer had a higher average chlorophyll concentration than plants grown in 

organic fertilizer (P= 0.000518, one tail unpaired T·test) 333.75 ± 9.28mg/L. The 

chlorophyll concentration in the leaves between both groups suggested that chemical 

fertilizers would yield taller, greener, and thus more tomatoes per plant. The height 

differences between the two groups were displayed after the second week of growth with 

inorganic fertilized tomato plants taller by an average of 5cm. Organic fertilizers have a 

slow release of nitrogen compared to chemical fertilizers that have readily available 

nitrogen levels. The results indicated that growth in tomato plants are greatly influenced 

by the amount of nitrogen available to the plants in their fertilizer. 

Introduction 

Fertilizer is nutrients added to soil to make it 

more fertile. Miracle Grow (chemical fertilizer) will 

produce taller, greener leaves, and heavier mass of 

tomatoes than Organic Fertilizer. Miracle Grow 

contains exact amounts of all three nutrients of 

nitrogen, phosphorus, and potassium. The amount of 

nitrogen that is readily available and easy to control 

results in greater efficiency of plant growth (Heeb et 

al., 2005). Organic Fertilizers consisting of cow, 

sheep, poultry, and horse manure contain low levels of 

each nutrient. They are dependant on microorganisms 

in the soil to break down and release the nutrients. 

Organic Fertilizers have a slower release of nitrogen 

which if needed immediately would not be able to 

provide (Chu et al., 2006). Nitrogen is the main 

nutrient required the most in tomato plant. The tomato 

plants grown in fertilizer with the most abundant 

amount of nitrogen will yield heavier masses of 

tomatoes (Wang et al., 2001). 

Two groups of sixteen tomato plants (eight 

with chemical fertilizer/eight with organic fertilizer) 

were placed outside receiving equal amounts of water 

and sunlight for growth monitoring. Averages of 

chlorophyll concentration, height of growth and 

number of blossoms were collected during a four week 

period. A hypothesis that the chemical fertilizer will 

produce taller and greener tomato plants over the 

tomato plants given organic fertilizer. 

Material and Methods 

64 




The sixteen Early Girl Tomato plants were 

purchased from Armstrong Gardens in San Juan 

Capistrano, California. The tomatoes were potted in 

plastic pots (11 cm in diameter) with four drainage 

holes at the bottom of each pot. The plants were placed 

together outside in direct sunlight and the temperature 

was dependent on the outside weather in the city of San 

Juan Capistrano, California. All sixteen tomato plants 

were centered between two sprinklers providing the 

equal amounts of water daily. Once a week, one group 

of eight tomato plants were given 9.92 ml of E.B. 

Stone Organic Fertilizer and the other eight tomato 

plants were given 9.92 ml of Miracle Gro Fertilizer 

(chemical). For one month each individual tomato 

plant was measured for height of stalk every two weeks 

(see Fig. 1). Also, counted were the numbers of 

blossoms per group at the end of week four for the Chi- 

Square analysis (see Fig. 2). There are group pictures 

displaying the progress of both groups of tomato 

plants. 

Chlorophyll concentration in tomato plant 

leaves between organic and chemical fertilizers were 

taken using the Beckmann Du 700 spectrophotometer. 

A total of three samples of chlorophyll extract were 

taken from each organic and chemical fertilizer groups. 

Each vial sample contained two 6.1 mm leaf discs 

(using a paper hole puncher) added to 5ml of 80 % 

(v/v) acetone. All forty-eight vials (24 of each group) 

placed into the refrigerator (4º C) for 24 hours. 

Concentration of chlorophyll was then measured using 

the Beckmann Du 700 spectrophotometer. An average 

of 3ml per sample in the vial were transferred into a 

cuvette for absorbance reading which is a measure of 

the light absorbed by the solution in nanometers (nm). 

The average total chlorophyll concentration of organic 

and chemical fertilizers was calculated in mg/L and a 

T· test was done (see Fig. 3). 

Results 

The tomato plants that were fed chemical 

fertilizer (Miracle Gro) grew to be taller in height (cm) 

than the tomato plants grown in organic fertilizer. Our 

experiment suggests that chemical fertilizer produces 

more fruit because the count of blossoms was 84 versus 

the organic tomato plants which produced 63 blossoms 

at the end of the four week period. Both groups of 

tomato plants initially started out at with an average 

height of 13cm. For the first measurement after two 

weeks, the average chemical fertilizer grew 10cm and 

the average organic fertilizer plants grew 9cm. The 

second measurement at the end of four weeks 

displayed a significant difference in that the chemically 

fed tomato plants with an additional average growth of 

19cm (total in 4 weeks = 29cm) while the organically 

65 



fed tomato plants with only an additional average 

growth of 14cm (total in 4 weeks = 23cm). We also 

measured the chlorophyll concentration of the different 

plants leaves. We found that there was a significant 

difference between plants grown chemically had more 

chlorophyll than organic fertilizer (P = 0.000518, one 

tail unpaired T · test). 

Chlorophyll concentration (mg/L) 

400 

350 

300 

250 

200 

150 

100 

50 

0 

Non-organic 

Organic 

Fertilizer Groups 

Figure 1. Plants grown in a chemical fertilizer had a 

higher average chlorophyll concentration that organic. 

(P= 0.000518, one tail unpaired T·test) 333.75 ± 

9.28mg/L. 

Average Height of Tomato Plants for 

One Month in cm 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

Figure 2. Average height growth of chemically fed 

tomato plants displayed a 5cm difference over 

organically fed tomato plants at the final measurement 

of week 4. 

Average Number of Blossoms 

Yielded 

12 

10 

8 

6 

4 

2 

0 

Chemical 

Non-Organic 

Figure 3. Tomato plants grown in a chemical fertilizer 

yielded more blossoms than tomato plants grown in 

organic (p=0.001804, one tail unpaired T ·test 11.25 ± 

0.647799 blossoms. 

Discussion 

Organic 

Organic


We found in our study that there is a significant 

difference between chemical and organic fertilizer. 

For taller, greener plants with more tomatoes in a 

shorter period of time, our experiment would 

suggest that chemical fertilizer indeed works best. 

Companies put nitrogen in the chemical fertilizer 

to allow for better growth. According to J. Heeb, 

high amounts of nitrogen result in faster plant growth 

(Heeb et al., 2005). Organic plants do not have 

this efficient production of nitrogen, they rely on 

microorganisms to break down and release 

nutrients so the process is much slower (Chu et al., 

2006). Over a month’s time, we did not have any 

actual full tomato growth, so we cannot say 

whether or not the tomatoes would be better 

tasting or larger in size, but the aspects of the 

chemically fed tomato plants help to indicate that 

the tomatoes might be larger in size. The 

chemically fed plants grew much larger in a short 

amount of time than the organically fed plants, 

they also, through the use of a chlorophyll 

concentration test, produced much greener leaves 

which helps to show how they would indeed be 

creating more sugars through photosynthesis thus 

possibly yielding more fruit. It has been tested in 

other studies that the more nitrogen produced 

during the growth of a plant the more it grows and 

chemical fertilizers add nitrogen to the soil the 

plant is growing in (Wang et al., 2001). In conclusion, 

we found, along with many other studies, that for larger 

more productive tomato plants, chemical fertilizer 

works the best compared to organic fertilizer. 


Chu, H., Fujii, T., Morimoto, S., Lin, X., Hu, J., and 

Zhang, J. 2006. Community Struction of Ammonia- 

Oxidizing Bacteria under Long-Term Application of 

Mineral Fertilizer and Organic Manure in a Sandy 

Loam Soil. Applied and Environmental Microbiology. 

73: 485-491 

Heeb, A., Lundegardh, B., Errcsson, T., and Savage, P. 

2007. Nitrogen form affects yield and taste of 

tomatoes. Journal of the Science of Food and 

Agriculture. 85: 1405-1414 

Wang, Y., Garvin, D., and Kochian, L. 2001. Nitrate- 

Inducted Genes in Tomato Roots. Array Analysis 

Reveals Novel Genes That May Play a Role in 

Nitrogen Nutrition. Plant Physiol. 127: 345-359 

66 




THE EFFECTS OF FIRE ON SOUTHERN CALIFORNIA PLANT SUCCESSION AND 

THE PREVALENCE OF ARTICHOKE THISTLE, CYNARA CARDUNCULUS. 

Lauren Ferris 




Southern California chaparral habitat is fire dependent for seed propagation and 

plant health. Artichoke Thistle, Cynara cardunculus, and Black Mustard, Brassica nigra, 

have been observed to thrive and out compete California native species in fire burned 

habitats. Plant plot sampling is used to determine the density, frequency, and coverage of 

each species of plant in a 100 m² chaparral sample. The two locations were selected as one 

burned in five to six years prior and the other as unburned in fifteen or more years. The 

results indicated that Artichoke Thistle is statistically greater (p= 0.0001) in prevalence in a 

chaparral habitat that has been burned than in unburned chaparral habitat; 58 Artichoke 

Thistle compared to 3 Artichoke Thistle respectively. Black Mustard is statistically greater 

in unburned chaparral habitat than in burned chaparral where competition against 

Artichoke Thistle is greater; 533 Black Mustard compared to 174 Black Mustard 

respectively. California wildfires are a significant factor in the increased prevalence of the 

invasive Artichoke Thistle in chaparral habitat because the fires clear the habitat of any 

plant species leaving the habitat open for recolonization by non-native species. 

Introduction 

Southern California wildfires are a common 

occurrence. Underbrush and dead plant matter are 

reclaimed quickly by fires and new plant succession 

follows in the replenished soil. Frequent Southern 

California fires have demonstrated a feedback response 

in selecting for plants with physiological mechanisms 

for fire tolerance (Naveh 1975). Post fire plant 

communities will demonstrate different ratios of plant 

species than areas that have not be recently burned, 

approximately 20-30 years. Three to four years after a 

fire in Southern California four categories of plant 

species can be found in a community; generalized 

herbaceous perennials, generalized annuals, specialized 

“fire-annuals”, and specialized “fire perennials” 

(Keeley et al. 1981). Locations sampled at different 

periods post fire should be seen to represent different 

ratios of plant species. As fire frequency increases, 

non-native weeds increase dramatically (in prevalence) 

(Haidinger and Keeley 1993). While Southern 

California Chaparral depends of fires for seed 

propagation and plant growth (Hanes 1971), the 

wildfires are also propagating non-native species that 

destroy the diversity of the habitat. 

The wild artichoke came to the United States 

in the mid – 1800s. Escape from cultivation and 

subsequent propagation by seed resulted in a reversion 

to of the aggressive and ‘wild’ characteristics (Kelly 

and Pepper 1996). Artichoke Thistle (Cynara 

cardunculus) is prevalent across much of Southern 

California and the eradication of the species is difficult. 

The seeds of Artichoke Thistle are wind-dispersed and 

can disperse over 40 m in non-vegetated sites, such as 

those opened by wildfires (Holt and Marushia 2006). 

There has been little research on mechanical control of 

C. cardunculus, although repeated cultivation has been 

reported as an effective control method (DeSimone and 

Ernie 2002). 

Black Mustard (Brassica nigra) was 

introduced into the United States with the cattle by the 

Spaniards in the 1700s. Brassicas can produce up to 

ten generations of seed per year (Williams and Hill 

1986). Rapid seed production lends itself readily to 

invasion of habitat that has been cleared by fire, either 

wildfire or human initiated fire, or by cattle grazing. 

Within California's inland grasslands, 

nonnative annual vegetation has changed seasonal 

patterns of resource availability (Dyer and Rice 1999). 

These changes affect the native Bunchgrass species 

such as Nassella pulchra. Chaparral native species 

include Scrub Oak, Chamise, Laurel Sumac, Coyote 

Brush, Yucca, and Bunchgrass. This natural diversity 

67 




is destroyed by the growth of Artichoke Thistle and 

Black Mustard. The purpose of this research is to 

demonstrate the increased prevalence of Artichoke 

Thistle in California chaparral habitat post wildfire 

when compared to chaparral habitat unaffected by 

recent wildfire. 


Two locations were selected for the 

experiment; both locations are relatively close in 

proximity along Antonio Parkway from Rancho Santa 

Margarita, California to Las Flores, California. The 

first location N 33º 36.510’ W 117º 36.950’ consists of 

chaparral habitat that experienced a fire on May 13, 

2002. The second location N 33º 34.227’ W 117º 

37.798’ was not affected by the May 13, 2002 fire or 

any fires five years prior to 2002 until November 25, 

2007. At each location ten plots measuring 2 m² by 5 

m² were selected and marked out with 100 m 

measuring tapes provided by Saddleback College. 

Each plant within the plot was counted and measured 

for diameter with a 5 m measuring tape and then the 

area covered by that plant was calculated. The total 

area for each species was summed and the density 

calculated from number of individuals of that species 

divided by the area sampled (100 m²). The relative 

density was then calculated for the number of 

individuals of that species divided by the total number 

of all the plant species. The frequency was calculated 

from the number of plots in which a species occurred 

divided by the total number of plots (10) and the 

relative frequency calculated from the frequency 

divided by the sum of the frequencies for all the 

species in that location. The coverage of each species 

was calculated from the total area covered by that 

species multiplied by the density of that species and 

divided by the total number of individuals for that 

species. The relative coverage was calculated from the 

coverage divided by the total coverage of all the 

species. Finally the importance value for each species 

was calculated from the sums of the relative density, 

relative frequency, and relative coverage. The 

importance value indicates the true prevalence of a 

species based on multiple factors instead of relying on 

frequency of a species only. These calculations were 

done for each species in both the burned and unburned 

locations. 

Results 

Data were collected on November 4, 2007 for 

the burned location and November 11, 2007 for the 

unburned location. Seven hundred and eighty plants 

were measured for diameter in 20 plots total. The 

importance value for Black Mustard (Brassica nigra) 

68 



in the unburned location is 2.8092 and the importance 

value for Artichoke Thistle (Cynara cardunculus) is 

0.1908. A paired one tailed t-test calculated the mean 

area covered for a 10 m² plot was 253291.7 cm² of 

Black Mustard and 4785.52 cm² of Artichoke Thistle 

(p= 1.14E-10). The importance value for Black 

Mustard in the burned location is 1.3484, for Artichoke 

Thistle it is 1.3416, and for the unknown species 0.31. 

The burned location contained 174 Black Mustard and 

58 Artichoke Thistle in the 100 m² section. The 

unburned location contained 533 Black Mustard and 3 

Artichoke Thistle in the 100 m² section. A chi square 

analysis for the plant count of Black Mustard and 

Artichoke Thistle yielded a two-tailed p value of p= 

0.0001 indicating the difference in Black Mustard and 

Artichoke Thistle in the two locations is statistically 

different. 

Importance Value 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

-0.5 

Wild Mustard Artichoke Thistle 

Plant Species 

Figure 1. Importance values of Black Mustard 

(Brassica nigra) versus Artichoke Thistle (Cynara 

cardunculus) at N 33º 34.227’ W 117º 37.798’. 

Artichoke Thistle is not prevalent in unburned habitat. 

Importance Value 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

-0.2 

Wild Mustard Artichoke Thistle Unknown 

Plant Species 

Figure 2. Importance values of Black Mustard 

(Brassica nigra) versus Artichoke Thistle (Cynara 

cardunculus) versus unknown species at N 33º 36.510’ 

W 117º 36.950’. Artichoke Thistle is prevalent in 

burned habitat.


Discussion 

Plant invasion in California chaparral habitat 

by plants such as Black Mustard (Brassica nigra) and 

Artichoke Thistle (Cynara cardunculus) are often the 

result of nature disturbances such as wildfires or 

human disturbances (Le Houerou 1991). After the fire 

on May 13, 2002 it has been demonstrated that the 

Artichoke Thistle species was able to grow in an equal 

ratio to Black Mustard (Figure 2). In the burned 

chaparral habitat the plant count indicated that there are 

more Black Mustard plants (174) than Artichoke 

Thistle (58). When the density and coverage of the 

Artichoke Thistle is taken in to consideration in the 

importance values, a nearly one-to-one ratio can be 

seen; 1.3484 Black Mustard and 1.3416 Artichoke 

Thistle. Since Black Mustard is a tall plant (~2 meters) 

but with a narrow diameter (< 1 meter) many 

individuals could grow in a single area forming a dense 

net of the species. Artichoke Thistle instead has a large 

diameter (> 1 meter) that prevents and other individual 

plant species to grow near it. One other species of 

plant was found in the burned 100 m² section but was 

not included in the chi square analysis because of the 

statistically low prevalence of the species. 

In unburned chaparral habitat the species 

count indicated that Black Mustard (533) is more 

prevalent than Artichoke Thistle (3). The importance 

values confirmed that Black Mustard (2.8092) is more 

prevalent than Artichoke Thistle (0.1908) with the 100 

m² section containing only 6.79% Artichoke Thistle 

(Figure 1). The absence of fire in the last fifteen years 

in this area maintained a dense coverage of plant 

species that Black Mustard with its narrow diameter 

could infiltrate but was more difficult for the large 

diameter Artichoke Thistle. No other species occurred 

in the 100 m² section. Pasture grassland areas, 

commonly found in Southern California, often show 

pure strands of this (Brassica nigra) species (Bell and 

Muller 1973). Non-native plant species invade the 

California grasslands and become significantly more 

prevalent after wildfires. 


Bell, D.T. Muller, C.H. 1973. Dominance of California 

Annual Grasslands by Brassica nigra. American 

Midland Naturalist. 90: 277-99 

DeSimone, S. Ernie, C. 2002. Mechanical Control of 

the Grassland Exotic, Cynara cardunculus, in a 

Southern Californian Nature Preserve. 

Dyer, A.R. Rice, K.J. 1999. Effects of Competition on 

Resource Availability and Growth of a California 

Bunchgrass. Ecology. 80: 2697-710 

Haidinger, T.L. Keeley, J.E. 1993. Role of High Fire 

Frequency in Destruction of Mixed Chaparral. 

Madrono. 40: 141-47 

Hanes, T.L. 1971. Succession After Fire in the 

Chaparral of Southern California. Ecological 

Monographs. 41: 27-52 

Holt, J.S. Marushia, R.G. 2006. The Effects of Habitat 

on Dispersal Patterns of an Invasive Thistle, Cynara 

cardunculus. Biological Invasions. 8: 577-94 

Keeley, S.C. Keeley, J.E. Hutchinson, S.M. Johnson, 

A.W. 1981. Postfire Succession of the Herbaceous 

Flora in Southern California Chaparral. Ecology. 62: 

1608-21 

Kelly, M. Pepper, A. 1996. Controlling Cynara 

cardunculus (Artichoke Thistle, Cardoon, etc.). 

California Exotic Pest Plant Council. 

Le Houerou, H.N. 1991. Biogeography of 

Mediterranean Invasions. Cambridge: Cambridge UP. 

333 

Naveh, Z. 1975. The Evolutionary Significance of Fire 

in the Mediterranean Region. Plant Ecology. 29: 199- 

208 

Williams, P.H. Hill, C.B. 1986. Rapid-Cycling 

Populations of Brassica. Science. 232: 1385- 89 

The Effect of Mycorrhizae on the Growth and Development of Bush Beans 

69 




Phaseolus vulgaris 

Gregory Nelson and Elena Novak 




The majority of plants growing under natural conditions contain fungal tissue in their 

roots called mychorrizae. Mycorrhizal fungus establishes a symbiotic relationship with 

plants. Many plants directly benefit from this connection because the fungus enhances 

nutrient uptake within the plant’s roots and improves growth of the plant. Mycorrhizal 

fungus, in turn, receives sugars which it extracts directly from the roots of the plant. This 

study examined whether or not mycorrhizal content would affect bean plant growth and 

development. Two groups of 14 bush beans were planted on October 15, 2007. The 

experimental group was planted in the organic soil rich in mycorrhizal (Ecto Mycorrhizae 

Inoculant: 3,000,000 viable organisms per cubic foot and Endo Mycorrhizae Inoculant: 

1,900 viable organisms per cubic foot). The control group was planted in organic potting 

soil. After 36 days of growth, plant heights were measured. Results showed that there was 

no significant difference between experimental and control group (unpaired t-Test: 

p=0.3058). The number of pea pods were counted from the experimental and control 

groups and the difference was significant. There was a greater amount of pea pods growing 

on control plants than on experimental plant (p=0.0340). These results did not support our 

initial hypothesis that bean plants grown with mycorrhizal compose will grow taller and 

would produce a greater amount of pea pods. Beans do not require mycorrhizea fungi for 

nitrogen fixation or mineral uptake which promotes growth and development. This may be 

the reason why our hypothesis was not proven. 

Introduction 

Plant growth and development is dependent 

on many factors, such as the nutrient abundance in soil, 

water availability, and light. A region of intense 

microbial activity exists in the soil surrounding plant 

roots. Soil-inhabiting organisms and more specialized 

parasitic organisms infect living roots and utilize them 

as a source of food. Plant tissue becomes disorganized 

in this process and the root system becomes infected 

(Gerdemann, 1968). 

Mychorrhizae obtain food from the host 

without destroying the functioning of root tissue, and a 

balanced relationship between host and microorganism 

is established. This type of symbiotic relationship can 

be regarded as the highest level of parasitic 

specialization (Daft, 1966). Plants that grow under 

natural conditions are symbiotic organisms which 

uptake water and nutrients through the root system and 

fungus tissue. These fungi like roots are called 

mycorrhizae. 

Relatively few plants are completely free from 

mycorrhizae. The root system of an individual plant 

70 



can be completely mycorrhizal or only a little portion 

of it can be infected. Mycorrhizal fungi colonize plant 

roots and the purpose of the following experiment is to 

compare plant growth with mycorrhizal and without it. 

Mycorrhizae often enhance the host plant’s growth and 

mineral uptake, particularly for plants grown under low 

nutrient and mineral stress conditions (Clark, 1997). 

The initial hypothesis for this experiment is: 

soil concentrated with mycorrhizae will enhance the 

plant’s ability to take up all the nutrients from the soil 

and will result in much healthier, larger plants than the 

soil which is not going to be treated with mycorrhizae. 


Two groups of Bush Beans (Phaseolus 

vulgaris) seedlings were planted, in two separate 

planting trays, with organic potting soil in the green 

house of Saddleback College on October 15, 2007. 

Fourteen seeds for each group were planted in a 

planting tray with the recommended distance between 

rows of 3 to 4 inches and a seed depth and spacing of 1 

inch. The experimental group was planted with


Monrovia® Custom Soil Blend (Ecto Mycorrhizae 

Inoculant: 3,000,000 viable organisms per cubic foot 

and Endo Mycorrhizae Inoculant: 1,900 viable 

organisms per cubic foot). The control group was 

planted in E.B. Stone Organics Potting Soil®. All 

plants were watered three times a week as directed by 

instructions. Equal portions of water were given to 

each group regularly to insure that the soil was evenly 

moist, but not soggy. 

Plant measurements, from the two groups, 

were taken at the end of 36 days of growth. The heights 

of the plants were measured with a metric ruler and 

recorded in centimeters. The number of pea pods that 

were produced by each plant were counted and 

recorded as well. 

Results among the two groups were compared 

using an unpaired T-test. Differences were considered 

significant at P


bacteria live in close association with the plant. In 

legumes the bacteria live in small growths on the roots 

called nodules. Within these nodules, nitrogen fixation 

is done by the bacteria, and the NH3 produced is 

absorbed by the plant. Nitrogen fixation by legumes is 

a partnership between a bacterium and a plant 

(Lindmann, Glover, 2003). Legumes do not need 

assistance from mycorrhizea in nitrogen fixation. This 

may be the reason our results did not match previous 

findings. Perhaps a different species of plant, other 

than Phaseolus vulgaris, would replicate the previous 

test results of Wilson and Hartnett. 


Baas, R., Van der Werf A. Root Respiration and 

Growth an Plantago Major as Affected by Vesicular- 

Arbuscular Mycorrhizal Infection. Plant Physiology. 

1989 Sep; 91(1): 227-232. 

Clark, R.B. 1997. Arbuscular Mycorrhizal Adaptation, 

Spore Germination, Root Colonization and Host Plant 

Growth and Mineral Acquisition at Low pH. Plant and 

Soil 192: 15-22, 1997. 

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Plant Growth. Department of Botany, Queen’s College, 

Dundee. 

Gerdemann, J.W. 1968. Vesicular-Arbuscular 

Mycorrhiza and Plant Growth. Department of Plant 

Pathology, Univrsity of Illinois, Urbana, Illinois. 

Kothari, S.K., Marschner H. Effect of VA Mycorrhizal 

Fungi and Rhizophere Microorganisms on Root and 

Shoot Morphology, Growth and Water Relations. New 

Phytology1990. 116, 303-311. 

Kytovita, M.M. Micorrhizal Benefit in Two Low 

Arctic Herbs Increases with Increasing Temperature. 

American Journal of Botany, 2007. 94:1309-1315. 

Lindmann, W.C., Glover, C.R. 2003. Nitrogen 

Fixation by Legumes. College of Agriculture and 

Home Economics. 

Malloch, D.W., Pirozynski, K.A., Raven, P.H. 1980. 

Ecological and Evolutionary Significance of 

Mycorrhizal Symbioses in Vascular Plants. National 

Academy of Science, 1980. 77:2113-2118. 

Smith, S.E. Physiological Interactions Between 

Symbionts in Vesicular-Arbuscular Mycorrhizal Plants. 

Plant Physiology, 1988. 39:221-44. 

Wilson, Gail, Hartnett, D.C. 1997. Effects of 

Mycorrhizae on Plant Growth and Dynamics in 

Experimental Tallgrass Prairie Microcosms. American 

Journal of Botany, 84(4): 478-482. 

Wilson, Gail, Hartnett, D.C., Smith, M.D., 2001. 

Effects of Mycorrhizal on growth and demography of 

Tallgrass prairie Forbs. American Journal of Botany, 

2001; 88:1452-1457 

72 




Exhaustion due to Mental Stress and Metabolism of Sugar and Caffeine in Energy Drinks 

Thomas Caldwell 




The average American consumes energy drinks on a regular basis to maintain 

energy levels throughout the day, but this consumption of unstable energy leads to extreme 

exhaustion throughout the day. Prior research shows that the caffeine in energy drink 

when combined with sugar can have negative effects on the body in the long run. The 

purpose of this research is to discover whether the exhaustion is due to the metabolism of 

sugar and caffeine or just caffeine alone. Six subjects were chosen to drink water, sugared 

energy drinks, and diet energy drinks on separate days. Over a period of time, their 

reaction times were measured and recorded. The data was analyzed for a time of crashing 

and it was found that the average time period of crashing for the water trial was 1.54 ± 0.18 

hours (MEAN ± SE, N = 6), the sugar energy drink was 1.50 ± 0.23 hours (MEAN ± SE, N 

= 6), and the diet energy drink was 1.71 ± 0.22 hours (MEAN ± SE, N = 6). A one-tailed, 

paired t-test showed that there was no difference between the results of the energy drink 

trials (p = 0.28). An ANOVA comparison of the results revealed that there were no 

significant differences in the three groups (p = 0.76). Caffeine alone is the major 

contributor to mental exhaustion from energy drink consumption. 

Introduction 

It is well known that sugar-based energy 

drinks give people a quick boost of chemical energy 

early in the day due to the ingestion high 

concentrations of sugar and caffeine. However, as 

soon as the body utilizes the chemical energy in the 

energy drinks, the body crashes and becomes 

exhausted due to a lack of energy. The reasoning for 

the exhaustion is that the energy drinks contain high 

concentrations of simple sugars, specifically sucrose 

and fructose, which the body metabolizes too quickly 

and gets depleted. Since people generally take energy 

drinks to maintain their energy levels to do work later 

on in the day, mental and physical activities will 

increase the rate of ATP expenditure. An increased use 

of ATP beyond normal capacity would lead to mental 

exhaustion, especially if the body is dependent on 

simple sugars as the main source of energy instead of 

more stable, energy-abundant carbohydrates, such as 

starch. 

Caffeine is a drug found in many consumer 

food products which stimulates the body’s functions 

such as the circulatory and nervous systems to keep the 

body alert and energized. It is one of the main 

ingredients found in high concentrations in energy 

drinks. It is not well known as to whether the caffeine 

contributes to the body’s exhaustion or if it is 

73 



beneficial to the body’s effort in utilizing energy. If 

caffeine does not directly cause mental exhaustion, 

then it must be the rapid metabolism of simple sugars 

that causes the exhaustion. However, there is plenty of 

information regarding the consumption of caffeine and 

its impacts on the healthy well-being of people who 

consume caffeine on a daily basis. 

Maughan and Griffin (2003) found that 

caffeine and methylxanthine compounds may have 

adverse effects on the body’s diuretic functions. It is 

possible that energy drinks may cause the body to 

excrete more of its own store of water and become 

dehydrated; the dehydration would in turn deprive the 

body of energy and cause it to become exhausted. This 

is one mechanism that explains how energy drinks 

effect the body’s utilization of energy. As theorized by 

the scientists, the abstinence from caffeine may 

actually increase the rate of exhaustion and dehydration 

due to a caffeine withdrawal and an increase in 

urination; this, however, has not yet been proven 

scientifically (Maughan and Griffin, 2003). 

MacGregor et al (1990) injected folatedeficient 

rats with caffeine and discovered that while 

the deficiency of folic acid in rats causes chromosomal 

damage in their DNA, caffeine actually increased the 

potential for the chromosomal damage by a significant 

amount. The scientists stated that folate deficiency and


caffeine consumption is extremely prevalent amongst 

the human population in the United States and may 

lead to serious health issues in the future (MacGregor 

et al, 1990). 

Caffeine has other effects on the body. It can 

stimulate a greater activity in the sympathetic nervous 

system, which could promote positive feedback of 

certain functions of the body like heart rate, but it does 

not promote lipid oxidation so there is no evidence that 

can show whether caffeine can also be used as a 

supplement to aid weight loss (Acheson et al, 2004). 

However, Lopez-Garcia et al (2006) found that even 

though caffeine does not aid weight loss, it does 

impede weight gain. So, caffeine taken in small 

amounts on a regular basis may be beneficial in 

preventing weight gain. Regardless, maintaining a 

good diet and exercising are the best ways of sustaining 

a healthy well-being. 

Parker and Ivorra (1991) conducted an 

experiment on Xenopus oocytes, where they examined 

the effects of caffeine on the release of calcium ions in 

the cells; they discovered that the caffeine increased 

the threshold amount of inositol triphosphate but did 

not elicit any clear calcium activated current. If so, 

then the caffeine may induce a negative effect that 

deactivates the action of this messenger. Furthermore, 

if more inositol triphosphate is being produced due to 

caffeine consumption and does not promote higher 

calcium activity, then ATP and GTP are wastefully 

consumed for the stimulation of the inositol 

triphosphate, which may be a contributing factor to 

mental exhaustion in energy drink consumption. 

Leijten et al (1984) conducted research on 

calcium stores in rabbit aortas after being injected with 

caffeine and found that caffeine inhibits noradrenalineinduced 

contractions in the heart and inhibits highpotassium-induced 

contractions by lowering its action 

potential in cardiac tissues. So caffeine may have a 

negative effect on the pumping rate of the human heart 

if taken regularly in high quantities. If the caffeine has 

an effect on the action of noradrenaline in the body, 

then it is possible that energy drink consumption over 

long periods of time may induce chemical imbalances 

in the brain, leading to depression, chaotic mood 

swings, and abnormal sleeping habits, since 

noradrenaline serves an important role in mood 

regulation and sleep. If so, then exhaustion from 

energy drink consumption may also be influenced by 

chemical imbalances in the brain by the inhibited 

action of noradrenaline. 

Finnegan (2003), uncovered the possibility of 

chronic health problems related to the long term use of 

high sugar-based energy drinks; problems range from 

exhaustion due to a lack of a healthier nutritional 

74 



source of energy to drug dependence on caffeine to 

heart failure in the long term. However, Noordzij et al 

(2005) found that regular caffeine intake in high 

amounts will increase blood pressure by about 2.04 

mmHg (systolic BP) and 0.73 mmHg (diastolic BP) 

over time, but when ingested through coffee, where the 

concentration of sugar is much more diluted than the 

amount found in most energy drinks, the effect of 

increasing the blood pressure is much, much smaller. 

So ingesting caffeine in moderate amounts can be 

beneficial in the morning so the body can maintain an 

alert, fully conscious state without causing too much 

problem. A higher blood pressure can play a role in 

quicker exhaustion because of the expenditure of ATP 

to extend the action of epinephrine. 

One study, concerned with exhaustive state of 

patients undergoing insulin therapy, found that as a 

patient was administered insulin, the insulin had a 

sedative effect on the mind, calming anxious patients 

and making them more tired (Bertin, 1945). When 

applied to energy drinks, however, it is understandable 

that when consuming energy drinks with high 

concentrations of sugar, the body’s blood-glucose level 

rises dramatically, stimulating the release of insulin to 

lower blood sugar. In which case, the release of insulin 

is also a contributing factor to reaching an exhaustive 

state from energy drink consumption. 

As far as understanding the effect of the sugar 

by itself is concerned, Lavin et al (1997) found that 

substituting decaffeinated drinks sweetened with 

sucrose for decaffeinated diet drinks sweetened with 

aspartame does not reduce energy intake or energy 

metabolism throughout the day. So drinking liquids 

with moderate to high concentrations of sugar without 

caffeine does not have a negative impact on the body’s 

utilization of chemical energy in the same way that 

energy drinks with high concentrations of both sugar 

and caffeine usually do. If this is true, then caffeine 

and sugar together may either have a negative effect or 

no effect on the body’s utilization of energy. 

In light of the prior research, caffeine must be 

a major contributor in rapid mental exhaustion when 

consuming energy drinks. But little is known of the 

effect of sugar consumption also. Therefore, the 

hypothesis of this experiment predicts that people who 

consume energy drinks with high concentrations of 

sugar and caffeine will reach mental exhaustion faster 

than those who consume diet energy drinks without 

sugar. 


Six human subjects were chosen for 

participation in this research. On the first day of 

experimentation, the participants were instructed to 

consume a high volume of water, directly proportional


to their body weight, for the control factor. For a three 

hour period, the participants worked on study material 

to expend their energy on mental stress to achieve total 

exhaustion, in order to simulate the working day of the 

average American. During this time, their reaction 

times were recorded at every 15 minute interval using a 

reaction time ruler (accurate to ± 12.5 ms). This 

process was repeated for the energy drink and diet 

energy drink trials on different days for the same six 

individuals; each individual had to consume equal 

volumes of the afore mentioned drinks as they did 

during the water trials. 

Results 

All data in this experiment will be given in 

the form of the MEAN ± the standard error. The 

average time of crashing from consumption for the 

water trial was 1.54 ± 0.18 hours. The average time of 

crashing from consumption for the sugar energy drink 

trial was 1.50 ± 0.23 hours. The average time of 

crashing from consumption for the diet energy drink 

trial was 1.71 ± 0.22 hours. 

Reaction Time 

(ms) 

250 

200 

150 

100 

50 

0 

R 2 = 0.2473 

0:15 

0:30 

0:45 

1:00 

1:15 

1:30 

1:45 

2:00 

2:15 

2:30 

2:45 

3:00 

Time (hours) 

Figure 1. This chart shows the average trend of reaction 

times throughout the 3-hour period. High peaks in this graph 

indicate times of exhaustion and low peaks in this graph 

indicate times of alertness. The solid black line is the best fit 

line for the water trial (control factor). 

Time from Consumption 

(hours) 

2.5 

2 

1.5 

1 

0.5 

0 

Water Rockstar 1 Diet Rockstar 

Selected Drinks 

Water Trial 

Sugar Energy Drink Trial 

Diet Energy Drink Trial 

Linear (Water Trial) 

Figure 2. This graph displays the average time to reach 

mental exhaustion from start of consumption for all drinks. 

Error bars represent the standard error in the data for each 

group. 

75 



In the previous graph above, the chi squared 

value for the closeness in relationship between the 

control factor and its linear trendline is 0.2473, 

indicating high variation between the average trendline 

and its data. The average times of crashing after 

consumption were not found to be significantly 

different between both sugar energy drink and diet 

energy drink trials (p = 0.28, one-tailed, paired t-test). 

There are no statistical differences in the three groups 

(ANOVA, single factor, p = 0.76). 

Discussion 

According to the statistics, there were no 

significant differences between the sugared drink and 

diet drink trials, indicating that the monosaccharides do 

not play a major role in the mental exhaustion due to 

crashing. The major contributor to the body’s 

exhaustion must be the caffeine. This confirms the 

finding made by Lavin et al (1997) that sugar 

consumption does not influence the body’s energy 

metabolism, even though the study was primarily 

focused on the effect of sugar itself without the 

participation of caffeine. 

Caffeine must be depriving the body’s energy 

by high ATP and GTP utilization. According to the 

findings of a previous study, caffeine is found to 

increase the amount of inositol triphosphate in heart 

muscle, but it does not seem to activate a higher 

calcium current (Parker and Ivorra, 1991). Thus the 

caffeine is wastefully consuming energy abundant ATP 

and GTP for the increase in the concentration of 

inositol triphosphate with no added effect on the action 

of calcium ions to stimulate further cellular actions. 

And more ATP is consumed as caffeine stimulates the 

action of epinephrine to increase the heart rate. This is 

one reason for the crash. 

However, the ANOVA test showed that all 

three groups were not different from each other (p = 

0.76). This arises some question as to why the data 

from the water trial was not different from the energy 

drink trials. The reason for this may be in a high 

amount of variation amongst the human population, a 

very small population size, especially for human 

subjects, and possibly insufficient metabolism of 

energy through mental stress, as was required for this 

experiment. This could explain why the chi squared 

value for the baseline average of the water trial was so 

low. In other words, the water group was already 

energy deprived from the start because the individuals 

were asked not to eat anything before starting the 

experiment, so the data fluctuates due to their hunger. 

In the other two groups, the test subjects may not have 

been studying to the full extent that they were 

instructed, which conserves their energy output. In


future experiments, this should be corrected by 

increasing the sample size and have the water group eat 

food before arriving. This should correct the problem 

in the experiment and it may show a difference in the 

results if the experiment is performed again with the 

aforementioned modifications. 

Besides this, there was a noticeably small 

difference between the averages of the sugar energy 

drink group (1.50 ± 0.23 hours) and the diet energy 

drink group (1.71 ± 0.22 hours). The subjects seem to 

last longer before they crashed on the diet energy drink 

than they did on the sugar energy drink by about 13 

minutes. Of course this is very peculiar because, as 

logic would dictate, there is a high concentration of 

carbohydrates in the regular energy drink than in the 

diet one, so there should be more energy output in the 

regular drink than in the diet drink. However, the 

carbohydrates in the sugar energy drink are in the form 

of simple sugars, and as stated before, they are not a 

stable form of energy, so they are metabolized too 

quickly. As found by a previous study, insulin has a 

sedative effect on the human mind (Bertin, 1945). So, 

it is a logical explanation that the reason why the 

individuals who consumed the energy drink with sugar 

crashed a little sooner was that the rise in bloodglucose 

levels stimulated the release of insulin, which 

caused them to get tired early on. The diet energy 

drink trials did not exhibit this kind of response in great 

detail because the individuals in this group did not have 

the blood sugar trigger for the release of insulin. More 

over, these individuals had a declining blood-glucose 

level, which may trigger the release of glucagon, and in 

turn trigger the breakdown of glycogen into glucose, 

thus there is more energy investment in the glycogen 

breakdown because it is a more stable source of energy 

than simple sugars. However, this is not a confirmed 

mechanism; several tests must be conducted to 

ascertain whether or not it is the insulin causing the 

crash and whether or not there is a significant 

difference between the two trials with a more improved 

design for the experiment to acquire better results. 

On another note, the consumption of caffeine 

can have a positive outcome. Although this experiment 

did not show it, it is understandable that the utilization 

of energy without energy drinks would be more 

conserved if the body refers to a balanced diet for a 

more stable source of energy. Based on the results of 

Acheson et al (2004) and Lopez-Garcia et al (2006), 

caffeine does not promote weight loss but does impede 

weight gain, thus it would not be an unhealthy choice 

to consume a controlled amount of caffeine each 

morning to make the body more alert and awake, 

especially for those with weight problems. In fact, this 

would be a good idea because caffeine ingested in 

76 



moderate, regular amounts without a high 

concentration of sugar can allow the body to process 

energy more efficiently (Noordzij et al, 2005). 

While caffeine has its positive aspects, it does 

present a more serious health issue to society. 

According to the research of Finnegan (2003) and 

MacGregor et al (1990), energy drinks subside in 

serious side effects that could impede and damage the 

body’s organ systems, leading to illnesses, such as 

chromosomal damage and chronic heart disease. 

Maghaun and Griffin (1990) studied into the effects of 

caffeine on the body’s renal activity and postulated that 

caffeine withdrawals can lead to increased urination. If 

true, energy drink consumption could cause the 

kidneys to overwork and possibly lead to renal failure. 


Acheson, K. J. et al. 2004. Metabolic Effects of 

Caffeine in Humans: Lipid Oxidation of Futile 

Cycling? The American Journal of Clinical Nutrition. 

79(1): 40 – 46. 

Bertin, M. K. 1945. Insulin Therapy in Combat 

Exhastion. The American Journal of Nursing. 45(12): 

1040 – 1041. 

Finnegan, D. 2003. The Health Effects of Stimulant 

Drinks. Nutrition Bulletin. 28(2): 147 – 155. 

Lavin, J. H. and French, S. J. and Read, N. W. 1997. 

The Effect of Sucrose- and Aspartame- Sweetened 

Drinks on Energy Intake, Hunger, and Food Choice of 

Female, Moderately Restrained Eaters. International 

Journal of Obesity. 21: 37 – 42. 

Leijten, P.A and Breemen, C van. 1984. The Effects 

of Caffeine on the noradrenaline-sensitive calcium 

store in Rabbit Aorta. The Journal of Physiology. 357: 

327 – 329. 

Lopez-Garcia, E. et al. 2006. Changes in Caffeine 

Intake and Long-Term Weight Change in Men and 

Women. The American Journal of Clinical Nutrition. 

83(3): 674 – 680. 

MacGregor, J. T. et al. 1990. Cytogenetic Damage 

Induced by Folate Deficiency in Mice is Enhanced by 

Caffeine. Proceedings of the National Academy of 

Sciences of the United States of America. 87: 9962 – 

9965.


Maughan, R. J. and Griffin, J. 2003. Caffeine 

Ingestion and Fluid Balance: a review. Journal of 

Human Nutrition and Dietetics. 16(6): 411 – 420. 

Noordzij, M. et al. 2005. Blood Pressure Response to 

Chronic intake of Coffee and Caffeine: a Meta- 

Analysis of Randomized Controlled Trials. Journal of 

Hypertension. 23(5): 921 – 928. 

Parker, I. and Ivorra, I. 1991. Caffeine Inhibits 

inositol triphosphate-mediated Liberation of 

Intracellular Calcium in Xenopus oocytes. The Journal 

of Physiology. 433: 229 – 240. 

Serial Lactate Levels Increase over Time under Anesthesia on Healthy Canine Patients 

Undergoing Elective, Minimally Invasive, Lower Abdominal Surgery 

Heather Rufino and Alexa Milman 




When assessing anesthetic patients, special attention must be given to 

physiological parameters affecting the intraoperative status of the patient. The 

increase in blood lactate concentration is the result of the disruption of balance 

in the production of lactate from pyruvate and the clearance by vital organs and 

physiological processes, and can be indicative of potential systemic issues. The 

purpose of this study was to support the suggestion that use of serial lactate 

testing, as an additional diagnostic parameter during anesthetic procedures, can 

help to reduce anesthetic risk as a factor signaling potential systemic 

compromise. Serial lactate levels were taken on 10 healthy canine patients 

undergoing elective, minimally invasive, lower abdominal surgery. Samples were 

taken in 20 minute intervals starting at 0 minutes to 100 minutes (the longest time 

elapsed under anesthesia during this study), using 0 minutes as a baseline 

parameter. For the data to be considered statistically significant, P must be 

≤0.008. The single-factor ANOVA test reported P= 9x10 -16 displaying a statistical 

significance between time intervals. The highest lactate level percentage increase 

(111%) occurred between the 0 minute (1.43mmol/L) and 100 minute (3.03mmol/L) 

time intervals. The increase in lactate did not arrive at the clinical value indicating 

lactic acidosis (>5.0 mmol/L) however, patients experiencing Hyperlactatemia 

with the highest mean value of lactate (3.03 mmol/L), were those under 

anesthesia for the longest time period (100 min). This study concluded that there 

was a significant difference in lactate over time under anesthesia, supporting the 

hypothesis that lactate levels will generally increase as time under anesthesia 

elapses. 

Introduction 

Lactate is a by-product produced from 

pyruvate by lactate dehydrogenase during anaerobic 

metabolism. It should be noted that blood lactate levels 

do not increase unless the production exceeds the rate 

of removal (Guyton and Hall, 1996). Furthermore it is 

important to recognize this increase in lactate as the 

symptom of potential problems concerning overall 

condition of systemic health, e.g. the increase in lactate 

itself, is not a disease; it is a symptom of disease. 

During most anesthetic procedures in a 

veterinary practice, the overall physiological status of a 

77 




patient under anesthesia is monitored by external 

diagnostic parameters. Utilizing external monitoring 

equipment in addition to peripheral palpation, 

clinicians and veterinary nurses observe the continuous 

status of the patients pulse rate, respiration rate, 

Saturation of Peripheral oxygen (SPO 2) and body 

temperature. Other important parameters include 

Electro-Cardiogram (ECG), Systolic (SBP), Diastolic 

(DBP), and Mean Arterial blood Pressures (MAP) as 

well as End-Tidal carbon dioxide (ETCO 2 ) levels. 

Lactate has also been established as a valuable test in 

the vast diagnostic repertoire of clinicians (Pang & 

Boysen 2007). However, the use of serial lactate 

monitoring in veterinary medicine during anesthetic 

procedures, is not common practice despite the recent 

increase of its use in human medicine and the 

convenience of handheld portable devices that are now 

available. 

Elevated blood lactate levels are typically 

associated with hypo-perfusion; that is the decrease in 

the process of nutritive delivery of arterial blood to 

capillary beds in biological tissue, when pyruvate is 

unable to enter the Krebs cycle as the cellular oxygen 

supply is insufficient (Pang and Boysen, 2007). This 

experiment focused on the change in lactate levels over 

time in canines undergoing elective, minimally 

invasive, lower abdominal anesthetic procedures. Prior 

observations in anesthetic research support evidence 

indicative of anesthesia having a significant effect on 

the decrease of systemic perfusion (hypo-perfusion) 

(Pang and Boysen, 2007). 

A previous study conducted at Lokmanya 

Tilak Municipal Medical College and Hospital, by 

Shinde et al. (2005), intended to establish serial blood 

lactate levels as a prognostic tool, in human patients 

receiving valvular heart surgery and undergoing cardio 

pulmonary bypass (CPB) during the procedure. The 

parameters that were evaluated were lactate levels and 

its correlation with preoperative clinical condition and 

the intra and postoperative outcomes, following CPB 

for valvular heart surgery. They concluded that there 

was a significant increase in lactate during CPB from 

0.8 as a baseline to the intraoperative level of 7.0 +/- 

2.3, also noting a decrease post operatively during rewarming 

of the patient immediately following CPB 

(Shinde et al., 2005). This raises the question of the 

necessity of serial lactate testing during anesthesia 

while setting precedence for further research as to the 

potential merit of the common use of serial lactate 

testing in veterinary medicine. 

Blood lactate is a dynamic balance involving 

production and clearance (Pang and Boysen, 2007). 

Production of blood lactate exists in low levels of 

concentration under normal conditions of aerobic 

78 



metabolism, in canines this level is considered normal 

when it is less than 2.5mmol/L. Ideally, with normal 

healthy individuals, lactate clearance is optimally 

maintained by the liver and kidneys. However, an 

imbalance in metabolic function, stress from surgery, 

and other conditions can alter this balance (Evans, 

1987). 

When the metabolic balance is disturbed, a 

condition known as hyperlactatemia (lactate levels 

>2.5mmol/L and


monitor. A primary blood gas and chemistry was run 

on the Nova® (Nova Stat Profile Critical Care Xpress 

(CCX), Nova biomedical Corp., Waltham, MA) blood 

gas analyzer for basic chemistry and blood gas 

analysis. Each sample was spun at an apex of 

approximately 6,000 rpm (2,000 x g). Serum was used 

to run a full chemistry panel using the Alpha 

Washerman® (Alfa Wassermann ACE; Clinical 

System, West Caldwell, NJ, USA) blood chemistry 

analyzer. These tests were used to establish the overall 

internal physiological health of each canine patient. 

Only patients found to be within normal ranges met the 

criterion for this study. The weight, sex, and breed of 

each canine patient was also recorded. 

Each experimental subject received a 

peripheral intravenous catheter in the left or right 

cephalic vein and subsequently placed on Normasol- 

R® (Normasol-R; hospera inc., Abbott Laboratories, 

Chicago, IL) crystalloid fluid therapy for 1-2 hours preanesthetically 

at 2.5 mL/kg/hr. 

Patients were anesthetized using a preanesthetic 

pain medication, Hydromorphone HCl (2.0- 

mg/mL) injectable, administered intravenously (IV), 

and dosed by weight at 0.1 mg/kg. Anesthesia was 

induced using the IV injectable anesthetic, Propfol 

(Proflo®, Abbott Laboratories, North Chicago, IL), at a 

dose of 5 mg/kg titrated to effect. Each test subject was 

then intubated with a sterile cuffed endotraecheal tube. 

Anesthetic was maintained using a gas anesthetic 

agent, Isoflurane (IsoFlo®; Abbott Laboratories, North 

Chicago, IL USA), which was delivered by a closed 

circuit system via a constant flow of oxygen at 2.0 

L/min passed through an Isoflurane vaporizer (Drager, 

Lubeck, Germany) initially at the rate of 3.0 % ± 1.5 % 

(dependent on weight and initial anesthetic depth). 

Crystalloid fluid therapy was increased 5 ml/kg/hr 

while under anesthesia to ensure proper profusion 

during the procedure. 

A 25 gauge tuberculin syringe was used to 

draw 0.10 mL of blood from the saphenous accessory 

vein of the hind limb and added to the lactate testing 

strip for lactate analysis. Samples were taken at 20 

minute intervals for the remainder of the procedure 

including one immediate post-induction sample and 

then processed immediately with the Accutrend® 

handheld Lactate monitor. Each study patient was 

monitored using the LifeWindow 6000V vital signs 

monitor (multi-parameter patient monitor, LW6000; 

Digicare Biomedical Technology, West Palm Beach, 

FL), in addition to visual supervision by the veterinary 

surgical nurse and veterinarian. Heart rate (Beats Per 

Minute), blood pressure (mmHg), respiratory rate 

(Breaths Per Minute), ETCO 2 (%), SPO 2 (%), 

temperature (ºF), IV fluid rate (mL/hr), anesthetic level 

79 



(%), O 2 level, and blood lactate levels were measured 

and recorded at specific time intervals throughout the 

procedure. Each patient received heat support via a 

warm air delivery system and a heated surgical table 

(Bair Hugger®; Arizant Healthcare Inc., Eden Prairie, 

MN), during the anesthetic procedure. 

Results 

The ten canine patients involved in this study 

showed a significant increase in average blood lactate 

levels as the time under anesthesia elapsed. A Singlefactor 

ANOVA (Analysis Of Variance) test was run to 

determine the average means and P-value of the blood 

lactate levels. To see where the difference was, a 

Bonferroni Correction test was run between all groups 

against the 0 minute time interval. To be considered 

statistically significant the P-value must be ≤0.008. The 

ANOVA results showed P=9x10 -16 indicating a 

statistically significant P-value. 

The initial mean blood lactate level (0 min) 

was 1.435mmol/L and was used as a baseline 

parameter to compare the increasing blood lactate level 

samples. In comparing the 0 min mean lactate level 

with the 20 min mean lactate level (1.89mmol/L), there 

was no statistical difference. However, when compared 

with the 40 min mean sampling (2.2mmol/L), there 

was a 53% increase in blood lactate level. When 

compared with the 60 min mean sampling 

(2.56mmol/L), there was a 78% increase. When 

compared to the 80 min mean sample (2.82mmol/L), 

there was a 96% increase. Finally, when compared 

with the 100 min mean sampling (3.03mmol/L), there 

was a 111% increase in blood lactate level (Figure 1). 

Figure 1. Graph displaying the percent increase of 

blood lactate levels over given time intervals with P= 

9x10 -16 . Blood lactate levels steadily increased as time 

under anesthesia elapsed. 

Discussion


During anesthesia, complications can often 

occur in spite of the many precautions taken such as, 

vital monitoring of blood pressure, heart rate, 

electrocardiography, body temperature and entidal 

CO 2 . These parameters are in addition to pre-anesthetic 

and interoperative precautions such as, the evaluation 

of pre-operative blood chemistries, heat support during 

anesthesia and the increase of interoperative 

intravenous fluid rate (intended to supplement 

perfusion). Due to overall systemic depression from 

anesthetic agents, such as inhalant gas anesthesia, 

hypoperfusion, although in mild proportions, is often 

still present (Guyton and Hall, 1996). The effects of 

inadequate perfusion on cell function can be 

detrimental to vital organ systems; however, if 

addressed in an appropriate and timely manner, can be 

reversed (Guyton and Hall, 1996). During this study, 

crystalloid fluid therapy was increased to 5 ml/kg/hr 

and external heat support was added while study 

patients were under anesthesia in order to maintain 

adequate profusion during the procedure. 

Ideally, in healthy organ systems, residual 

blood lactate is metabolized in part by the liver (50%) 

and kidneys (20%). Liver function and liver blood flow 

can influence hepatic lactate clearance. Clinically 

healthy patients were utilized during the study to insure 

that pre-anesthetic hypo-perfusion and systemic 

compromise were not factors in the increase of lactate 

intraoperatively. Pre-anesthetic blood chemistries 

including lactate levels, as well as pre-operative, interoperative 

and post-operative vitals were noted during 

this study, however were not included as statistical 

variables with respect to our results, as the specific 

values were not the focus of this experiment. Further, 

more specific exploration of these parameters should 

be tested to establish any existing correlation with vital 

signs and blood chemistries in relation to anesthesia 

and the outcome of the increase in lactate. 

With respect to the purpose of this study 

regarding the importance of continual lactate 

monitoring, Steele and Elliott (2002) conducted a 

similar study examining the importance of the serial 

lactate testing in humans. The study involved 137 

SICU patients who had serial lactate and blood gas 

measurements taken intraoperatively, 24 hours before, 

and 24 hours post operatively. Patients were 

categorized by absolute lactate and base deficit values 

as well as time to lactate clearance. The results 

indicated an initial and 24-hour lactate level that was 

significantly elevated in non survivors versus 

survivors. The study concluded that the elevated initial 

and 24-hour lactate levels are significantly correlated 

with mortality and appeared to be superior to 

corresponding base deficit levels (Steele and Elliott, 

80 



2002). Thus the significance of lactate monitoring was 

supported. 

The danger of increased lactate levels lies 

with the causes of the decrease in clearance. The 

increase in lactate level is not the cause of systemic 

damage, it is the indicator of the decrease in systemic 

function and a warning sign of potential damage which 

may be occurring or has already occurred as a result. 

An increase in lactate of greater than 5mmol/L is 

classified as lactic acidosis. Furthermore lactic acidosis 

is sorted into Type A and Type B lactic acidosis and 

subcategorized thereafter. The principal feature of 

these conditions is the incapacity to use pyruvate in 

oxidative metabolic pathways. The result is the 

accumulation of pyruvate, with subsequent production 

of lactate (Marko et al., 2004). 

Elevated lactate levels resulting from 

inadequate tissue perfusion is classified as Type A 

lactic acidosis (Marko et al., 2004). The accumulation 

of pyruvate and hydrogen ions, along with NADH, 

drives the production of lactate, however once 

oxygenation is improved, aerobic metabolism should 

resume and acidosis should resolve provided the organ 

systems are functioning at adequate levels. The type of 

lactic acidosis, in which tissue perfusion is adequate 

however enzyme systems are not functioning 

adequately, is classified as Type B lactic acidosis 

(Guyton and Hall, 1996). While Lactate levels during 

this study showed a maximum mean of 3.03 mmol/L 

during the 100 minute interval, indicating moderate 

Hyperlactatemia, lactic acidosis did not occur. It can 

be deduced that the type of lactic acidosis that would 

most likely associated with this study, had it occurred, 

would be Type A lactic acidosis. 

It should be noted that this study was 

randomized to evaluate patients in a clinical 

environment. A controlled study using a controlled test 

colony would be indicated to eliminate certain random 

variables. This was not the intended design of this 

study, as true clinical parameters during anesthesia 

were believed to be of vital importance in enhancing 

future knowledge of this subject. Further studies may 

be indicated in non-clinical studies that would include 

non-surgical anesthetic patients without the use of fluid 

therapy, or analgesia throughout the study, which may 

or may not alter overall results. In this study, the 

patients were limited to the standard anesthetic 

protocol used during surgical procedures developed by 

the board certified veterinarians at Advanced Critical 

Care and Internal Medicine. Test subjects were clinical 

patients and not a controlled test colony. 

This study concluded that there was an 

increase in lactate over time during the anesthetic 

procedures; however the increase in levels during this


study where not found to be exceedingly high; the 

highest value at 100 min time interval (3.03mmol/L). 

While lactate levels in canine patients elevated only 

mildly during the non-invasive and relatively short 

anesthetic procedure times, the elevation in the levels 

was statistically significant (p= 9x 10 -16 ). This 

suggests warrant for further research, examining serial 

blood lactate measurements in more lengthy and 

critical anesthetic procedures, as very limited 

information readily exists on the topic. Furthermore, 

the results of this study support the routine use of serial 

lactate monitoring during anesthesia, as part of a well 

rounded anesthetic monitoring protocol, especially in 

previously compromised or critically ill patients. 


Evans, G.O. 1987. Plasma lactate measurements in 

healthy beagle dogs. Am J Vet. 48: 131–132. 

Guyton, A.C. and Hall, J.E. 1996. Textbook of medical 

physiology. 9th edition. W. B. Saunders Company, 

London: 471-489. 

Lagutchik, M.S., Ogilvie, G.K., Hackett, T.B., 

Wingfield, W.E. 1998. Increased Lactate 

Concentrations in III and Injured Dogs. J of Vet. 

Emerg. and Crit. Care. 8(2): 117–127. 

Levraut, J.L., Ciebiera, J.P., Chave , S.A., Rabary, 

O.L., Jambou, P.S., Carles, M.F., Grimaud, D.B. 1998. 

Mild hyperlactatemia in stable septic patients is due to 

impaired lactate clearance rather than overproduction. 

Am J Respir. Crit. Care Med. 157: 1021-1026. 

Marko, P.R., Gabrielli, A.M., Caruso, L.J. 2004. Too 

much lactate or too little liver. J of Clin. Anesth. 16: 

389-395. 

McMichael , M.A., Lees, G.E., Hennessey, J.L., 

Sanders, M.A., Boggess, M.D. 2005. Serial plasma 

lactate concentrations in 68 puppies aged 4 to 80 days. 

J Vet Emerg. Crit. Care. 15: 17–21. 

Meregalli, A.L., Olivieri, R.P., Friedman, G.S. 2004. 

Occult hypoperfusion is associated with increased 

mortality in hemodynamically stable, high-risk, 

surgical patients. Crit Care Med. 8: R60–R65. 

Pang, D.S., Boysen, S.R. 2007. Lactate in veterinary 

care: pathophysiology and management. J Amin Hosp 

Assoc. 43: 270-279. 

Santosh , B.R., Shinde, S.D., Kumud, K.B., Golam, 

P.K., Neela, D.P. 2005. Blood lactate levels during 

CPB. Annals of Cardiac Anesthesia. 8: 39–44. 

Steele, S.R., Elliott, D.C. 2002. Serum lactate and base 

deficit as predictors of mortality and morbidity. Am J 

of Surgery. 185: 485-491. 

Thorneloe, C.R., Bedard, C.B., Boysen, S.D. 2007. 

Evaluation of a hand held lactate analyzer in dogs. Can 

Vet J. 48: 283–287. 

Vincent, J.L., Dufaye, P.S., Berre, J.V., Leeman, M.G., 

Degaute, J.P., Kahn, R.J. 1983. Serial lactate 

determinations during circulatory shock. Crit Care 

Med. 11: 449–451. 

The Correlation Between the Vertical Jump Height and Calf Length in Athletes and 

Non-athletes 

Jasmine Mitchell and Tony Schofer 




The type of muscle in the lower body and certain training regimens has been 

known to improve vertical jumping. In this study, a correlation between a person’s 

calf length and vertical jump height was tested for athletes and non-athletes. 

Athletes jump vertically higher than non-athletes. The reach of the participants, 

81 




which included athletes (N=18) and non-athletes (N=25), was measured. With their 

feet flat beneath them and a piece of masking tape wrapped around their index, 

middle, and ring finger, the participants jumped vertically placing the tape on the 

wall at their apex. This was repeated two more times. The distance was measured 

from the reach height to the vertical jump height. The maximum vertical jump 

height was used for analysis. From the Lateral Malleolus up to Popliteal Fossa, the 

6 

calf lengths were measured. Athletes (55 2.13 cm) significantly (p= 1.3 10 ) 

jumped higher vertically then non-athletes (37.5 2.22 cm). Between the calf lengths 

of the athletes (38 0.90 cm) versus the non-athletes (37.5 0.74 cm), no statistical 

difference was shown. There was no correlation found comparing the athletes’ and 

non-athletes’ vertical jump heights versus calf lengths (R 2 =0.40, R 2 =0.58, R 2 =0.59). 

Introduction 

Among sports, vertical jumping is common 

and varies for its purpose. It has been used as a way to 

measure lower body power and to test one’s athletic 

ability. A vertical jump consists of a force being 

applied to the body’s mass, while the body is still in 

contact with the ground, in order to accelerate it to the 

maximum (Kreighbaum and Barthels, 1996). This 

movement of vertical jumping produces its power from 

the quadriceps group, ankle plantarflexors, and hip 

extensor muscles (Kowalski, 2003). Furthermore, the 

muscle type in these areas will aid in a higher vertical 

jump for most cases as studied by Curley (2000). There 

are two muscle types: fast twitch and slow twitch 

muscle. Fast twitch muscle is the most idle for vertical 

jumping because the neurons in the muscle fire at a fast 

rate causing the sudden rush of power to the lower 

body. The slow twitch muscle does not generate the 

same amount of power; thus, the vertical jump height 

will not be as high. Another study showed that during 

the last part of pushing-off, the compliance of 

tendinous structures would allow muscle-tendon 

complex to generate a moderately large power at a high 

joint angular velocity region, which would help with 

the vertical jump height (Kurokawa et al., 2000). 

However, in this study, a correlation between 

calf length and vertical jump height will be compared 

with the participants, athletes, and non-athletes to 

determine if having a longer calf length will increase 

vertical jump height. It will be done without a warm up 

because Koch and associates found that warm-up has 

no effect on jumping performance (Koch et al., 2003). 

A second study will be done comparing the vertical 

jump height of the athletes and non-athletes. It is 

expected that athletes jump higher than non-athletes, 

and that there is a correlation between calf length and 

the vertical jump height. 


Athletes (N=18) and non-athletes (N=25) 

ranging from 16 to 38 years old participated in this 

study. The participants wrapped a piece of masking 

tape around the index, middle and ring finger and 

placed it as high on the wall as their reached allowed. 

Without warming up, each participant jumped 

vertically with their feet flat beneath them, placing the 

tape on the wall at their apex. This was repeated two 

more times. The highest vertical jump was used for 

each participant. The distance between the reach height 

and vertical jump height was measured. The calf 

lengths were measured from the Lateral Malleolus up 

to Popliteal Fossa, the region behind the knee. 

The maximum vertical jump height versus calf 

lengths for all subjects were plotted to determine if 

there was a correlation between the calf length and 

one’s vertical ability to jump higher. A second 

correlation was done between athletes and non-athletes. 

A student unpaired t test was run comparing vertical 

jump height; p ≤ 0.05 was considered statistical 

difference between athletes versus non-athletes. 

Results 

Athletes (55 2.13 cm) had a statistically 

6 

(two tailed, p = 1.3 

10 ) higher vertical jump than 

non-athletes (37.5 2.22 cm) as shown in Fig 1. There 

was no difference between the calf length of the 

athletes (38 0.90 cm) versus the non-athletes (37.5 

0.74 cm), which is seen in Fig 2. Between the 

participants’ vertical jump height (44.82cm, N=43) and 

their calf lengths (38cm), no correlation was found 

(R 2 =0.40) as seen in Fig 3. Into athletes and nonathletes, 

both shown together in Fig 4, there was no 

correlation found for both (R 2 =0.58, R 2 =0.59) between 

the vertical jump height and calf length. 

82 




60 

Average Vertical Jump Height (cm) 

Figure 1. The average vertical jump height between 

athletes (55 2.13cm, N=18) and non-athletes (37.5 

2.22cm, N=25). Athletes significantly (two tailed, p= 

6 

1.3 

10 ) jumped vertically higher than nonathletes. 

Average Calf length (cm) 

Figure 2. The average calf lengths between athletes 

(38 0.90 cm, N=18) and non-athletes (37.5 2.22 

cm, N=25). There was no significant difference found. 

Vertical Jump Height (cm) 

50 

40 

30 

20 

10 

0 

50 

45 

40 

35 

30 

25 

20 

15 

10 

80 

70 

60 

50 

40 

30 

20 

10 

5 

0 

0 

athletes 

athletes 

non-athletes 

non-athletes 

28 30 32 34 36 38 40 42 44 46 48 

Calf Length (cm) 

Figure 3. The vertical jump heights (44.82cm) of the 

participants (N=43) compared with their calf length 

(38cm) to determined if the longer the calf length the 

higher the vertical jump. There was no correlation 

found (R 2 =0.40). 

Vertical Jump height (cm) 

80 

70 

60 

50 

40 

30 

20 

10 

0 

30 32 34 36 38 40 42 44 46 48 

Calf Length (cm) 

athletes 

non-athletes 

Linear (athletes) 

Linear (non-athletes) 

Figure 4. A comparison between the athletes’ (N=18) 

vertical jump height (55cm) versus their calf lengths 

(38cm) in order to see if a higher vertical height is 

achieved because of a longer calf length. There was no 

correlation found (R 2 =0.58). The same comparison 

was done between non-athletes’ (N=25) vertical jump 

height (37.5cm) and calf length (37.5cm). No 

correlation was also shown (R 2 =0.59). 

Discussion 

Athletes were found to significantly jump 

higher than non-athletes. However, the average calf 

length of the athletes and non-athletes had no statistical 

difference; as a result, the vertical jump height was not 

influenced by the calf length. This can be due to two 

main factors: muscle mass in the leg and specific types 

of training. Golomer et al. (2004) and Harley et al. 

(2002) both found in their study of ballet dancers that 

muscle mass in the leg is directly linked to jump 

height. The dancers with the greater quantity of muscle 

in the leg jumped significantly higher than those with 

less amounts. 

Specific types of training are another major 

factor, which are used to increase vertical jumping 

when more muscle mass is not the main focus and less 

desired. Plyometrics and vibration are some of these 

training regimens that sports have being using in order 

to increase vertical jump height, which have been 

shown to be successful. This type of training 

concentrates on the neuromuscular aspects of 

development in power, which is beneficial for sports 

similar to dance and basketball (Luo et al., 2005; 

Radcliffe and Farentinos, 1999; Wyon et al., 2006). 

Leg muscle mass and training can also explain 

why no correlation was found between the vertical 

jump height and calf length for the athletes, nonathletes, 

and all the data put together. Because 

depending on the type of athletes and how they train 

will determine whether they are able to jump vertically 

83 




as high as someone who trains in order to vertically 

jump. 

In conclusion, athletes jump higher than nonathletes; 

and no correlation was shown between the 

calf length and the vertical jump height of the 

participants, athletes, and non-athletes, with muscle 

mass in the legs and training having a great influence. 

In future studies, a more in depth look comparing 

different types of athletes with the same and different 

training regimens will be looked into. 


Curley P. Special test: How Powerful Are Your Legs? 

Bicycling, 2000; 41.6: 98 

Golomer E, Keller J, Eery Y, Testa M. Unipodal 

performance and leg muscle mass in jumping skills 

among ballet dancers. Percept Motor Skills, 

2004;98(2):415-428 

Harley Y, Gibson A, Harley E, Lambert M, Vaughan 

C, Noakes T. Quadriceps strength and jumping 

efficiency in dancers. J Dance Med Sci, 2002;6(3):87- 

94. 

Koch AJ, O’Bryant H, Stone M, Sanborn K, Proulx C, 

Hruby J, Shannonhouse E, Boros R, Stone M. Effect of 

warm-up on the standing broad jump in trained and 

untrained men and women. Journal of strength and 

conditioning research, 2003;17(4):710-714. 

Kowalski C. Correlation between time to peak torque 

and peak torque to vertical jump in college athletes. 

Thesis, 2003. 

Kreighbaum E, and Barthels K.M. Biomechanics: A 

Qualitative Approach for Studying Human Movement. 

Needham Heights, Massachusetts: Allyn and Bacon, 

1996. 

Kurokawa S, Fukunaga T, Kukashiro S. Behavior of 

fascicles and tendinous structures of human 

gastrocnemius during vertical jumping. J Appl Physiol, 

2001;90:1349-1358. 

Luo J, McNamara B, Moran K. The use of vibration 

training to enhance muscle strength and power. Sports 

Med, 2005;35(1):23-41 

Radcliffe J, Farentinos R. High-Powered Plyometrics. 

Champaign, III: Human Kinetics, 1999. 

Wyon M, Allen N, Angioi M, Nevill A, Twitchett E. 

Anthropometric Factors Affecting Vertical Jump 

Height. J Dance Med Sci, 2006;10(3-4):106-110. 

Comparison of Chlorophyll Content of Leaves in a Green House and their normal 

environment of a Cyclamen Plant (Cyclamen Persicum) 

Chris Yang and Josue Mandujano 

Department of Biology 



Chlorophyll is fundamental for photosynthesis, which obtains its energy 

from the sunlight. The chlorophyll content varies between plants and the light 

exposure with sun. Given that photosynthesis occurs more efficient in a green house, 

it was predicted that the leaves inside a green house would contain higher 

chlorophyll content than plants that are in their normal environment. A 

spectrophotometer was used to determine the amount of chlorophyll content from 

the leaves from greenhouse and normal environment. Five mL of 80% concentrated 

acetone were mixed with leaf, two 6mm leaf chads in scintillation vials. Three mL 

solution was inserted in a cuvette into the spectrophotometer for further analysis. It 

was discovered that there wasn’t a significant difference (p = 0.41) in chlorophyll 

content between cyclamen leaves inside the greenhouse and normal environment. 

Therefore, the results rejected the hypothesis, which stated that the cyclamen leaves 

84 




inside the greenhouse would contain higher chlorophyll content than the leaves in 

their normal environment. 

Introduction 

Pigments are chemical compounds which 

reflect only certain wavelengths of visible light (Speer 

95). Chlorophyll a is the type of chlorophyll that makes 

photosynthesis possible. It does this by passing on its 

energized electrons on to molecules which will 

manufacture sugars (Speer 95). A second type of 

chlorophyll, chlorophyll b only occurs in plants and 

green algae that transfer energy to chlorophyll a. 

Photosynthesis is divided into two different and distinct 

stages – the Light Reaction, and the Calvin Cycle. 

These two stages of photosynthesis are dependent of 

each other. The light reactions depends on the NADP+ 

and ADP and P, that the Calvin cycle generates and 

Calvin cycle depends on the NADPH and ATP that the 

light reactions generates (Campbell 05). 

A previous paper deal with the estimation of 

chlorophyll in plant extract by application of 

absorption of chlorophyll in plant. In aqueous acetone 

dried solid chlorophyll components are dissolved and 

made to volume with solvent of identical composition 

with extract from wavelength 680nm to 540nm 

(McKinney 1941). From his experiment, molar 

absorbance coefficients among the range of 

wavelengths were found. 

Chlorophyll content varies with different 

environmental factors. A plant that is healthier will 

have a higher amount of chlorophyll overall (Cate 

03). The amount of chlorophyll in a leaf is directly 

related to the amount of direct sunlight it receives 

(Wells 2000). The main purpose of this experiment to 

determine which leaves, either the ones in the green 

house or the ones in their normal environment, will 

contain higher chlorophyll content. It is predicted that 

the leaves inside the greenhouse will contain more 

chlorophyll than the leaves in their normal 

environment. Green house could control factors such as 

temperature, carbon dioxide (CO2) concentration and 

relative humidity that lead plants as accurately as 

possible for optimum crop growth (Bio Medicine). 


In collecting data for analysis of chlorophyll content 

inside a greenhouse and their normal environment of 

cyclamen plant (Cyclamen persicum), samples were 

collected. Twenty leaves were collected: ten leaves 

from inside the greenhouse, and ten leaves from their 

normal environment. The greenhouse was built out of 

transparent vinyl and wires in form of a bucket. On 31 

85 



October 2007, the greenhouse was put on top of plant 

and was set up for the experiment. The area from 

which samples were taken was from a garden located 

in Mission Viejo. 

Leaves were prepared for chlorophyll analysis 

on 21 November 2007 at Saddleback College. Twenty 

scintillation vials were filled with 5 mL of 80% 

acetone, measured with the use of a pipette. Two leaf 

chads, each with a diameter of 6 mm were added to 

each vial and labeled according to their category. All 

vials were placed in a 4°C environment for 48 hours. 

Chlorophyll readings were taken on 26 

November 2007 using a Beckman DU 730 

spectrophotometer, calibrated for measurement of 

chlorophyll content in acetone at a two wavelengths in 

nm. Three milliliters of an 80% acetone was pipetted 

into a cuvette, to zero out the spectrophotometer. Then, 

three milliliters of each of the sample mixtures were 

pipetted into cuvettes, and the readings of chlorophyll 

content were taken individually for total combined 

chlorophyll content at mg/L. 

The Beckman DU 730 spectrophotometer was 

set up with the incorrect program and inaccurate 

measurements were given. Leaves were prepared for 

chlorophyll analysis on 28 November 2007 once again. 

This time the machine was calibrated with the correct 

wavelengths in nm, and the readings of chlorophyll 

content were taken individually for total combined 

chlorophyll content at mg/L. We used K 1 A 1 +K 2 A 2 

equation to calculate the concentration of chlorophyll a 

and b. 

Results 

The cyclamen leaves inside the green house 

did not contain more chlorophyll than the leaves from 

their normal environment. The total average 

measurement of milligrams of chlorophyll per liter of 

80% concentrated acetone of the leaves inside the 

green house of the cyclamen plant were 3.754 mg/L 

(+0.15 se, N=10). The total average measurement of 

milligrams of chlorophyll per liter of 80% concentrated 

acetone of the leaves in their normal environment of 

the cyclamen plant were 3.822 mg/L (+0.24 se, N=10). 

The difference between the average amounts of 

chlorophyll between the two leaves was not 

significantly different (p value = 0.41). Figure 1 shows 

the graph of the average of chlorophyll concentration 

of the leaves inside the greenhouse and their normal 

environment.


Ave. Chlorophyll Concentratio 

4 

3 

2 

1 

0 

Inside 

Figure 1. Bar graph showing the mean + SE values for 

chlorophyll concentration. 

Discussion 

In measuring and comparing the chlorophyll 

content of leaves inside the greenhouse and their 

normal environment of a cyclamen plant, the results 

showed there was no difference in amount of 

chlorophyll between the two. 

The experiment attests that the major factors 

contributing to chlorophyll concentration of leaves 

inside the greenhouse and their normal environment is 

overall location of the plant (Lafferty 2001). In the 

experiment concerning the monitoring of chlorophyll 

in sugar maples tree leaves (Cate and Perkins, 2003), it 

was stated that angle of incidence and PAR irradiance 

affect chloroplast distribution and angle (Haupt 1982). 

In addition, in an experiment to determine the 

absorption of light by chlorophyll solutions (G. 

MacKinney, 1941), it was found that solvents, 

including 80% anhydrous acetone, have an affect on 

the absorption coefficients of chlorophyll a and b, 

altering them. All of these factors come into play with 

chlorophyll analysis. 

Given that the majority of the readings of the 

samples taken from the leaves were not significantly 

different to each other. The average chlorophyll 

content was very similar to each other. After running 

several analyses, it appears that there was not a 

significant difference between leaves inside the 

greenhouse and from their normal environment. This 

1 

Outside 

may be due to the fact that since the leaves inside the 

greenhouse might receive nearly the same amount of 

sunlight exposure to undergo photosynthesis as the 

leaves from the normal environment. The vinyl that we 

used for the experiment was transparent. Therefore, the 

sun light went through the vinyl and did support the 

production of chlorophyll (Farabee 2001). 

A Greenhouse is a better environment for 

plants to grow, and it support insulating heat, keeping 

moisture inside and preventing from herbivores to eat 

(Hershey 2001). No significant difference appeared. 

This may be due from the temperature and moisture. 

Further research of correlations of temperature and 

moisture would be required to verify this. 


Campbell, N. A. and Reece, J. B. 2005. Biology: 

Seventh Edition. San Francisco, CA: Pearson 

Education, Inc. 

Cate, T. M. and Perkins, T. D. 2003. Chlorophyll 

content monitoring in sugar maple (Acer saccharum). 

Tree Physiology. 23, 1077−1079 

Farabee, M. J. 2001. Photosynthesis. 

. 

Hershey, David (2001) Botany, The Greenhouse 

environment, New York 

Knudson, Linda L. (1977) Department of Horticulture, 

University of Wisconsin, Madison, Wisconsin 

Lafferty, Kenneth (2001) Plant Biology, Science 

buddies, New York 

MacKinney, G. 1941. Absorption of Light By 

Chlorophyll Solutions. The Journal of Biological 

Chemistry. 132, 315-322 

Speer, B. R. 1995. Photosynthetic Pigments. 

. 

Wells, Kenneth (2000) Department of agriculture, 

University of Kentucky, Kentucky "Something New 

Under the Sun." Bio Medicine 10 December 2007 

The Effects of Ginkgo Biloba on the Cognitive Thinking of Mus musculus 

Milad Danesh 

86 







Cognitive thinking improvement is a highly sought out activity in today’s society. 

One method of improving cognitive thinking is through the use of Ginkgo biloba, a method 

that dates back to Ancient China. Ginkgo biloba has frequently been associated with mind 

enhancement, but recent studies have shown conflicting results. An experiment was carried 

out to accumulate more knowledge on the matter. A group of ten mice (Mus musculus) 

underwent a series of three trials to test the effects of Ginkgo biloba. The first trial involved 

the mice being exposed to a maze, under normal conditions, in which their ability to reach 

the goal box was measured by time. A second trial was administered in a similar manner, 

but with the mice being under the influence of Ginkgo biloba. A third and final trial was 

administered, after the second trial, in which the mice were free of Ginkgo biloba. The 

completion time for each mouse was recorded for each trial. Data analyses comparing the 

data for each trial showed that there was no significant increase in performance when the 

mice were under the influence of Ginkgo biloba. The results of the experiment have shown 

that there is no positive correlation between Ginkgo biloba use and cognitive thinking in 

mice. 

Introduction 

In an increasingly competitive world, society 

is always looking for ways to increase their cognitive 

thinking ability. Such an increase can be particularly 

helpful in schooling, and can be used as possible 

treatments for various mental disorders. One such 

method that has been used since Ancient China is the 

consumption of Ginkgo biloba extract (Carson-DeWitt 

2007). Potential benefits of consuming the extract 

include improvement of cognitive memory, processing 

speed, attention, and concentration (Elovic and Zafonte 

2001). 

In a study that examined the effectiveness of 

Ginkgo biloba, it was concluded that the extract could 

improve cognitive performance in patients with 

dementia (Huffman 2002). However, some studies 

have also concluded that Gingko biloba has no effect at 

all. One study examined the effects of Ginkgo biloba 

on subjects over the age of 60. Results came out 

negative, and the study concluded that there was no 

significant increase in cognitive memory after taking 

the extract (Huffman 2003). Another study, though, has 

shown that Ginkgo biloba can be beneficial for the 

treatment of Alzheimer’s disease (Sierpina, 

Wollschlaeger, and Blumenthal 2003). 

One study concluded that Ginkgo biloba 

extract had no effect on the memory of healthy adults, 

but conceded that it might be helpful in larger dosages 

(Frankish 2002). The recommended dosage ranges 

from 120 to 240 milligrams a day (Heffel 2006). 

87 



However, in larger amounts, Ginkgo biloba has been 

known to carry side effects. Ginkgo can thin-out blood 

if taken with medication and can also adversely affect 

blood-glucose levels in diabetics (Gale 2007). It is 

important to know the effects that Ginkgo biloba 

extract has on cognitive thinking in order to gain 

knowledge on how it could be used for human benefit. 

Since the ginkgo plant is long-lived and can withstand 

areas with strong pollution, the extract can be made 

readily available in large amounts (Dilcher 2007). 

The hypothesis that will be tested is that 

consumption of Ginkgo biloba extract has a positive 

effect on the cognitive thinking in mice (Mus 

musculus). A similar study has been done that showed 

positive results, but further examination is needed for a 

more solid conclusion (Gajewski and Hensch 1999). 

The results of this experiment could potentially show 

that ginkgo has a positive effect on cognitive thinking. 

In this event, the leaf extract could be used to improve 

cognitive thinking in students or could even be used in 

the treatment of dementia. 


Mice (Mus musculus) were received from 

Petco, Mission Viejo, California. The mice were kept 

in well-ventilated containers, where they remained 

until the initiation of the first trial. The treatment of


the mice was identical from mouse to mouse 

throughout the entire experiment. The mice were fed 

mouse food mix provided by Petco, and were given 

water through a drip-feed container. Each of the ten 

mice was weighed on a balance provided by Canyon 

Lake Urgent and Family Care, Canyon Lake, 

California. The average weight of adult humans, the 

recommended dosage of ginkgo for humans, and the 

average weight of the experimental mice group were 

used to calculate roughly how much Ginkgo biloba 

extract would be needed for the experiment. The 

recommended dosage for Ginkgo biloba in humans was 

micro scaled at around 0.1 milligrams for each mouse 

to more closely simulate a human experiment. 

The mice were to be tested on their cognitive 

thinking, so a maze had been constructed specifically 

for the experiment. The maze was constructed using 

poster board received from Office Max, Mission Viejo, 

California. At the end of the maze, or the goal box, 

there was a piece of cheese that gave the mice 

incentive to reach the goal box. The first trial the mice 

endured in the maze was the pre-ginkgo trial. Each 

mouse was introduced to the maze, and was timed on 

how quickly each mouse could complete the maze 

using a stopwatch supplied by the experimental 

investigator. The maze was thoroughly cleansed 

between each of the mice trials throughout the entire 

experiment so as to eliminate confounding variables 

and to avoid an increase in completion time due to 

memory. The mice were not given any training with 

the mice preceding the pre-ginkgo trial, so as to 

minimize the effect on completion times due to 

prolonged exposure to the maze. 

The mice were then placed back in their wellventilated 

container for the next three days. During this 

three day period, the mice were fed as usual, but were 

also given a daily 0.1 mg portion of a Ginkoba, a 

dietary supplement containing Ginkgo biloba. The 

Ginkoba was provided by Canyon Lake Urgent and 

Family Care. Ginkoba was administered to the mice by 

crushing it and placing it in front of each mouse, 

individually, in a separate small container. The extract 

would sometimes need to be placed in mouse food in 

order for it to be consumed by the mice. After the three 

day period, the mice were put through a second trial, 

the ginkgo-induced trial. Each mouse was once again 

placed in the maze and was timed on how fast they 

could reach the goal box. After the second trial, each 

mouse was placed back in their container. The maze 

was again cleansed between each of the mice trials. 

The mice were kept in their well-ventilated 

container for another three days after the ginkgoinduced 

trial. During this second three day period, the 

mice were once again fed normally, but without the 

88 



daily 0.1 mg portion of Ginkoba. The mice were then 

put through a third and final trial, the post-ginkgo trial. 

The mice were again put through the maze and timed 

on how fast they could reach the goal box. The maze 

was cleaned between mice trials. All data were 

transferred to MS Excel (Microsoft Corporation, 

Redmond, Washington) where all further calculation 

and statistical manipulations were performed. All 

experimentation and calculations were done in the 

month of November 2007. 

Results 

The average weight of the mice was recorded 

at 28.87 ± 0.26 g (±se, N= 10). The average maze 

completion time for the mice during the first trial was 

shown at 21.50 ± 0.86 seconds. The second trial, the 

ginkgo-induced trial, showed similar completion times. 

The average maze completion time for the second trial 

came out at 20.81 ± 0.83 g (±se, N= 10). The third trial, 

the post-ginkgo trial, also resulted in similar results to 

the both previous trials. The average maze completion 

time for the third trial came out at 21.04 ± 0.80 g (±se, 

N= 10). 

Although the averages seemed to stray away 

from the tested hypothesis, further analysis was needed 

to confirm the results. An ANOVA statistical analysis 

test comparing the three performed trials revealed that 

there was not a significant decrease in completion time 

between the three trials (F =0.180, P = 0.84). The F- 

value is a measurement of distance between individual 

values, so the low value indicates no significant 

difference between the calculated means. The average 

completion times for each trial did not show enough 

variance to prove a significant difference between the 

three trials (Figure 1). These results show that Ginkgo 

biloba did not have a significant effect on the cognitive 

thinking in mice. 

Time (seconds) 

25 

20 

15 

10 

5 

0 

Pre-Ginkgo Ginkgo-Induced Post- Ginkgo 

Trial 

Figure 1. The average completion times are shown for 

each trial run. The figure shows that Ginkgo biloba did


not produce a significant decrease in completion time 

for the maze (F=0.180, P = 0.84). Error bars indicate 

a 95% confidence interval. 

Discussion 

The hypothesis stated for this experiment was 

that extracts of Ginkgo biloba should have a positive 

effect on the cognitive thinking of mice. The results of 

the experiment have shown that the mice failed to 

perform significantly faster in the maze when under the 

influence of ginkgo than when ginkgo played no part in 

the mice. The mice were unable able to reach the goal 

box faster during the ginkgo-induced trial, because 

Ginkgo biloba seemed have no effect on the cognitive 

thinking of the mice. This proves the tested hypothesis 

implausible. 

The relationship between cognitive thinking 

and Ginkgo biloba is not well understood, according to 

most studies. Studies often contradict each other on the 

subject. Some studies have concluded that Ginkgo 

biloba has no effect on healthy adults at all (Huffman 

2003). However, a similar study to the one performed 

has seen an improvement in their samples after the use 

of Ginkgo biloba (Gajewski and Hensch 1999). Some 

studies have even stated that Ginkgo biloba does work, 

but only on those with memory problems or dementia 

(Sierpina, Wollschlaeger, and Blumenthal 2003). 

The experiment performed provides evidence 

against the relationship between Ginkgo biloba and 

improved cognitive thinking, but it is questionable how 

relevant this data is to humans. Although there have 

been mixed results about studies concerning the matter, 

the general consensus seems to be that Ginkgo biloba is 

more beneficial to those with below average cognitive 

thinking or any type of dementia. The increase in 

cognitive thinking may be only minimal. So as a result, 

an increase in cognitive thinking may only be detected 

significantly in those with below average thinking. An 

increase in cognitive thinking may not be so visible in 

those with already good cognitive thinking. 

Future research could examine humans’ 

performance under the same condition as the 

performed trials. The effects of Ginkgo biloba on 

cognitive thinking can be studied by comparing its 

effects on those with dementia and those with good 

cognitive thinking. A significantly larger sample size 

and perhaps more exposure to the experimental activity 

would be needed to get the most accurate results. The 

amount of ginkgo given to subjects may be increased 

as well, but this route must be cautioned because of the 

possible side effects seen from increased Ginkgo biloba 

use (Gale 2007). If positive results are seen in a followup 

experimentation it could prove beneficial towards 

humans, because Ginkgo biloba extract can be readily 

89 



available in large amounts due to the plant’s 

availability due to its strong withstanding of pollution 

and its long lifespan (Dilcher 2007). Dementia can be 

slowed down or even stopped, and Ginkgo biloba can 

be a first step in the process of stopping it for good. 


Carson-DeWitt, Rosalyn, M.D. (2007), Ginkgo biloba, 

Gale Encyclopedia of Mental Disorders, Gale Virtual 

Reference Library 

 

Dilcher, David (2007), Ginkgo, Plant 

Sciences, Vol. 2: 179-181 

 

Elovic, Elie P., and Zafonte, Ross D. (2001), Ginkgo 

Biloba: Applications in Traumatic Brain Injury, The 

Journal of Head Trauma Rehabilitation, Expanded 

Academic ASAP 

< http://find.galegroup.com/ips/start.do?prodId=IPS > 

Frankish, Helen (2002), Ginkgo biloba does not 

enhance memory in healthy adults, US study finds, The 

Lancet, Expanded Academic ASAP 

 

Gajewski, Ann, and S.A. Hensch (1999), Ginkgo 

Biloba and Memory for Maze, Psychological Reports, 

Expanded Academic ASAP 

 

Gale (2007) Ginkgo biloba may offer benefits, but be 

wary of risks: if you're taking this popular herbal 

supplement, use it with caution, especially if you take 

medications that thin your blood.(Over-the-counter), 

Men’s Health Advisor, Expanded Academic ASAP 

 

Heffel, Rianne (2006), Ginkgo biloba: feeling 

forgetful? Here's how this herb can help lift your brain 

fog, Better Nutrition, Expanded Academic ASAP 

 

Huffman, Grace Brooke (2002) Efficacy of Ginkgo 

Biloba in Treating Dementia, American Family 

Physician, Expanded Academic ASAP. 

< http://find.galegroup.com/ips/start.do?prodId=IPS> 

Huffman, Grace Brooke. (2003) Ginkgo Ineffective for 

Memory Enhancement, American Family Physician, 

Expanded Academic ASAP. 

< http://find.galegroup.com/ips/start.do?prodId=IPS>


Sierpina, Victor S., Bernd Wollschlaeger, and Mark 

Blumenthal. (2003) Ginkgo Biloba.(Complementary 

and Alternative Medicine)(Health Benefits and Dosage 

Information), American Family Physician, Expanded 

Academic ASAP. 

< http://find.galegroup.com/ips/start.do?prodId=IPS> 

90 




Increased performance level due to carbohydrate vs. carbohydrate-protein composition in 

sports drinks 

Catherine Pearson 




This study tested the effects of carbohydrates and proteins on performance and 

exhaustion levels during strenuous cycling exercise. Four male participants were tested, all 

in good athletic shape. All four participants tested for each liquid (water, Original 

Gatorade, Accelerade and Cytomax), cycling outdoors for 2 hours straight and recording 

data each half hour. Their distance traveled, ounces of liquid consumed and perceived 

exertion levels were recorded every half hour. After the data was collected, a single-factor 

ANOVA was used for statistical analysis of the average distance traveled and the average 

ounces of liquid consumed. The P values for each liquid were over 0.05 and therefore did 

not show statistical significance for any of the four liquids. Even though the tests were not 

statistically significant, there were slight changes in exhaustion levels between each liquid 

and participants did feel a difference in overall physical effect. The results of this study did 

not support the previous carbohydrate-protein studies that showed a significant increase in 

performance when protein was included in the sports drink formula. 

Introduction 

With the growing concern for health and 

fitness these days, the sports drink industry has become 

very competitive. Instead of water, there is now an 

abundant amount of specialized drinks that key into 

energy, endurance and re-hydration. Beyond the 

obvious benefits of hydration, water just doesn’t seem 

to cut it these days in the fitness world. What is so 

different and special about the various sports drinks 

available today? This study explored that question, 

especially concentrating on the difference between 

carbohydrate only sports drinks compared to 

carbohydrate-protein sports drinks. These ingredients 

in sports drinks (sodium, carbohydrate and proteins) 

have been shown to aid in the body’s recovery rate and 

endurance level during and after exercise. Godfrey et 

al. tested the impact of ultra-purified water on a 40 km 

cycling trial and the results supported the importance 

of electrolyte and carbohydrate infused drinks. After 

testing 8 highly trained athletes during four 40 km 

trials of cycling the performance enhanced as a result 

of ingesting Penta[R] (purified water which included 

carbohydrates and electrolytes) compared with tap 

water (Godfrey et al., 2005). A study done at St. Cloud 

University in 2006 tested the effects of Accelerade, 

Gatorade and water after exercise (Cycling), focusing 

on the difference of the carbohydrate only drink 

(Gatorade) compared to the carbohydrate-protein drink 

91 



(Accelerade). This study concluded that Accelerade 

was 15% more effective in re-hydration than Gatorade, 

and 40% more effective than water (Seifert et al., 2005) 

Athletes usually consume carbohydrate-rich 

food because the body metabolizes faster when 

exercising. Carbohydrates provide the fuel necessary 

for muscle contraction and prevent the body from 

breaking down proteins to be used for energy. Along 

with carbohydrates, proteins are a crucial part of 

athletes’ diet. Proteins help to build and repair muscle 

tissue in athletes along with increasing carbohydrate 

storage in the form of glycogen. During exercise, the 

main goal for nutrient consumption is to replace lost 

fluid and maintain carbohydrate levels or blood glucose 

levels. (CJDPR, 2000) Ivy et al. tested 9 cyclists with 3 

different liquids solutions consisting of a placebo, a 

carbohydrate solution and a carbohydrate-protein 

solution. This study demonstrated increasing plasma 

glucose and insulin levels during prolonged exercise 

reduced the loss of muscle glycogen and increased 

endurance. The results of this study showed an 

enhanced endurance performance with the 

carbohydrate-protein solution compared to the placebo 

and carbohydrate only solution (Ivy et al., 2003). An 

additional study was conducted at Springfield College 

that tested CHO (carbohydrate) solutions against CHO- 

PRO (carbohydrate-protein) solutions with ten male 

runners. This study resulted in the CHO-PRO drink


enhancing the release of insulin which is known to 

facilitate in the uptake of glucose into skeletal muscles. 

The CHO-PRO also resulted in a longer time to 

exhaustion when compared to the PRO drink. (Niles et 

al., 2001) 

The four drinks tested in this experiment 

were: water, Gatorade, Accelerade and Cytomax. 

Although each drink (other then water) contains similar 

ingredients focused on energy and endurance, the ratios 

differ whether it been a stronger protein level or a 

higher amount of carbohydrates. The first liquid tested 

was water, which contains no carbohydrates and no 

proteins. The next liquid is Original Gatorade, which 

contains 14g of carbohydrates per serving (8 fl oz.) and 

no proteins. Accelerade is the third liquid which has 

15g of carbohydrates per serving (8 fl oz) and 4g of 

protein per serving. The last liquid is Cytomax which 

has 22g of carbohydrates per serving (8 fl oz) and no 

proteins. 4 Cyclists were tested in this experiment, each 

drinking all four liquids during four separate cycling 

sessions. Their distance traveled, ounces of liquid 

consumed and exertion level, were recorded every 30 

minutes for 2 hours. The purpose of this study was to 

test the different liquids against each other to determine 

whether protein does increase performance and 

endurance levels during long term, strenuous exercise. 

Based on previous studies, it was expected that the 

protein-carbohydrate solution would result in the best 

performance and lowest exertion levels. 

Procedures and Materials 

This experiment consisted of 4 male cyclists. 

The participants exercised on a daily basis and were in 

good athletic shape. Each cyclist used their own bikes 

for the four separate endurance tests and they were 

provided the four different liquids. They were given 

two 16 ounce water bottles, two 20 ounce original 

Gatorade bottles, two 20 ounce Accelerade bottles and 

two 20 ounce Cytomax bottles. Four index cards were 

printed out for the participants to record their 

information (Figure 1). After each 30 minute segment 

of exercise, the participants were asked to record the 

distance they traveled, the amount of liquid they drank 

and their level of energy based on a weak, moderate, 

strong level. Due to certain restrictions and time 

delays, the participants were not all tested on the same 

days. The tests were performed over a month time 

period. An ANOVA was used to run the statistical 

analysis for average distance traveled and average 

ounces of liquid consumed, using a p value of p< 0.05. 

Results 

Each liquid was tested for all four athletes 

using a single-factor ANOVA. The distance traveled 

92 



was tested to see if there were any significant 

differences in mileage between each sports drink over 

the period of two hours (30 minute increments). The 

water sample for distance traveled resulted in a P value 

of P = .9199. Gatorade was tested next (P = .9977), 

followed by Accelerade (P = .9989) and Cytomax (P = 

.9878). The mean scores for distance traveled are 

shown in figure in table 1. Figure 2 shows the 

comparison of distance traveled for all four liquids 

during the 30 minute time segments, each notch 

representing a mean score. After the distance traveled 

was tested, I ran an ANOVA for the amount of liquid 

consumed over the 2 hour time period for each 30 

minute segment. The water data resulted in (P = .6444), 

followed by Gatorade (P = .8066), Accelerade (P = 

.9555) and finally Cytomax (P = .7461). The mean 

scores for ounces consumed are shown in table 2. 

Figure 3 is a graph showing the ounces consumed for 

each liquid in 30 minute segments. Along with the data 

on distance traveled and ounces consumed, data was 

also recorded for exertion levels throughout the ride. 

Based on 3 levels of exertion (weak, moderate and 

strong) water overall produced the weakest exertion 

levels compared to the other 3 sports drink liquids. 

When comparing the exertion of the sports drinks, 

Cytomax was the overall strongest level of exertion. 

Participants felt strongest while drinking Cytomax 

(Carbohydrate only) over the 2 hour cycling time. 

30 

Min 

1 1/2 

Hr 

1 Hr 

2 Hr 

(Liquid) 

Distance (mi) 

Oz. 

consumed 

Energy Level 

(1) Weak 

(2) Moderate 

(3) Strong 

Table 1. Sample data card used during each liquid 

trail 

Distance (miles) 

9.5 

9 

8.5 

8 

7.5 

7 

6.5 

Average Distance Traveled 

30 60 90 120 

Time (minutes) 

Water 

Gatorade 

Accelerade 

Cytomax


Figure 2. Line graph displaying the mean values for 

distance traveled for each of the four liquids tested 

during 30 minutes, 60 minutes, 90 minutes and 120 

minutes 

Ounces 

12 

10 

8 

6 

4 

2 

0 

Average Liquid Consumed (ounces) 

30 60 90 120 

Time (minutes) 

Water 

Gatorade 

Accelerade 

Cytomax 

Figure 3. Line graph displaying the mean values for 

ounces of liquid consumed during 30 minutes, 60 

minutes, 90 minutes and 120 minutes, for all four 

liquids tested 

Discussion 

Carbohydrates and proteins provide the 

necessary fuel that humans need during exercise. 

Although water is a good source of hydration for the 

body, people exercising for more then 30 minutes need 

more then H20 to keep them energized. The main goal 

of this experiment was to test different sports drinks 

during a period of long exercise to see if the difference 

in carbohydrate and protein levels had an effect on the 

cyclist’s performance and exertion level. The data for 

the water sample showed a decrease in distance 

traveled compared to the other drinks (Figure 2). This 

is expected over a 2 hour time period when there are no 

carbohydrates or proteins being consumed and 

therefore the body is not receiving the energy it needs 

to provide endurance. The water sample also showed 

an increase in the amount of liquid consumed over the 

2 hours of exercise concluding that the participants 

needed to drink more water to keep cycling (Figure 3). 

Next I compared the individual sports drinks 

with the mean scores for water. There are many 

previous studies that examine the effects of 

carbohydrates and proteins on endurance and time-toexhaustion 

rates during strenuous exercise. A study 

published in the Journal of Sports Science tested the 

difference between the carbohydrate only sports drink 

and the carbohydrate-protein sports drink on 13 cyclists 

over 120 minutes exercise period. This study resulted 

in a significant difference for the carbohydrate group 

93 



(P < 0.05) when compared to the placebo group, but 

not a significant difference for the carbohydrate-protein 

group meaning there was no added effect of protein 

during exercise (Osterberg et al., 2007). After reading 

about these studies I was interested to find out if the 

added protein really did have an effect on performance 

and exertion during long periods of exercise. 

The first sports drink, Original Gatorade, has 

14g of carbohydrates per serving (8 fl oz) and 0g of 

protein per serving. Although participants did show a 

slight increase in distance traveled per 30 min segment, 

the mean scores were not significant (P = .997). The 

next drink tested was Accelerade, which has 15g of 

carbohydrates per serving (8 fl oz) and 4g or protein 

per serving. The combination of carbohydrates and 

protein was expected to increase the participants 

performance level based on pervious studies; however 

the data did not result in a significant difference (P = 

.998). Finally Cytomax was tested, which contains 22g 

of carbohydrates per serving (8 fl oz) and 0g of protein 

per serving. The data for Cytomax did show increased 

mileage per 30 minute segment and a decreased 

amount of liquid consumed, however it was not 

statistically significant (P = .988). Overall there did 

not seem to be a difference of performance level when 

comparing the carbohydrate only solutions to the 

carbohydrate-protein solutions. The added protein in 

the Accelerade sports drink did not provide the 

participants with increased performance levels when 

compared to the carbohydrate only sports drinks. 

Along with the distance traveled and ounces 

of liquid consumed, the participant’s general exertion 

levels were recorded and resulted in moderate 

differences among the liquid. Based on 3 exertion 

levels (weak, moderate, and strong), the participants 

recorded feeling weaker during the water trial than any 

other liquid tested. When the three sports drinks were 

compared, Cytomax (Carbohydrate only) scored with 

the strongest level of exertion, participants felt best 

throughout the 2 hours while drinking this liquid. 

Cytomax has a significantly higher amount of CHO 

compared to the other drinks, which may be the cause 

of the improved exertion levels. Based on these 

findings, the protein did not show any added effect to 

the endurance or exertion level of the participants 

compared the non-protein drinks. 

Based on my data collected and the statistical 

analysis, my original hypothesis was not reached in this 

experiment. The protein-carbohydrate sports drink, 

Accelerade, did not prove to be any more efficient 

during strenuous exercise than the carbohydrate only 

sports drinks (Gatorade and Cytomax). Although 

protein did not result in improved performance, 

carbohydrates did add to the participant’s performance.


The effects of added protein to energy drinks may not 

be evident in physical exertion or endurance but still 

plays a role in building and maintaining mussel tissue 

which is equally important for training athletes. 

There are a few ways that this study could be 

improved. Increasing the sample size might lead to 

more significant data along with increasing the amount 

of trails. I only tested each drink once for each person. 

Certain factors such as weather, meals eaten before the 

experiment took place and daily physical conditions 

could have played a part in the results of my study. 

Increasing the amount of tests for each liquid would 

cancel out the effects of external conditions. 


Godfrey, R., Shave, R., Franco, A., and Flemming, P. 

(2005). The impact of ultra-purified water on 40 km 

cycling time trial performance. Journal of Sports 

Sciences. 23.11-12 (Nov-Dec): 1285(2). 

Harmon J.H., Burckhard J.R., Seifert J.G. (2007). 

Ingestion of a carbohydrate-protein supplement 

improves performance during repeated bouts of high 

intensity cycling. Medicine & Science in Sports & 

Exercise. 39(5):S363 

Ivy, J.L., Res, P.T., Sprague, R.C., Widzer, M.O. 

(2003) Effect of a carbohydrate-protein supplement on 

endurance performance during exercise of varying 

intensity. International journal of sport nutrition and 

exercise metabolism. Department of Kinesiology and 

Health Education, The University of Texas, Austin, TX 

Sep;13(3):382-95. 

Niles E. S., Lachowetz, T., Garfi, J., Sullivan, W., 

Smith, J.C., Leyh, B.P., Headley, S.A.(2001). 

Carbohydrate-Protien Drink Improves Time to 

Exhaustion after Endurance. Exercise Journal of 

Exercise Physiology. Vol. 4 Number 1, (Jan) 

Osterberg, K.L., Zachwieja, J.J., Smith, J.W. (2007). 

Carbohydrate and carbohydrate + protein for cycling 

time-trial performance. Journal of Sports Sciences, 

99999 (1), 0264-0414. 

Rehrer, N. J., Brouns, F., E. J. Beckers, W. H. M. Saris 

(1994) The influence of beverage composition and 

gastrointestinal function on fluid and nutrient 

availability during exercise. Scandinavian Journal of 

Medicine & Science in Sports 4 (3), 159–172. 

Saunders, M.J., Kane, M.D., Todd, M.K. (2004) 

Effects of a carbohydrate-protein beverage on cycling 

endurance and muscle damage. Med Sci Sports Exerc 

7(36):1233-1238, 2004 

Van Essen, M., and Gibala, M.J. (2006). Failure of 

protein to improve time trial performance when added 

to a sports drink. Medicine and Science in Sports and 

Exercise vol. 38.8 (August): 1476-1483. 

The Position Statement from the Dietitians of Canada, 

the American Dietetic Association, and the American 

College of Sports Medicine, Canadian Journal of 

Dietetic Practice and Research (CJDPR), winter of 

2000, 61(4): 176-192 

Seifert, J.G., Harmon, J., DeCiercq, P., (2005). Fluid 

retention properties of carbohydrate/protein and 

carbohydrate-only sports drinks. St. Cloud State 

University 

Comparison of Drinks and Their Effects on Exercise Performance 

Shengchieh Chang 




This study used a simple fitness monitor and an elliptical trainer to compare 

the effects of three drinks: the sports drink, the vitamin enhanced water, and the 

protein enhanced sports drink, with water on the exercise performance. Average 

heart rates, peak heart rates, and calorie output of twenty five minute exercise with 

drinking four different liquids were recorded for three participants. Both positive 

94 




and negative effects on calorie outputs and heart rates were found for the drinks 

tested. However, no statistical significance was found between water and three 

drinks, for each fitness index, from the further analysis by the single factor ANOVA 

and the following post hoc analysis. 

Introduction 

The sports drinks replenish the water, 

sugar, electrolytes, and other nutrients lost in the 

exercise for recreational and professional 

athletes. Several studies indicate that the sports 

drinks could enhance performance and prevent 

fatigue (Ivy et al., 1988; Burke, 1993; Sugiura et 

al., 1998, Ivy et al., 2003). Most sports drinks 

contain only water, electrolytes, and 

carbohydrates; some drinks claim containing 

special ingredients, such as proteins, which could 

improve the endurance and performance 

(Saunders et al., 2004; Valentine et al., 2006; 

Harmon et al., 2007), while other researchers 

were in doubt about the effect of protein addition 

(van Essen et al., 2006). 

This study compared the effects of 

different drinks on the fitness performance of 

participants. The parameters of fitness 

monitored in this study are calories output, 

average heart rates, and peak heart rates. The 

calorie output is the most commonly monitored 

parameter in the exercise. The oxygen 

consumption rate is directly related to the 

metabolism but it requires advanced equipment 

and therefore the heart rates were monitored 

instead since the oxygen uptake is proportional 

to the heart rate (McArdle et al., 2005). The heart 

rate data is used to interpret the fitness condition 

of the participants within the exercise. The peak 

heart rate was adopted to express the intensity of 

the exercise and the average heart rate was used 

as the index of the performance. 

Methods and Materials 

An elliptical fitness crosstrainer, EFX 

556i, manufactured by Precor, was used to 

simulate the running condition. The program on 

the cross-trainer can be adjusted according to the 

weight, age, and the preferential exercise mode 

of the participants. The manual mode was 

selected and each participant entered his or her 

own age and weight. 

A Suunto t1 performance monitor watch 

measured the fitness data of athletes within the 

workout. The watch stores the personal data such 

as the birthday, weight, height, etc. Then the 

performance, calorie output, was calculated 

instantaneously based on the heart rate 

monitored and personal information entered. The 

data recorded from the exercise included calorie 

95 



outputs, peak heart rates, and average heart rates. 

Since the watch can only store one data entry, 

the watch was reset after the record was recorded 

down in the notebook. 

Four drinks were tested and their major 

ingredients are listed in table 1. Twenty ounces 

of each drink were consumed for one running 

session. For each session, participants drank 3 

portions, 7oz, 7oz, and 6oz of the desingated 

liquid, at the time before the start, 50% complete, 

and 80% complete of the running. 

There were three participants, two 

males and one female, for this study and their 

basic physical information is listed in table 2. 

Their ages range from nineteen to thirty seven 

while the activity levels spanned between 

“medium active (5.0)“ and “atheletic (8.0) 

(Suunto, 2006). The fitness performance data, 

including calories, peak heart rate, and average 

heart rate, of twelve sessions (for four drinks and 

three persons) were recorded for later analysis. 

Drink/Calorie Key Nutrients Key Ions 

Bottled Water 

(0) 

Carbonhydrate 0g 

Protein 0g 

Sodium 0mg 

Potassium 0mg 

Enhanced Water 

(130) 


Protein 0g 

Sodium 65mg 

Potassium 0mg 

Sports Drink 

(130) 


Protein 0g 

Sodium 270mg 

Potassium 75mg 

Protein Sports 

Drink (180) 


Protein 9g 

Sodium 290mg 

Potassium 40mg 

Table 1. Drinks tested in the study and the major 

components 

Participant 

# 

Gender Age Weight 

(kg) 

Height 

(cm) 

Activity 

(1-10)* 

1 Male 20 68 191 8 

2 Female 19 66 168 5 

3 Male 37 69 175 7 

Table 2. Physical condition of participants 

*Activity levels are defined by the monitor 

manual (Suunto, 2006) 

Results 

The calorie outputs, average heart rates, 

and peak heart rates of three participants were 

give in figures 1, 2, and 3, separately, for each 

drink tested. The fitness data with consuming 

only water were used as the baseline to contrast 

the effect of other three drinks. As the participant 

number is small, only simple analysis was 

conducted first. The means and difference 

percentage for each drink from the water were


listed below in table 3 for the calories output, 

average heart rate, and peak heart rate. 

It was found that the protein enhanced 

sports drink increased 9.2% of calorie outputs. 

The effect of sports drink to improve the fitness 

performance, i.e. the reduction of average heart 

rate, was observed; however, the value, 1.0%, 

was small. These two observations showed the 

carbohydrate only and the protein enhanced 

sports drinks enhanced the exercise performance. 

For the negative effect, the vitamin enhanced 

water decreased the energy output by 6.8% while 

raising the peak heart rate for 2.7%. For the peak 

heart rates, the differences range from 1.8% to 

14% for the three drinks when being compared 

with water. 

The single factor analysis of variance 

(ANOVA) was used to identify the difference 

among drinks for the three fitness indexes. The F 

values were listed in table 4, indicating that the 

difference existed in the calorie output for drinks 

tested while no difference was observed for other 

two indexes about heart rates. This insignificance 

on peak heart rated illustrated that drinking 

different fluids did not drastically change the 

intensity of exercise. The post hoc analysis was 

adopted to detect the significance of difference 

for water and the drinks in the calorie output. 

The t values were listed in table 5, demonstrating 

that no statistical significance was found 

between water and each of the drinks studied for 

all the fitness indexes. 

Calorie Output (kcal) 

290 

280 

270 

260 

250 

240 

230 

220 

210 

200 

Water Sports Drinks Enhanced Water Protein Drinks 

Drink Type 

Figure 1. The calories outputs for four drinks. 

The three bars of each drink from left to right are 

for the three participants (#1, #2, and #3). 

Average Heart Rate (BPM) 

200 

190 

180 

170 

160 

150 

140 

130 

120 

110 

100 


Drink Type 

Figure 2. The average heart rates for four drinks. 


for the three participants (#1, #2, and #3). 

Peak Heart Rate (BPM) 

200 

190 

180 

170 

160 

150 

140 

130 

120 

110 

100 


Drink Type 

Figure 3. The peak heart rates for four drinks. 


for three participants (#1, #2, and #3). 

Calories Average HR Peak HR 

Mean ∆P% Mean ∆P% Mean ∆P% 

Bottled 

Water 246.33 135.67 152.33 

Sports 

Drink 251.67 2.2 134.33 -1.0 155.00 1.8 

Enhanced 

Water 229.67 -6.8 139.33 2.7 163.00 7.0 

Protein 

Drink 269.00 9.2 140.00 3.2 174.67 14.7 

Table 3, Fitness performance analysis: mean and 

the deviation (difference of percentage from the 

baseline) 

Calorie Average HR Peak HR 

6.911 

0.073 

0.408 

(Yes) 

(No) 

(No) 

Table 4, F statistics of ANOVA and statistical 

significance for fitness Data. The F-value, 

f(0.05,3,8)=4.066 

Calorie Average HR Peak HR 

Sports Drink 0.613 

(No) 

0.092 

(No) 

0.120 

(No) 

Enhanced 

Water 

2.221 

(No) 

0.253 

(No) 

0.481 

(No) 

Protein Drink 2.600 

(No) 

0.299 

(No) 

1.007 

(No) 

96 




Table 5, t statistics and the significance of post 

hoc analysis between water and the other three 

drinks tested. 

Discussion 

Positive effects were observed for two 

drinks tested: The reduction of average heart rate 

by the sports drinks and the increase of calorie 

outputs by the protein drink. For the negative 

effect, the vitamin enhanced water was not a 

proper exercise drink since it lowered the calorie 

outputs while raising the average heart rates. 

However, these influences were not found 

conclusive as the statistically significance was 

unable to be recognized by the further analysis. 

Comparing with the exercise time in the 

previous studies (Saunders et al., 2004; Harmon 

et al., 2007), twenty five minutes of exercise 

might be too short for muscles to metabolizing 

all the creatine phosphate and glycogen, and start 

to source externally, i.e. the sugars and/or 

proteins in drinks, to supply the muscle 

functioning. Also, the sugar composition also 

affects the participants’ absorption since one 

research found that chocolate milk had more 

significant effect on cyclists’ muscle recovery 

and performance than drinks containing protein 

(Karp et al., 2006). Moreover, the rising of heart 

rate for the protein enhanced sports drinks might 

be related to the digestion of protein and 

therefore the effect of carbohydrate 

replenishment was neutralized. 

To be able to study the problem more 

thoroughly, some modifications can be done for 

the experimental methods. First, the size of 

participants should be increase and therefore 

other statistical methods can be used to analyze 

the data and the data can be more representative. 

Second, when choosing study participants, those 

having similar fitness background and activity 

levels should be located and therefore the 

physiological variation can be minimized. Third, 

the exercise time should be elongated to allow 

the muscle to reach the exhaust state. Fourth, the 

challenge exercise intensity should be adjusted to 

the same for all the athletes in order to calibrate 

the calorie output. Furthermore, the measures to 

remove the psychological effect due to the 

product names, such as using drinks of the same 

flavor and unlabeled bottle, should be utilized. 


Burke LM (1993). Dietary supplements in sports. 

Sports Med, 15:43-65. 

Harmon JH, Burckhard JR, and Seifert JG 

(2007). Ingestion of a carbohydrate-protein 

supplement improves performance during 

repeated bouts of high intensity cycling Med Sci 

Sports Exerc. 39:S363. 

Ivy JL, Lee MC, Brozinick JT, and Reed MJ 

(1998). Muscle glycogen storage after different 

amounts of carbohydrate ingestion. J Appl 

Physiol 65:2018-2023. 

Ivy JL, Res PT, Sprague RC, and Widzer MO 

(2003). Effect of a carbohydrate-protein 

supplement on endurance performance during 

exercise of varying intensity. Int J Sport Nutr 

Exerc Metab. 13:382-395. 

97 




Karp JR, Johnston JD, Tecklenburg S, 

Mickleborough T, Fly A, and Stager JM (2006) 

The efficacy of chocolate milk as a recovery aid. 

Int J Sport Nutr Exerc Metab. 16:78-91. 

McArdle WD, Katch FI, and Katch VL (2005). 

Essentials of Exercise Physiology, Lippincott 

Williams & Wilkins, Philadelphia, PA, USA. 

352-355. 

Saunders MJ, Kane MD, and Todd MK (2004). 

Effects of a Carbohydrate-Protein Beverage on 

Cycling Endurance and Muscle Damage. Med. 

Sci. Sports Exerc., 36:1233-1238. 

___ (2006). Suunto t1 User’s Guide, Sunnto 

USA, Inc., Ogden, UT, USA. 

van Essen MJ, and Gibala MJ (2006). Failure of 

protein to improve time trial performance when 

added to a sports drink. Med Sci Sports Exerc 38: 

1476-1483. 

Valentine RJ, St. Laurent TJ, Saunders MJ, Todd 

MK, and Flohr JA (2006). Comparison of 

responses to exercise when consuming 

carbohydrate and carbohydrate/protein 

beverages. Med Sci Sports Exerc. 38:S341. 

Sugiura K, and Kobayashi K (1998). Effect of 

carbohydrate ingestion on sprint performance 

following continuous and intermittent exercise. 

Med Sci Sports Exerc 30:1624-30. 

Correlation between attractiveness of male body odor and facial feature of 

Homo. sapiens 

Tarick Sheikh and Chris Glendinning 




Evolutionary fitness is determined by many factors. In Homo sapiens, fitness 

is largely based on certain aspects of attractiveness. Individuals found more 

attractive to the opposite sex than their peers have a larger pool of potential mates, 

and therefore a proportionately larger probability of genetic contribution to the 

next generation. Pheromones and facial features are the main cues of male 

attractiveness. There is evidence that both of these features cue and attract females 

to males with high degrees of genetic dissimilarity, focusing largely on dissimilar 

genetic immune systems increasing the likelihood of genetic diversity and offspring 

fitness. Expectantly a correlation is likely to exist between a female’s determination 

of a male’s (H. sapiens) attractiveness, facial features, and his pheromones. Nine 

males wore shirts while sleeping for three nights, 15 females rated the shirts to test 

for a correlation between appearance and body odor. Correlation was measured by 

averaging out mean ratings of appearance and body odor. The results showed that 

the null hypothesis was rejected (p > 1 x 10 -1 ), and a significant correlation 

coefficient (r= 3.6 x 10 -1 ) was found between facial appearance and scent in (H. 

sapiens) males. 

Introduction 

A multitude of factors contribute to the 

degree of attractiveness of an individual or more 

precisely what an individual finds attractive in a 

potential mate. The common saying “beauty is in 

the eye of the beholder” is not quite the whole 

98 




story. Many attempts have been made to quantify 

the nature of beauty, specifically the elements of 

attraction which influence mate selection. Thus far 

the study of attraction has identified several 

influential components of heterosexual attraction 

specifically body form, pheromones, and 

reproductive cycles. Body form studies have 

focused mainly on distinct facial features in males 

and females, certain facial features have been 

hypothesized to have a direct correlation to 

physical attractiveness. Facial symmetry has been 

directly linked to attractiveness in both male and 

female faces (Rhodes 1998). Male facial features 

with high degrees of masculinity were found to 

confer attractiveness, a large square jaw was found 

to be a visual cue of attractiveness in male faces 

(Grammer & Thornhill 1994). However some 

research has concluded that it is a mixture of 

masculine and feminine traits, not the 

predominance of either one that is often found 

attractive (Cummingham 1990). 

Hypothesizes have also proposed that females 

find certain male features attractive to varying 

degrees during distinct phases of their reproductive 

cycles (Penton-Voak & Perrett 2000). Never the 

less, a correlation has been established between 

certain male facial features and attractiveness. 

Additionally a man’s distinct body odor has been 

linked to his attractiveness and desirability as a 

mate (Hertz & Cahill, 1997). Appropriately it is a 

combination of a man’s pheromones “his scent” 

and his facial features that determine his level of 

attractiveness to potential mates. The evolutionary 

significance of attractiveness seems to play a role 

in “good gene” selection; one such gene appears to 

be the major histocompatibility gene complex 

(MHC) (Thornhill et al. 2003). MHC genes play a 

key role in pathogen recognition and immune 

response and therefore evolutionary fitness of 

couples’ offspring. The MHC gene has a direct 

influence on body odor and therefore an influence 

on mate choice (Thornhill et al 2003). Pheromones 

have also been linked to traits of facial 

attractiveness. Studies have shown that the body 

odor of men with symmetrical facial features was 

found to be more attractive to women during their 

menstrual cycle (Rikowski & Grammer, 1999). 

The objective of this experiment is to study the 

correlation between a man’s level of attractiveness 

based on his facial features and his attractiveness 

based on his body odor. It is expected that there 

will be a significant positive correlation between a 

man’s attractiveness based on his physical features 

and body odor. 


Three packages of large plain white t- 

shirts (n=9) were purchased from Wal-mart in 

Laguna Niguel, California. Each shirt was then 

washed to remove any scent absorbed during 

production. The shirts were then packaged into 

Zip-lock double zipper plastic bags numbered 1-9. 

Each t-shirt was distributed to 9 different males 

ranging in age from 18-23. The shirts were worn 

for 3 nights by each male participant, allowing 

proper time for their scent to infuse into the 

material. Each shirt was then collected along with 

a current photograph of the participant, allowing 

for proper current portrayal of each individual to 

test for a correlation. Each male individual was 

numbered based on the t-shirt they were given, and 

each shirt correlated with a differently numbered 

picture to maintain consistent results throughout 

the rating process. 

The following day, 15 female participants 

were asked to rate each of the 9 male participants 

at random on a scale of 1 to 5 based on physical 

appearance, and then based on the scent of their t- 

shirts. The results were recorded by each female 

participant on a provided survey sheet. The 

following day, each male participant’s appearance 

and scent ratings were totaled up and averaged out 

to the nearest hundredth. 

Results were calculated and compared 

using Spearman’s rank correlation coefficient test. 

Data significance would be determined by having 

an r 2 value greater than 0 and less than 1, 

demonstrating a correlation in ratings between 

appearance and scent of each male individual 

rated. 

Results 

The correlation in mean ratings of each 

male individual based on appearance and scent 

(Fig 1). Results were calculated using Spearman’s 

rank correlation coefficient formula as: 

A correlation coefficient of (r = 3.6 x 10 -1 ) was 

calculated using the formula shown above, 

supporting our hypothesis. Raw averages for each 

individual male (Table 1) shows the range of 

scores. A value of p was calculated as (p > 1.0 x 

10 -1 ), rejecting the null hypothesis and supporting 

our hypothesis. Mean ratings based on appearance 

were 2.87, and mean ratings based on scent were 

2.82 (Table 1). Overall difference in mean values 

was + 0.05. Positive linear correlation between 

99 




both variables (r 2 = 1.3 x 10 -1 ) rejects the null 

hypothesis (Fig 2). Results supported our 

hypothesis that there would be a positive 

correlation between appearance and scent ratings 

of males when rated by females. 

Average 

Rating 

Appearance 

Average 

Rating 

Scent 

Person 

1 3.86 3.33 

Person 

2 2.46 3.33 

Person 

3 2.73 2.8 

Person 

4 3.33 2.6 

Person 

5 2.53 2.73 

Person 

6 3.06 2.53 

Person 

7 2.13 2 

Person 

8 2.33 2.8 

Person 

9 3.46 3.26 

Mean 2.87 2.82 

Table 1. Displaying mean values for each 

individual’s appearance and scent ratings. Values 

calculated based on ratings by 15 females. Mean 

values are shown at the bottom of each column, 

with an overall mean difference of 0.05 between 

the two variables.. 

Mean Ratings 

3 

2.5 

2 

1.5 

1 

0.5 

0 

1 

Appearance and Scent Values 

Appearan 

Scent 

Figure 1. Positive correlation of mean ratings 

between appearance (Filled bar) and scent 

(Slashed bar) of male individuals. (N 1 =9) based on 

ratings from females (N 2 =15). An r value of 3.6 x 

10 -1 shows positive correlation between the two 

variables. 

Average Rating 

5 

4.5 

4 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

0 2 4 6 8 10 

Individuals 

Figure 2. Mean ratings of scent and facial 

appearance in each individual on a scatter plot. 

Trend line shows linear correlation (r 2 = 1.3 x 10 - 

1 ) between both variables. 

Discussion 

The findings of this experiment support 

the hypothesis that a correlation exists between the 

attractiveness of a man’s facial features and the 

attractiveness of his body odor. There is a 

statistically significant relationship between these 

two factors in determining a male’s level of 

attractiveness to potential mates. This is shown by 

the correlation coefficient value of (r = 3.6 x 10 -1 ). 

Positive correlation is considered as any r-value 

such that 0 < r < 1. An r-value of 1 would indicate 

100% positive correlation between each of the 

variables being compared. 

Mate preference in H. sapiens has 

evolved in part because it produces MHC 

heterozygous offspring with enhanced levels of 

immune competency, which guards against a 

greater range of pathogens. Because MHC alleles 

are co dominant, the greater the difference 

between the alleles carried by the parent’s the 

greater the scope of antigen recognition by their 

offspring’s immune system (Brown, 1997). 

Passing on beneficial genes to offspring largely 

influences a females mate choice and her judgment 

of male attractiveness. Genetic dissimilarity is 

conveyed through both a man’s body odor and his 

facial features. Potential mates pickup these visual 

and pheromone cues, perceiving genetic 

dissimilarity as attractive. The attractiveness of a 

man’s facial features and the attractiveness of his 

body odor carry overlapping information; women 

prefer the smell of facially attractive men 

(Rikowski & Grammer 1999). There is a direct 

correlation between the attractiveness of a man’s 

odor and his facial attractiveness as perceived by 

females. 

d 


Looks 


Smell 

100 




Pheromones and facial features can also 

act as cues of avoidance, preventing potential 

problems inherent in inbreeding, these cues 

function as an evolutionary mechanism 

discouraging males and females from mating with 

those with similar genes (Penn & Potts, 1999). It is 

the genetic variation of the offspring that attraction 

works to promote. At the level of mate selection, 

attraction functions with natural selection in mind. 

By discouraging mating between closely related 

individuals, pheromones and facial cues influence 

genetic variation. Genetic variation may also drive 

another curious finding in women during the most 

fertile phase of their menstrual cycles consistently 

rated men with more masculine facial features as 

more appealing. Females were also more likely to 

demonstrate this increased desire for masculine 

traits if they were already involved in long-term 

relationships (Penton-Voak et al. 1999). 

A female’s desire for a mate with 

masculine facial features evidently increases if she 

is already pair-bonded with a male, a man with 

presumably less masculine traits. Mating with 

males out side of a female’s primary relationship 

increases the chances of genetic diversity within 

her brood while still maintaining a stable inflow of 

resources provided by her long-term mate. 

Evolution and morality may see eye to eye on the 

notion of inbreeding, but only because the former 

is served by the latter. In a mating situation like 

ours where genetic diversity is likely to increase 

out side of monogamy, morality is on its own. 

The correlation between a man’s facial features 

and body odor is related not only by attractiveness 

but also an underlying genetic dissimilarity. The 

androgens that influence bone structure and facial 

features during human development also present 

themselves as androgen-derived substances in 

body secretions (Grammer, 1993). The 

pheromones that are released by the body are in 

part derived from the androgens coded for by an 

individual’s genome, these cues convey 

information to potential mates, information that 

overlapped with the information convey by a 

man’s facial features. The combination of a man’s 

facial features and body odor advertise certain 

distinct genetic components, particularly his 

immunity genes. The overlap of information 

conveyed by body odor and facial features is a 

determining factor in the correlation that exists 

between the attractiveness of a man’s body odor 

and the attractiveness of his facial features. In this 

study the most physically attractive males were 

found to produce the most attractive body odor as 

determined by 15 college age females in Mission 

Viejo, CA. 


Brown JL, (1997). A theory of mate choice based 

on heterozygosity. Behavioral Ecology 8:60–65. 

Cunningham, M. R., Barbee, A. P. & Pike, C. L. 

(1990). What do women want? Facialmetric 

assessment of multiple motives in the perception 

of male facial physical attractiveness. Personality 

and Social Psychology 59, 61-72 

Grammer, K. & Thornhill, R. (1994). Human 

(Homo sapiens) facial attractiveness amd sexual 

selection: the role of symmetry and averageness. 

Comparative Psychology 108, 233-242. 

Herz, R. S., & Cahill, E. D. (1997). Differential 

use of sensory information in sexual behavior as a 

function of sex. Human Nature, 8, 275–286. 

Penn DJ, Potts WK, (1999). The evolution of 

mating preferences and 

major histocompatibility genes. American 

Naturalist 153:145–164. 

Penton-Voak, I. S., & Perrett, D. I., (2000). 

Female preference for male faces changes 

cylically. Evolutionary Human. Behavior 21, 39- 

48. 

Penton-Voak, I. S., & Perrett, D. I., Castles, D., 

Burt, M., Koyabashi, T. & Murray, L. K. (1999). 

Female preference for male faces changes 

cyclically. Nature 399, 741-742. 

Rhodes, G., Proffitt, F., Grady, J. & Sumich, A. 

(1998). Facial symmetry and the perception of 

beauty. Psychonomic Bulletin & Review 5, 659- 

669. 

Rikowski A, Grammer K, (1999). Human body 

odor, symmetry and 

attractiveness. Proceedings of The Royal Society 

Biological Sciences. 266:869–874. 

Thornhill, S.W. Gangestad, R. Miller, G. Scheyd, 

J. Knight and M. Franklin. (2003). MHC, 

symmetry, and body scent attractiveness in men 

and women. Behavioral Ecology 14:668-678. 

101 




1. THE EFFECT OF SODIUM CHLORIDE CONCENTRATION ON THE GROWTH OF THE 

COMMON BEAN (Phaseolus vulgaris). Alexandra M. Franco and Earl-Eugene E. Ringpis. 

Department of Chemistry, Saddleback College, Mission Viejo, California 92692. 

The majority of plants, including most crop plants, are glycophytes, which can only tolerate relatively 

small amounts of salt in the soil from which they grow. It is not expected that glycophytes will tolerate 

increases in salt concentration due to the resultant cellular water loss. Therefore, we hypothesized a 

negative, nonlinear relationship between salinity and growth in the glycophyte Phaseolus vulgaris. We 

tested the effect of increasing NaCl concentration on the growth of the common bean, P. vulgaris. Each 

of eight plant groups (N group = 3) was watered every 48 hours with 15 mL of a specific saltwater solution 

containing a different NaCl concentration (0.00% - 3.03% NaCl) over an experimental period of seventeen 

days (N = 24). Plant survival was significantly reduced when salinity was 1.68% and higher. The only 

experimental group to remain unaffected by its salt concentration was the group watered with a 0.20% 

NaCl solution. Each plant was measured for dry mass at the end of the experiment. Plant growth was 

significantly affected, and the effect of the increasing salt concentrations on growth produced a negative, 

nonlinear trend (R 2 = 0.212, F 1, 22 = 5.93, p-value = 0.023, linear regression). These results indicate a low 

salt-tolerance in P. vulgaris, thus supporting our hypothesis. 

2. PREFERENCE OF DOGS (Canis familiaris) FOR HIGH FAT DOG FOOD VERSUS LOW FAT DOG 

FOOD Chris J. LaCroix and Jocelyn A. Finley. Department of Biological Sciences, Saddleback College, 


The general purpose of this study was to discover if Canis familiaris, the common domestic dog, has a 

preference of high fat dog food over a low fat dog food. The high fat dog food chosen had a minimum of 

17% crude fat, while the low fat dog food had a labeled 6% crude fat content. The dogs were tested in a 

period of three trials, in which the time was recorded on how long it took the dog to favor one food over 

the other. After each trail was concluded, a twenty minute recess was given to reset the dogs mind about 

the food he or she had just received. The average time for the seven dogs to favor the high fat dog food 

over the low fat dog food was 4 minutes and 14 seconds. After collecting data, the discovered conclusion 

was that dogs actually favor a high fat dog food over a low fat; in addition, C. familiaris can distinguish 

between the two when presented with both at the same time. 

3. TESTING FOR INTELLIGENCE IN THE SLIME MOLD Physarum polycephalum Raiff Josey and 

Christopher Luna. Department of Biological Sciences, Saddleback College, Mission Viejo, California 

92692 

Intelligence has only ever been associated with a relatively small list of multicellular animals, and for 

plants, it is a debate between calling their behaviors intelligence or thoughtless instinct. The slime mold, 

Physarum polycephalum, an amoeboid organism, was tested for intelligence in an attempted replication 

of a 2000 experiment in which the Physarum polycephalum was “run” through a plastic maze atop a plate 

of plain agar gel between two agar blocks of oat nutrient. According to the 2000 experiment, the 

Physarum polycephalum is not only capable of navigating the maze, but “chose” the shortest route out of 

four possible, connecting the two agar blocks of oat nutrient, thus expressing a form of intelligence. Our 

hypothesis predicted that the slime mold would select one of the four routes between the two agar blocks 

of oat nutrient, thus displaying as the 2000 experiment did, that the slime mold expresses intelligence. 

However, due to the inadequate placement of Physarum polycephalum in the agar gel mazes, the slime 

mold did not interconnect to itself; therefore, it did not select any of the available routes between the two 

agar nutrient blocks. The failure of the slime mold to connect and “choose” a route between the agar gel 

nutrient blocks failed to yield any data that would allow us to test our hypothesis. 

102 




4. EFFECT OF pH ON RATES OF CLOSURE IN VENUS FLY TRAPS (Dionaea Muscipula). Jason R. 

Riggio Department of Biological Sciences, Saddleback College, Mission Viejo, California 92692 

Venus flytraps are peculiar in the fact that they can close their leaves completely in times of under a 

second. This does not seem very remarkable until one steps back and understands that plants do not 

have any muscle tendons or a nervous system. Scientists are still not exactly sure how this phenomenon 

occurs, however, the current (leading) hypothesis is a combination of acid growth, turgor pressure, and 

rapid cell division. The hypothesis tested in this paper, the effect of two pH’s on the rate of closure of the 

leaves, was simply to test the first part of the leading hypothesis, acid growth. This was done by picking 

16 leaves or “traps” from four different plants and randomly placed into sixteen 10ml beakers, eight 

beakers each filled with 5mL of HNO 3 , at a pH of four, and eight more each filled with 5mL of NaOH, at a 

pH of 10. The leaves were left in the solutions for 24 hours and then timed on individual rates of closure. 

Timing was done by use of digital camera and further analyzed on computer. Average rate of closure of 

leaves in HNO 3 was 5.487(±0.654) seconds and average rate of closure in NaOH was 1.814 (±0,654) 

seconds. Surprisingly, the leaves in the basic solution closed much more quickly than the leaves left in 

the acidic solution, putting the leading hypothesis into question. Several factors may account for this 

difference, such as age of the leaves or defective leaves. 

5. EFFECT OF TEMPERATURE ON PHOTOSYNTHETIC RATE OF Elodea canadensis. Aubrey Michi 

and Jeremy Ward. Department of Biological Sciences, Saddleback College, Mission Viejo, California 

92692 

We designed an experiment to measure the rate of oxygen production of Elodea canadensis at 

various temperatures. We hypothesized that as temperature increased, photosynthetic rate would 

increase. Three test tubes containing 0.1M NaHCO 3 solution and Elodea were kept in water baths of 5ºC, 

22ºC, and 37ºC. A light was shined on the test tubes for 30 minutes and oxygen production was 

measured by water displacement on each attached manometer. On average, during the 30 minute 

interval, the Elodea in 5ºC water produced 0.10mL of oxygen; the plant in 22ºC water produced 1.03mL of 

oxygen; and the plant in 37ºC produced 3.10mL of oxygen. Therefore, oxygen production increased with 

an increase in temperature. This arises from the principle of Brownian Motion, which explains that the 

random movement of molecules is related to their overall kinetic energy. We expect that an increase in 

photosynthetic rate will continue with an increase in temperature until a limit is reached either due to the 

stoma in the plant closing or the plant’s tissues denaturing from excessive heat. Nevertheless, our results 

suggest that increases in environmental temperature, such as those caused by global warming, may 

affect the photosynthetic rate of various aquatic plants. 

6. THE EFFECT OF GUAVA Psidium guajava AGAINST Escherichia coli.. Anne Kathreane Ebol and 

Hannah Rae Manuel. Department of Biological Sciences, Saddleback College, Mission Viejo, California 

92692 

The “poor man’s apple of the tropics”, guava, has been known to be a great natural remedy for 

diarrhea in various countries. For this reason, an experiment was set up to find out the effectiveness 

and strength of guava Psidium guajava leaves extract against one of the common bacteria that causes 

diarrhea, Escherichia coli, also known as E. coli. The experiment aims to verify the efficacy of guava 

extracts in combating bacteria and intends to find a nonchemical substitute to antiseptics in efforts to 

prevent an increase in dangerous, resistant strains of bacteria. The strength of the guava extracts was 

evaluated against the strength of isopropyl alcohol, a common household antiseptic, with a 91% 

concentration. Water was used a determinant in bacterial growth inhibition. Cultures of Escherichia coli 

were isolated in order to evaluate guava’s anti-microbial effect. After measuring inhibition zones, E. coli 

showed about equal propensity to the isopropyl alcohol and the guava extract. The guava extract 

returned an average inhibition zone of 1.1cm ± 0.15cm (±se), n=16, and the isopropyl alcohol returned 

an average inhibition zone of 1.1cm ± 0.07cm (±se), n=24. The guava extract was not able to inhibit 

bacterial growth entirely; however, according to the data collected, guava extract is just as strong as the 

91% isopropyl alcohol when it comes to bacterial growth inhibition. There was no significant difference 

in mean inhibition zones (p= 0.5; one-tail t-test). 

103 




7. THE EFFECT OF TEMPERATURE ON BACTERIAL COLONY FORMATION IN Escherichia coli. 

Anoush A. Garakani and Cassra Minai. Department of Biological Sciences, Saddleback College, Mission 

Viejo, California 92692 

Rapid bacterial growth is usually associated with warm temperatures. Although each species of 

bacteria have their own optimal environments for growth, it is critical to learn about those bacteria which 

can cause harm to humans. Escherichia coli is a very well known bacterium which has plagued the food 

industry due to its rapid generation time. It is crucial to know which temperatures are able to inhibit the 

growth of bacterial colonies in Escherichia coli in order to prevent sickness from food products both in 

restaurants and at home. Temperature is known to have a significant effect on bacterial growth, and since 

there is evidence that the optimal growth temperature for Escherichia coli is 37o C, it was predicted that 

the least bacterial colony inhibition would be at 37o C and the greatest inhibition would be at eight 

degrees centigrade. Means of colony formation for 37o C were (1655.5 ± 134.19, ± SEM), no colony 

formation was found for those incubated at eight degrees. To verify the presence of the bacteria in the 

eight degree plates, after complete inhibition was verified the eight degree plates were incubated at 37o C 

for 24 hours (1364.8 ± 128.32, ± SEM) there was no significant difference 37o C incubation and those 

incubated at 37o C after incubation at eight degrees (p > 0.05). These differences support the hypothesis 

that Escherichia coli can be inhibited at very low temperatures and can be of great use in the food 

preparation industry. 

8. CYNIPIDAE GALL DISTRIBUTION ON YOUNGER AND OLDER COAST LIVE OAK TREES, 

QUERCUS AGRIFOLIA. Brooke A. Hargis and Candice B. Archer. Departments of Chemistry and 

Biological Sciences, Saddleback College, Mission Viejo, California 92692 

The Gall Wasp (Hymenoptera, Cynipidae) induces the formation of plant galls. A gall is an 

abnormal plant tissue growth formation that results from the plant reaction to a growth-regulating 

chemical released by the wasp as it feeds on plant tissue. This newly formed gall serves as a safe 

shelter and a food source for the gall wasp larvae to develop. The “Plant Vigor Hypothesis” states that 

insect performance is better on faster-growing plants. Since younger trees have increased levels of 

cellular mitosis and overall vitality when compared to older trees, we predicted that gall counts would be 

higher among younger trees than among older trees. Galls were counted up to 2.13 meters above 

ground on a total of 30 trees. Of these 30 trees, 15 had a circumference of 50 cm or less, thus 

considered “younger”, and 15 had a circumference of 90 cm or more, thus considered “older”. The 

average number of galls per tree was significantly greater on older (5.7 ± 4.6 s.d.) trees than on younger 

(1.4 ± 0.74 s.d.) trees (p=1.2 x 10 -3 , one-tailed). Therefore, our results rejected the “Plant Vigor 

Hypothesis”. Instead, they supported an alternative hypothesis for the preference pattern of gall wasps, 

the “Plant Stress Hypothesis”. This hypothesis states that slower-growing plants contain more nitrogen 

and thus favor insect performance. We discuss our results within the context of this hypothesis. 

9. EXTRACTION OF IRON FROM BREAKFAST CEREALS. Claudia M. Shuldberg and Ximena M. Horne. 

Department of Biological Sciences, Saddleback College, Mission Viejo, California 92692 

Maintaining a nutritional diet is an important topic for every person’s health. Iron is a very important 

component to the human body since it affects all the bodies’ functions. Iron can be found in different kinds of 

food and it is advertised as a primary component on breakfast cereals. Since iron is a ferromagnetic element, 

it can be extracted from Total Whole cereal and Kellogg’s Product 19 cereal using a Teflon covered magnet. 

The iron extracted from both cereals was compared to test if both cereals have the same amount of iron per 

serving size. Out of 10g of Total Whole cereal 0.00601g ± 0.000130g (±se) of iron was extracted and 

0.00573g± 0.000130g (±se) of iron from Kellogg’s Product 19 cereal. A two‐tailed unpaired t‐test showed that 

there is not a significant difference of iron when comparing the two cereals (p= 0.0806). 

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10. OPERCULAR PUMPING RATES OF GOLDFISH (Carassius auratus) IN DIFFERENT 

CONCENTRATIONS OF GLUCOSE. Andrew J. Shires and Christopher J. Walsh. Department of 


It is widely known that temperature is one of the most important environmental factors affecting 

heterothermic ecotherms; this is demonstrated by Van’t Hoff’s Q10 rule. In our experiment we chose to 

focus on how different treatments of water affect the physiological states of goldfish. We believed that by 

treating water with glucose, through regular ingestion and respiration, there overall opercular pumping 

rate would raise. In day one, two tanks were used; one with regular treated water and the other treated 

with 2% glucose (300g). The tank containing regular water showed an average opercular pumping rate of 

69.13 ± 4.97 (S.E.M), while the glucose treated tank had average of 106.88 ± 4.18 (S.E.M). After letting 

the fish acclimate for 24 hours the average opercular rates decreased slightly in the glucose treated water 

to 106.25 ± 3.21 (S.E.M). On the final day after the tank had been treated with 4% glucose (600g) the 

opercular pumping rate decreased to 99.71 ± 2.95 (S.E.M). There was a significant difference between 

the glucose treated and regular treated water, (P=0.0098). 

11. EFFECTS OF TABLE SUGAR AND STEVIA SWEETNER ON BLOOD GLUCOSE LEVELS IN 

HUMANS. Shayda Haghgoo and Erin Kang. Department of Biological Sciences, Saddleback College, 


Obesity, diabetes, and heart disease take a larger role everyday in lives of Americans. What foods 

people eat can impact their health severely especially the consumption of sugars. Stevia sweetener is 

part of a plant that stands at a zero on the glycemic index. Given this information we proposed that 

Stevia to be a better alternative than table top sugar. Blood glucose meters were used to achieve this 

prediction. Testing of blood glucose on eleven subjects took place before and a half hour after the 

consumption of three teaspoons (13.08g) of table top sugar and one teaspoon (2.19g) of stevia 

sweetener (One teaspoon of stevia is equivalent to three teaspoons of sugar) each mixed with 150mL of 

water. In addition, there was a standards test in which the subjects only consumed 150mL of water. They 

were tested on different mornings, unable to eat breakfast to ensure that their blood glucose levels were 

low. Blood glucose levels resulted in table top sugar to have a mean difference of 18.09mg/d ±2.84 while 

stevia had a mean difference of -0.8182mg/dl ±2.10. There was a significant differenc in the blood 

glucose levels from table top sugar and stevia sweetener (p=1.31E-05). Through this experiment Stevia 

sweetener can be thought of as a noteworthy and beneficial substitute than sugar. 

12. GROWTH INHIBITON OF HYDROGEN PEROXIDE ON THE PATHOGENIC BACTERIA, 

PSEUDOMONAS AERUGINOSA. Angel R. Vargas. Department of Biological Sciences, Saddleback 

College, Mission Viejo, California 92692 

Hydrogen Peroxide (H 2 0 2 ), a household disinfectant and anti-fungal, has been recently approved by 

the EPA for use as the active ingredient in pesticides in concentrations not exceeding strengths of 35%. 

Pseudomonas aeruginosa, an opportunistic pathogen that induces dermatitis and pneumonia in humans 

and soft rot in some plants was the subject of this study in which hydrogen peroxide was tested to inhibit 

bacterial growth. Four concentrations of H 2 0 2 were tested (35, 20, 10, 3%), using D.I. water as a negative 

control. It was predicted that all concentrations of H 2 0 2 would limit some bacterial growth and that higher 

concentrations of H 2 0 2 would equate to greater inhibition. Growth inhibition was determined by measuring 

circular zones of inhibition (cm). All H 2 0 2 concentrations inhibited the growth of P. aeruginosa; the control 

showed no inhibition. Average inhibition measurements used to construct a regression analysis (R 2 = 

0.767 ± 0.541 SE, RF 1, 34 = 108.4 cm, P= 5.8 x 10 -12 cm ± .007cm SE) show a linear relationship between 

H 2 0 2 concentration and growth inhibition diameters in support of my hypothesis. These results support 

H 2 0 2 as an effective inhibitor against P. aeruginosa. 

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13. COMPARISON OF LACTATE DEHYDROGENASE ACTIVITY BETWEEN THE THRESHER SHARK 

(Alopias vulpinus) AND THE PIKE MACKEREL (Cololabis saira). Robert Powers. Department of 


During lactic acid fermentation that occurs in all animals and some plants, the enzyme Lactate 

dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate as NADH is oxidized to NAD + . 

Muscle samples were taken from A. vulpinus and C. saira to compare LDH activity between the two 

epipelagic fish. Both species are quite fast and powerful swimmers, however this species of shark is 

considerably larger than the pike mackerel, and because LDH activity has been found to increase as the 

mass of an organism increases, it was expected that the LDH activity would be considerably higher in A. 

vulpinus muscle than in C. saira muscle. A UV light spectrophotometric assay was applied to measure 

∆Absorbance/minute as the substrate NADH was oxidized, and from this data LDH activity could be 

calculated as mol of substrate converted per minute per gram of muscle extract. A. vulpinus had a 

mean LDH activity of 635.1 mol/min/g ±66.4 mol/min/g (±SE), N=5. C. saira had a mean LDH activity 

of 770.1 mol/min/g ±37.0 mol/min/g (±SE), N=5. The data actually shows that C. saira has a higher 

LDH activity than A. vulpinus. Although a one tailed t-test reveals that these results are not scientifically 

significant (p=0.063), it certainly seems that the original hypothesis has been invalidated. It appears that 

size is not the only factor that needs to be taken into account for varying LDH activity among different 

species, but furthermore a species’ unique habits and skills that nature has selected for as well. 

14. THE EFFECT OF A HAIR GEL ON THE GROWTH OF MALASSEZIA FURFUR. Hanwool Park. 

Department of Biological Sciences, Saddleback College, Mission Viejo, CA, 92677. 

Malassezia Furfur (syn. Pityrosporum ovale), a yeast-like fungus, occurring in human skin can cause 

diseases such as dandruff, pityriasis versicolar, seborrheic dermatitis, etc. Use of hair styling products 

may promote the growth of the fungus and cause the diseases. However, some manufacturers claim the 

preservative system, used to prevent the products from contamination by microorganisms, inhibits the 

growth of the microorganisms in the scalp. A hair gel product was predicted to inhibit the growth of M. 

furfur as producers claimed. Microorganisms were collected from a man’s scalp, and M. furfur was 

selectively cultivated on Sabouraud’s dextrose agar medium. By using disc diffusion method, a hair gel 

product was screened against the growth of the fungus. The fungi grew well, but unlike the prediction, no 

inhibition zones appeared around the discs. It was suggested that a hair gel did not inhibit the growth of 

M. furfur. 

14. THERE IS NO SIGNIFICANT OXYGEN PRODUCTION OF SPINACIA OLERACEA PLACED INTO 

CALCIUM CARBONATE SOLUTION. David Guzman. Department of Biological Sciences, Saddleback 


Calcium and Carbonate ions are important for plants. Plants can use Carbonate for CO 2 , and 

Calcium is an important nutrient of plants. If the Calcium and Carbonate combines it becomes insoluble 

in water, and this would not be beneficial for plants, so it might be logical if plants have mechanisms to 

dissolve the CaCO 3 if their ions ever combined in nature. Plants need CO 2 to photosynthesize and 

produce oxygen and sugars. The experiment tests whether CaC0 3 can be dissolved into ions and 

produce CO 2 by an unknown mechanism in spinach. If this happens, I predict that oxygen will be 

produced, and the experiment will test this idea that spinach can dissociate Calcium Carbonate. In the 

experiment, Spinach disks that were cut out by cork borers and then put Petri dishes. Then 0.05 M 

Calcium Carbonate was placed onto the Spinach to see if there was significant amount of oxygen 

produced during a twenty minute period while expose to artificial light. There was no significant 

difference between the control, de-ionized water, and the 0.05 M Calcium Carbonate solution in O 2 

produced (paired t-test, p=1.00.) 

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15. THE EFFECT OF MODERN ROLLER COASTER RIDE ON HUMAN HEART RATE. Yoko Kamei and 

Diana Nguyen. Department of Biological Sciences, Saddleback College, Mission Viejo, California 92692 

The human heart has an average heart rate of 72 beats per minute. In our experiment, seven females 

and seven males between the ages of eighteen and twenty-eight were selected in order to determine 

whether riding a modern roller coaster has a significant effect on human heart rate before and after the 

ride. The fourteen human subjects being tested had no history of cardiac disease. We measured the 

human heart rate using a chest and wrist heart rate monitor on each human subject. We recorded the 

heart rate beats/min before and after riding Disneyland’s California Adventure’s California Screamin’ roller 

coaster. As a result, the average heart rate beats/min of the tested human subjects before riding the roller 

coaster was 87.50 beats/min ± 3.47 beats/min (±se). The average heart rate beats/min after riding the 

roller coaster was 171.85 beats/min ± 6.64 beats/min (±se). In our experiment, the higher heart rate 

beats/min after riding the roller coaster is the tachycardia heart rate. As a result, the average human heart 

rate beats/min is significantly higher after riding the roller coaster than the average human heart rate 

beats/min before riding the roller coaster (p=3.66 x 10 -9 , two-tailed t-test). 

16. THE AFFECT OF ROUNDUP ® ON OXYGEN PRODUCTION OF RED ALGAE (RHODOPHYTA) 

Robert E. Maloney and Bianca Christensen. Department of Biological Sciences, Saddleback College, 


Red algae have been established by biologists to be an efficient oxygen producer. The effect of a 

pollutant such as the presence of a commonly used weed killer, on the ability of Rhodophyta to continue 

producing oxygen at normal levels sparked an interest for our study. It was predicted that the RoundUp 

(Scotts Co, Marysville ,OH) will affect the photosynthetic efficiency of Rhodophyta and that the 

experimental group would produce signifgantly lower rates of oxygen production than that of the control 

group. Rates of oxygen production from the control group were 6.64 ± 0.702 ppm and from the 

experimental group were 4.86 ± 0.575 ppm. These results do support our hypothesis by showing that the 

Experimental group does produce significantly lower rates of oxygen production than that of the control 

group. 

17. THE EFFECT OF SOIL pH ON THE GROWTH OF PHASEOLUS VULGARIS AND RAPHANUS 

SATIVUS. Lindy A. Ackerman and Brittany N. Lincoln. Department of Biological Sciences, Saddleback 


The pH of the soil greatly affects plant growth, affecting the availability and uptake of nutrients by the 

roots of the plant. In this experiment, the effects of different pH solutions on the growth of two common 

food crops: the common pole bean plant (Phaseolus vulgaris) and radishes (Raphanus sativus) were 

studied to determine the optimum pH for cultivating these crops. It was hypothesized that there would be 

a significant difference between the growth of these plants, separately, at pH 3 and pH 9. It was 

determined that there was no significant difference between the growth of beans (Phaseolus vulgaris) at 

pH3 and pH9 (p=0.41, unpaired, two-tailed t-test). It was also determined that there was no significant 

difference between the growth of radishes (Raphanus sativus) at pH 3 and pH 9 (p=0.05, unpaired, twotailed 

t-test). 

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18. IMPACT OF RED AND GREEN LIGHT ON GROWTH RATE OF SNAPDRAGONS (Antirrhinum 

majus). Nicholas Schmidt and Bobby Stangl. Department of Biological Sciences, Saddleback College, 


One type of electromagnetic radiation is visible light. Visible light is absorbed by pigments in the 

chlorophyll of plants as the energy source for photosynthesis. These chlorophyll pigments absorb red and 

blue light and reflect green light. The purpose of our experiment was to observe and record the affects of 

red and green light on the growth rate of Antirrhinum majus over a time period of thirty days. We used a 

total of twelve Antirrhinum majus plants organized into four pots. Using a random numbers table we 

assigned two pots to constructed red and green cellophane tents. For each plant soil to top of stem 

measurements where recorded before and after the thirty days time period. After thirty days each plant 

was then unearthed to its roots end and dryed at 37.0 C for twenty four hours. Mass measurements 

where recorded using a gram balance after 24 hour drying period. We than ran a two-tailed t-test, which 

showed there was not a statistically significant difference, p=0.262, between the two colors of tents. We 

can conclude from our collected data that red and green light where not significant factors on the growth 

rate of Antirrhinum majus’. 

19. EFFECTS OF ENVIRONMENTAL TEMPERATURE ON THE SPEED OF COMMON GARDEN 

SNAILS (Helix aspersa). Grady S. Counts and Eric T. Rueda. Department of Biological Sciences, 

Saddleback College, Mission Viejo, California 92692 

The common garden snail, Helix aspersa, is often found in temperate environments. It’s affinity for 

dank, cool conditions did not surprise us, since our specimens were most active in the early morning 

around 0600 to 0700 hours. Studies on terrestrial snails have been steadily increasing over the past few 

years focusing on speed, mating habits, diet and often general observation. The main factor in our study 

was the effect of temperature on the speed of the snail H. Aspersa. Fifty snails were acquired and tested 

under two environmental conditions, a mean cold temperature of 9.7ºC ± 0.07ºC (±se), and a mean hot 

temperature of 38.2ºC ± 0.1ºC (±se). Distance was measured (cm) over a period of sixty seconds. The 

average speed was calculated for each snail. The mean speed for the cold environment was 0.1 cm/s ± 

4.0x10 -3 cm/s (±se) and the mean speed for the hot environment was 0.07 cm/s ± 3.0x10 -3 cm/s (±se). 

The mean cold temperature speed was significantly greater than the mean hot temperature speed (p= 

2.59x10 -6 , paired one-tailed t-test). 

20. THE EFFECT OF PH ON THE RESPIRATION RATE OF GOLDFISH (Carassius auratus). Hilda 

Gonzalez and Natsumi Iwata. Department of Biological Sciences, Saddleback College, Mission Viejo, 

California 92692 

In nature, fish species face many difficulties including those of gas exchange due to variable oxygen 

tension in the aquatic habitat in which they live in. Under poor conditions like oxygen depletion or low 

environmental temperatures, some fish species can slow down their breathing rate in a manner as to 

develop their own special adaptation abilities to aid them psychologically over time of drastic changes. 

Oxygen concentration shows the most concern as a consequence in aquatic habitats, and such like the 

goldfish, Carassius auratus, it was expected that the pumping rate of goldfish under acidic conditions, 

where oxygen saturation is low, would be lower than that of the basic conditions, where oxygen saturation 

is high. To explain these close couplings between acidic and basic, techniques were performed. The 

goldfish were first placed in an aquatic tank with low oxygen saturation being acidic that of which consists 

of a pH of 4.0, and after two hours of acclimation were used to determine their pumping rate. The same 

procedure was also used to assess the pumping rate of goldfish in an aquatic tank of basic solution with a 

pH of 9.5, where oxygen saturation is higher. Rates of respiration among the goldfish, C. auratus showed 

significant changes between the acidic pH (mean = 52.59±0.01 rate/min, p = 13.52± 0.01) and the basic 

pH (mean = 138.95±0.01 rate/min, p = 6.92±0.01). The pumping rate of goldfish resulted in significant 

changes due to hypoxia known to occurs in aquatic environments. This statistical analysis indicated that 

the rate of respiration in goldfish was determined by environmental oxygen concentration or oxygen 

consumption in the water. 

108 




21. DETERRENT EFFECTS OF PEPPERMINT OIL IN MICE. Tommy L. Roberts and Emmanuel 

Romero. Department of Biological Sciences, Saddleback College, Mission Viejo, California 92692 

Evolutionary changes between prey and predators cause continual changes in species in order to 

survive in their environment. Peppermint (Mentha x piperita) plants produce oil that has been used for 

medicinal purposes in the human society, but what is the original intention for this substance? The 

following experiment observed behavioral choices of mice to support the hypothesis that peppermint oil, 

found in peppermint plant leaves, has the ability to protect the plant by deterring potential predators, 

which may be harmful to the plant. It was predicted that when mice were placed into a maze which 

contained paths with and without peppermint oil, that the mice would avoid the path containing 

peppermint oil. The mean frequency of paths chosen with no peppermint oil was 0.75 + 9.9x10 -2 while 

the mean frequency for paths with peppermint oil was 0.25 + 9.9x10 -2 (two-tailed p=1.0x10 -3 ). Tests 

showed a significant difference in choice between paths containing no peppermint oil versus those that 

do. 

22. THE EFFECT OF ROLLER COASTERS ON HEART RATE IN RELATION TO GENDER. Samantha 

T. Dinh and Cole P. Lyou. Department of Biological Sciences, Saddleback College, Mission Viejo, 


The hypothesis being tested was to determine whether or not there was a change in heart rate in 

relation to gender on the Silver Bullet at Knotts Berry Farm Amusement Park, Buena Park, CA. Twentyfive 

males and females were selected and their heart rates were taken using the palpation method before 

and after riding the Silver Bullet. If there was a difference in heart rate between males and females, there 

may need to be different precautionary warnings for theme park attendees before ridings any rides. From 

the results, different amusement parks can determine whether or not it would be a liability to include 

certain rides from their theme parks because of extreme changes in heart rate for those who have 

underlying cardiac disease could raise risks. Also, regulation and awareness can be raised by the theme 

park companies warning riders of any risk for riding the roller coaster based on their gender. Average 

change in heart rate for males, from before to after the roller coaster, was 21.8 bpm ± 1.1bpm (±se), 

N=25. Average change in heart rate for females, from before to after the roller coaster, was 29.2 bpm ± 

1.3bpm (±se), N=25. From the data there is not a significant change in the mean of the heart rates, from 

before to after the ride, between males and females. 

23. THE COMPARATIVE COST OF LOCAMOTION IN FEEDER MICE (Mus musculus) AND 

ROBOROVSKI HAMSTERS (Phodopus roborovskii). Harris M. Elhan and Tracy L. Kubas. Department of 

Biological Sciences, Saddleback College, Mission Viejo, California USA. 

While running, mice and hamsters exhibit different mechanical approaches to locomotion. Hamsters 

were observed running with higher stride frequency and less vertical movement then the mice. While 

studies have shown that the cost of locomotion is related to the size of an animal, the biomechanical 

difference present between mice and hamsters may indicate a difference in the cost of locomotion. The 

VO 2 of mice (n=8) and hamsters (n=8) was determined by running each species on a treadmill until a level 

percent O 2 consumption was reached. The average metabolic rate for mice was found to be 0.548 mL 

O 2 /g/min ± 0.0650 (S.E.) while for hamsters the average metabolic rate was 0.717 mL O 2 /g/min ± 0.104 

(S.E.). The metabolic rates observed demonstrate no significant difference in the cost of locomotion for 

mice and hamsters. This would indicate that differences in locomotive mechanics, between the species, 

do not effect metabolic rate. 

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24. EFFECT OF CHLORINE ON ALGAL GROWTH. Bobby Fujimoto. Department of Biological Sciences, 

Saddleback College, Mission Viejo, California 92692 

Algae growth is one of the principle problems of swimming pool care. Sodium hypochlorite 

(NaOCl) is generally used to keep free chlorine levels at three parts per million (3ppm) to lyse algae cells. 

The aim of this experiment is to discover if chlorine is significantly more effective than chloramine at killing 

algae. Six experimental tubs were filled with 500mL of chlorinated water. One additional control tub of 

500mL of the original creek water was also used in the experiment. The creek water came from where 

the algae was sitting in before it was collected. The rest of the water came from a pool and was 

measured with a pH of 7.6 and 3ppm free chlorine. The first three tubs were left with 500 mL of the 

aforementioned water. In addition to the 500mL of aforementioned water 10mL of ammonia was added 

to the last three tubs and they were labeled accordingly. Then, 50g of algae was added to each of the 

tubs. No algae died in the control tub. The mean weight of dead algae in the pure free chlorine tubs was 

26.960 +3.201g where N=3. The mean weight of dead algae in the free chlorine and ammonia tubs was 

4.983 + 3.432g where N=3. A two sample t-test run between the two experimental groups showed 

significance. The null hypothesis that pure free chlorine is as effective as or less effective than 

chloramines was rejected where p


27. THE EFFICACY OF GARLIC (ALLIUM SATIVUM ) AS A BIOLOGICAL CONTROL OF MOSQUITO 

LARVA. Roky Coria. Department of Biological Sciences, Saddleback College, Mission Viejo, California 

92692 

Garlic (Allium sativum) has been cultivated by humans for thousands of years and has been valued by 

many civilizations as an important spice with medicinal properties. Allium sativum has evolved a defense 

mechanism to protect itself from bacteria, fungi, insects and other animals. These properties of A. sativum 

suggest a potential application as an effective mosquito larvicide. The objective of this study was to 

evaluate the rate of effectiveness of raw garlic against mosquito larva and compare this to the rate of 

effectiveness of a commercial larvicide. Data was collected on the deaths per day of mosquito larva 

exposed to a garlic solution, and mosquito larva exposed to commercial larvicide. Both garlic and the 

commercial larvicide, MosquitoDunks®, were toxic to the mosquito larva. However, the mean number of 

deaths per day in the larva exposed to garlic solution was significantly less than the number of deaths per 

day in the group exposed to MosquitoDunks®. The results of the study indicate that the rate of 

effectiveness of the garlic solution is not significant compared to the effectiveness of MosquitoDunks® 

(p=.92409). The rate of effectiveness of the garlic solution would vary depending on the concentration 

used. Therefore, further research on the effectiveness of garlic at different concentrations is necessary to 

evaluate garlic’s larvicidal properties against mosquitoes. 

28. EFFECTS OF AUDITORY AND CHEMICAL STIMULI IN MAMMALIAN MEMORY (Mus musculus). 

John Lowd and Charles Steinfeld. Department of Biological Sciences, Saddleback College, Mission Viejo, 


Memory is increasingly vital to production and efficiency in today’s society. There are several over the 

counter remedies for memory enhancement, we tested the product Leovit produced by Leovit Nutrio 

(Russia). We also tested the effect of predatory stimuli on mouse memory. It was hypothesized that the 

mean time to complete the maze for the mice treated with the Leovit memory enhancing supplement 

would be significantly lower than for the group of mice that received no treatment. It was also 

hypothesized that the group of mice that were exposed to Western Screech Owl (Megascops kennecotti) 

calls would have a significantly higher mean time comparative to the control group receiving no treatment. 

Mean time for the control mice to complete the maze was calculated (200.2 seconds ± 25.3 s.e.) After 5 

days of treatment the mice treated with Leovit achieved a mean time (154.2 seconds ± 19.2 s.e.) this 

difference was not considered statistically significant (p=0.141, two-tailed t-test assuming unequal 

variance). After receiving treatment for ten days, the Leovit Group’s mean time (118.9 seconds ± 6.1 s.e.) 

was considerably lower than the control group’s mean (159.7 seconds ± 13.2 s.e.). These values are 

considered to be statistically significant (p=.006, two-tailed t-test assuming unequal variance). The mean 

time for the group of mice receiving auditory stimuli for the 2 nd and 3 rd trials (206.8 seconds ± 21.6 s.e. & 

182.4 seconds ± 11.7 s.e.) respectively, was higher than the control group’s mean, however, these values 

were not considered statistically significant (p=1.8, two-tailed t-test assuming unequal variance for both 

trials). 

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29. A COMPARISON OF SUCROSE AND STEVIA EXTRACT AS A SUBSTRATE FOR YEAST 

METABOLISM. Sally A. Hutson. Department of Biological Sciences, Saddleback College, Mission Viejo, 


Stevia is a natural non-caloric sweetener widely used in Japan. Its use is growing among many 

countries including the United States. Stevia is gaining popularity because of the positive effects it has on 

health. In this study, support for the use of Stevia by individuals prone to chronic yeast infections was 

researched. Fermentation reactions were carried out in 24 tubes inside an incubator set at 37°C for an 

hour. The volume of CO 2 produced from a 10% sucrose solution and yeast was compared to the volume 

of CO 2 produced by a 10% solution of Stevia and yeast. The mean amount of CO 2 produced for sucrose 

was 2.58mL ± 0.98mL (±se), n=8, and the mean for Stevia was 0.68mL ± 0.45mL (±se), n=8. The 

amount of CO 2 produced by the reaction between sucrose and yeast was significantly greater than the 

amount of CO 2 produced by Stevia and yeast (p=2.9x10 -4 , one-tailed t-test). One interesting note is that 

Stevia which is known to have antimicrobial properties, actually reacted with yeast in small amounts, and 

the time it took the yeast to metabolize Stevia was longer in comparison to sucrose. The conclusion of 

the study was that Stevia is still a good alternative sweetener. 

30. EFFECTS OF PESTICIDE ON OXYGEN PRODUCTION RATES OF RED ALGEA (Aharghiella 

tenera). Natasha V. Polanski. Department of Biological Sciences, Saddleback College, Mission Viejo, 


I predicted that the exposure to pesticide would significantly decrease oxygen production rates in the 

common red algae, Aharghiella tenera. Ortho Basic Solutions Lawn & Garden Insect Killer, purchased at 

Home Depot, was used as the pesticide. Two separate tanks were set up with 1800mL of seawater, each 

containing 15g. of red algae and an aerator to circulate the water. A blue ultraviolet light, set on a timer to 

simulate natural daylight, was positioned above the tanks. 12mL of Ortho Basic Solutions Lawn & Garden 

Insect Killer pesticide was introduced into the environment of the experimental tank. Readings were 

conducted daily for seven days to test pH levels, salinity, visual inspection, and dissolved oxygen. pH 

levels were measured using a Hanna meter and maintained an average of 8.4 ± 0.01(S.D). A hand 

refractometer was used to check salinity of the water and its density maintained an average of 1.024 

4x10 -7 (S.D.). Temperature was steady at 23°C. Dissolved oxygen was measured using a Winkler Titration 

kit. Dissolved oxygen in the control began at 11.4ppm and ended at 7.2ppm with an average of 8.9 

2.1(S.D.). Dissolved oxygen in the exposed tank began at 10.8ppm and ended at 5ppm with an average 

of 6.7 4.6(S.D.). The color of the exposed algae lost significant amount of color. It turned from red to a 

faint pink, with some tips fully white, and a slippery milky film developed over the plant. The exposure to 

pesticide significantly negatively affected the visual health of the algae, but only moderately negatively 

affected the oxygen production rates. 

31. POST FIRE PLANT FREQUENCY IN NORTH AND SOUTH SIDED HILLS IN SANTIAGO CANYON. 

Department of Biological Sciences, Saddleback College, Mission Viejo, California 92692 

Scientific papers note that north facing hills appear to be less affected by fires than south facing hills in 

Southern California fires. If north facing hills tend to be less affected by forest fires than south sided hills, 

it would be advisable to build on north facing hills rather than south sided hills to prevent loss of private 

property. To test this, plant frequencies in north and south sided hills affected by the 2007 October 

Santiago Canyon fires were measured using squared meters. Sixty plots were randomly measured, 30 

from north sided hills, and 30 form south sided hills, at an altitude of approximately 2,200 feet. The 

average number of plants per square meter in north sided hills was 26 and in south sided hills the 

average was 11. There’s a significant difference between the two sides (p= 0.000722), with north sided 

hills having higher plant densities in Santiago Canyon after the forest fire. 

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1. EFFECTS OF SPIRULINA ALGAE VERSUS REGULAR FISH FLAKES ON METABOLISM IN BETTA 

FISH (Betta splendens). Thao Nguyen and Nelson Huang*. Department of Biological Sciences, 

Saddleback College, Mission Viejo, California, 92692, U.S.A. 

The betta fish, Betta splendens, is one of the world’s most popular species of freshwater aquarium 

fish, and are in popular demand in markets due to their attractive, vibrant aggression. Respiration is the 

driving mechanism behind all animal activity. Oxygen absorbed into the animal body allows oxidative 

respiration to be received by electrons freed in the degradation of glucose and other energy rich 

carbohydrates. With this objective, two types of fish foods were tested: Algae discs (Wardley Premium 

Spirulina Algae, Wardley), and regular tropical fish food (TetraColor, Petsmart). Six betta fishes were 

used this study and divided into two groups of three. One were fed Algae discs while the other group 

were fed regular Tetra fish flakes, over a feeding trial of once a day for one week. The opercular pumping 

rate was measured afterwards, and results showed modest significant difference in metabolism between 

the two food types. The purpose of this study was to show that Spirulina Algae discs has a potential 

natural supplement rich in raw protein and seven major vitamins that could improve immune function in 

both human and fish species. 

2.ASCORBIC ACID CONCENTRATION OF ORGANIC AND CONVENTIONALLY GROWN PRODUCE. 

Janelle Reed and Irina Alexandrova*. Department of Biological Sciences, Saddleback College, Mission 

Viejo, California, 92692, USA 

There are increasing numbers of health concerns surrounding diet and life style. Organic food has 

quickly become a preferred choice for those concerned with eating a healthy and balanced diet. The 

purpose of this research is to show that organically grown fruits and vegetables provide higher 

concentrations of ascorbic acid (Vitamin C) than inorganically grown. Ascorbic acid is an essential 

antioxidant and is required for the synthesis of collagen, carnitine, and neurotransmitters, which are vital 

for proper brain function. Vitamin C is produced by most mammals except humans; therefore, it is 

essential to obtain the vitamin through food molecules. Samples from Frageria ananassa (strawberries), 

Malus domestica (red apple), and Spinacia oleracea (spinach) were measure for ascorbic acid 

concentration via redox titration. Results for organic strawberry yielded the most significant difference with 

97.4% more ascorbic acid absorption than their inorganic counterparts. Spinach measured the least 

amount of difference for ascorbic acid with 25.8% in favor of organic. Results demonstrated that organic 

foods have a much higher absorption rate of vitamin C than conventionally grown produce. The 

significance of this study is to provide conclusive evidence that ascorbic acid concentration of various 

fruits and vegetables grown organically and inorganically differ significantly. 

3. ANTIBACTERIAL EFFECTS OF RED WINE ON ORAL BACTERIA. Kevin Murray and Arash 

Moghaddam. Department of Biological Sciences, Saddleback College, Mission Viejo, California, 92692, 

USA 

This study investigates the effect of red wine on oral bacteria. Red wine is known to have certain 

other health benefits. Red wine has some antibacterial effects. If red wine could reduce the amount of 

oral bacteria it could have some more health benefits such as the prevention of oral born pathogens. The 

claim is that things other than alcohol in the red wine are responsible for its antibacterial activity. A 

control was run using ethanol which was 13% alcohol by volume, the same as the red wine used. Twenty 

dishes were colonized with bacteria, ten contained discs soaked in red wine and the other ten contained 

discs soaked in the ethanol. There were four discs in each disc, 40 red wine discs and 40 ethanol discs 

(80 discs total). Only two dishes, out of the twenty, had zones of inhibition. Dish two, which had red wine 

discs in it, had an average zone of inhibition of 0.525mm ± 0.17mm (s.e.m.). Dish twelve, which had 

ethanol discs in it, had an average zone of inhibition of 0.150mm ± 0.03mm (s.e.m.). Although, the red 

wine had a greater average zone of inhibition a t-test gave a value of 0.06, which indicates no significant 

difference. Due to the low N value the results were determined to be insignificant and inconclusive. The 

isolated bacteria was found to be a Gram negative coccus. 

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4. THE EFFECT OF PYRUVATE ON OXYGEN CONSUMPTION OF MUS MUSCULUS. Izaak Miller and 

Lancelot Beier. Department of Biological Sciences, Saddleback College, Mission Viejo, California, 92692, 

USA 

Seeking to increase metabolism, pyruvate has been used in recent years to aid weight loss. Pyruvate 

is a primary intermediate molecule in oxidative metabolic pathways. Supplementation of pyruvate 

through ingestion should increase metabolic rate and therefore increase oxygen consumption in Mus 

musculous. The oxygen consumption of seven mice was tested before and after pyruvate 

supplementation. Oxygen consumption was measured via high flow respirometry. The oxygen 

consumption of the mice after supplementation was not significantly greater (P = 0.194) than that of the 

mice before supplementation. 

5. EFFECT OF LIGHT ON CHLOROPHYHLL CONCENTRATION IN ENGLISH IVY (Hedera helix). 

Michael G. Moeller. Department of Biological Sciences, Saddleback College, Mission Viejo, California, 

92692, USA 

Not all leaves can receive equal amounts of sunlight through out the day. Some plants grow in the 

shade, some have a large amount of leaves hidden within their own foliage and some have a large 

amount of light all day. Differences in the amount of light received by the groups of leaves may account 

for differences in chlorophyll amounts. Overall, leaves growing under intense light display the following 

characteristics: a higher mesophyll width, cuticle and photosynthetic rate. Ivy was examined that had 

been grown in the shaded areas and also in areas receiving sunlight. After extracting the chlorophyll from 

the leaves samples were ran using a spectrophotometer giving the data that the average chlorophyll 

concentration in shaded plants was 2.38 mg/L (SE ± 0.10) and plants grown in the sun had an average of 

1.74 mg/L ( SE ± 0.06). The data obtained showed that shaded English Ivy has a significantly high 

chlorophyll content than English Ivy that grows in the sun (P < 0.05, P=0.0002). 

6. SENSITIVITY OF PATHOGENIC BACTERIA STAPHYLOCOCCUS AUREUS TO HONEY. Anne 

Merielle Ebol and Shanon Carney*.Department of Biological Sciences, Saddleback College, Mission 


The ability of Staphylococcus aureus to change its chemical makeup made it become one of the 

antibiotic-resistant pathogens present in time. Its increased resistance to most commonly used antibiotics 

such as Penicillin, Methicillin and Vancomycin led to efforts in developing new compounds that would 

prevent its further proliferation. The use of honey in ancient times for treatment of diseases brought 

renewed interest in the utilization of the substance for the management of infections. Manuka honey from 

the wild tea tree Manuka bush in New Zealand has been recognized for its therapeutic properties and its 

inhibitory effect on the growth of different types of pathogens. The antibacterial action of honey is 

dependent on its high osmolarity. The amount of sugar content of the solutions used was a determinant 

in bacterial growth inhibition. It was predicted that any type of pathogen would be susceptible at a certain 

extent to the bactericidal effect of honey. Cultures of Staphylococcus aureus were used to assess this. 

The bacterial species showed more susceptibility to the sugar concentration of sucrose than to the sugar 

concentration of Manuka honey. Although honey was not able to completely inhibit bacterial growth, it 

still had some effect on the sample tested. The average zone of inhibition measurement for sucrose was 

0.35 cm ± 0.10 cm (±se) and the average measurement for honey was 0.15 cm ± 0.07 cm (±se). 

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7. THE EFFECT OF TEMPERATURE ON PHOTOSYNTHETIC RATE OF ELODEA CANADENSIS. 

Kaung Ko and Spencer Roberts. Department of Biochemistry, and Department of Biological Sciences, 

Saddleback College, Mission Viejo, California, 92692, USA 

Photosynthesis is a biochemical process necessary for survival of plants. During the initial process of 

photosynthesis light reactions use water and sunlight to produce ATP and NADPH. This energy is used 

to power the following Calvin Cycle process where CO 2 consumed by carbon fixation is then used to 

produce glucose, energy for plants, and oxygen, energy for other life forms. Temperature is one of the 

most critical environmental factors affecting the rates of photosynthesis of various plants. In this 

experiment the oxygen production rates of eight aquatic plants, Elodea canadensis, were measured at 

warm (25 o C) and cold (5 o C) temperatures to test the hypothesis that the oxygen production rates will be 

significantly higher for the Elodea in warmer temperature than in lower temperature. In the warm 

temperature, oxygen was produced at 0.35 mL/min, 0.46 mL/min, 0.40 mL/min, 0.64 mL/min, 0.63 

mL/min, 0.71 mL/min, 0.79 mL/min, and 0.86 mL/min for the eight plants. Under the cold temperature the 

plants produced 0.02 mL/min, 0.09 mL/min, 0.07 mL/min, 0.29 mL/min, 0.09 mL/min, 0.11 mL/min, 0.18 

mL/min, and 0.08 mL/min. Comparing the oxygen production rates at the two different temperatures, the 

two-tailed T-test gives p=8.39x10 -6 (N=8). The p-value less than 0.05 indicate the oxygen production rate 

at the higher temperature is significantly higher than that at the colder temperature. 

8. THE EFFECT OF WEIGHT LOAD ON THE OXYGEN CONSUMPTION OF PARAKEETS DURING 

TERRESTRIAL LOCOMOTION. Y Leho and Christopher Triana. Department of Biological Sciences, 

Saddleback College, Mission Viejo, California, 92675, USA. 

Birds are one of many species of animals which must hunt and gather food, and during the process, 

carrying weight is often times necessary. Melopsittacus undulatas is a small bird species and were used 

in this study. Five parakeets were induced to walk on a treadmill without any additional weight, then a 

4.01 g weight was added and the parakeets were walked on the treadmill again. The oxygen consumed 

by the parakeets (labeled 1-5) without weight was 0.994 ± 0.0591(±se), 1.476 ± 0.0101 (±se), 2.414 ± 

0.0181 (±se), 1.426± 0.0171 (±se), and 1.469 ± 0.0107 (±se), respectively. The amount of oxygen 

consumed with added weight was 1.093 ± 0.0327 (±se), 1.479 ± 0.0079 (±se), 2.409 ± 0.0165 (±se), 

1.444 ± 0.0163 (±se), and 1.461 ± 0.0124 (±se) for the parakeets (labeled 1-5), respectively. All values 

were measured in ml of . There was a general increase in the metabolic rate; however there was not a 

significant increase. The one tailed t-test yielded a p-value of 0.219 which suggests that metabolic rate 

was not significantly affected due to weight load increase. 

9. EFFECT OF SUCRALOSE AND SUGAR ON BLOOD GLUCOSE IN HOMO SAPIENS. Valerie 

Bowen and Samantha Lopez, Department of Biological Sciences, Saddleback College, Mission Viejo, 

California, USA 

Sucralose is a known sugar substitute that is 600 times sweeter than sucrose, but doesn’t affect 

blood glucose levels. To determine if the change in blood glucose levels after the consumption sucrose is 

significantly higher than the change in blood glucose levels after the consumption of sucralose in Homo 

sapiens. Seven H. sapiens were used in the study and the blood glucose levels were recorded after 

consumption of sucrose and sucralose. After comparing the average change of blood glucose levels after 

consumption of sucrose and sucralose it was determined that the change in blood glucose levels after 

consumption of sucrose was not significantly higher than the change in blood glucose levels after the 

consumption of sucralose (p=0.07146, t-test: two-sample assuming unequal variances). 

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10. COMPARISON OF LACTATE DEHYDROGENASE ACTIVITY IN SKELETAL MUSCLE OF BLUE 

ANCHOVY (ENCRASICHOLINA DEVISI) AND COW (BOS TAURUS). Sherri Burnett* and IxChel Cruz- 

Gonzalez*. Department of Biological Sciences, Saddleback College, 28000 Marguerite Parkway, Mission 

Viejo, California, 92692 USA 

Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the reaction of pyruvate to lactate 

during anaerobic respiration. Muscle was collected from both B. taurus, hind leg muscle and from a E. 

devisi to compare LDH activity rates. B. taurus is a large homeothermic endotherm and E. devisi is a 

small homeothermic ectotherm. Glycolytic enzyme activity in both endotherms and ectotherms has been 

found to increase as the mass of the animal increases. Due to relative size of both animals, it was 

expected that the muscle B. taurus should display a higher level of LDH activity then the muscle of E. 

devisi. A spectrophotometer was used to conduct an assay that measured the oxidation of NADH. LDH 

activity was calculated from the results and expressed as µmol of substrate converted per minute per 

gram of wet weight of muscle sample. Conducting three trials for each muscle, the B. Taurus had an 

average rate of 13.7 µmol/min/g ± 2.8 µmol/min/g (±se), N=3. The E. devisi had an average rate of 14.2 

µmol/min/g ± 1.5 µmol/min/g, N=3. A one-tailed t-test revealed that the E. devisi did not have a 

significantly higher LDH rate than that of B. taurus (p=.44). The results indicate that scaling between 

endotherms and ectotherms do not follow the scaling patterns found between animals of similar types. 

11. EFFECT OF PH ON GERMINATION RATE IN LIMA BEANS. Krystle Salazar and Matt Apke, 

Department of Biological Sciences, Saddleback College, Mission Viejo, California, 92675, USA. 

Seed Germination is a process in which plants emerge from dormancy. Germination is completed 

when part of the embryo and the radicle break out of the seed. We are testing if acids, pH 3 and pH5 will 

affect the germination rate of lima beans. However, pH 5 is less acidic than pH 3. This suggests that if 

pH 5 does in fact germination rate it may be the acid levels that make the seeds dormant. Although the 

phenomenon of dormancy is studied by many researchers, their studies are broken due to the fact that a 

different form of germination exists in different species. Before considering that the lima bean is dormant, 

we must first know the process that makes the seed germinate. Germination begins when the dry seed 

takes in water and is completed when the radicle penetrates through the walls of the seed surrounding it. 

We can assume that the pH 7 will make the seeds grow, but can we also assume that the pH 3 and/or the 

pH 5 is absorbed into the seed or will the seed take what it needs from the acids to allow itself to 

germinate. 

12. OXYGEN PRODUCTION OF BROWN ALGAE (Egregia laevigata) AND RED ALGAE (Gelidium 

robustum) IN DIFFERENT COLORS OF LIGHT. Saori Shimamoto and Yuriko Kayama. Department. of 

Biological Sciences, Saddleback College, Mission Viejo, California 92692, USA. 

Algae produces oxygen by the photosynthesis. In the deep sea, longer wavelength red light is absorb 

in shallow water, and only shorter wavelengths, such as blue light penetrate into deep water. Therefore, 

algae should have a variety of pigments adapted to absorb a wavelengths associated with their depth. 

The hypothesis is that the red algae, Gelidium robustum, will utilize red light efficiently. In brown algae, 

Egregia laevigata, which occurs at greater depth, we predicted that they will use blue and red light. The 

results showed that red algae produced the largest amount of oxygen in red light 0.554 ± 0.220 ml/g 

(±se). In brown algae, the most efficient oxygen production was in blue light (0.193 ± 0.066 ml/g ±se) and 

green light (0.189 ± 0.103 ml/g ±se). The results showed the participation of two photosynthetic pigments: 

phycoerythrin in red algae and fucoxanthin in brown algae. Pigments in algae are important to carry out 

efficient photosynthesis. 

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13. THE EFFECT OF SODIUM BICARBONATE ON TIME TO EXHAUSTION IN THE WESTERN FENCE 

LIZARD (Sceloporus occidentalis). Kyle Lutz and Yohsuke Kobayashi. Department of Biological 

Sciences, Saddleback College, Mission Viejo, California, 92692, USA 

We have seen claims that, when used before strenuous physical activities (running, swimming, etc.), 

taking sodium bicarbonate supplements will lead to increased performance by reducing the effects of 

lactic acid buildup. We used sodium bicarbonate injections to try to neutralize the lactic acid built up in the 

blood in an attempt to reduce muscle fatigue as measure by time to exhaustion (N = 6). The mean time to 

exhaustion in our saline injection control group was 177.0 ± 8.5s and the mean time to exhaustion in the 

sodium bicarbonate group was 177.3 ± 20.2s (means ± SE). We found that the sodium bicarbonate 

supplements had no significant effect on the time to exhaustion in the lizards (p = 0.49, one-tailed t-test). 

14. EFFECT OF CAFFEINE ON MEMORY IN MICE (Mus musculus). Deepa A. Thaker and Stanley Lin. 


The purpose of this experiment is to determine whether caffeine has the ability to enhance and sustain 

memory. The experiment will test the memory of mice after they are given a specified dosage of a 

caffeinated sugar water solution against the control, a sugar water solution. Prior studies suggest that 

caffeine acts as a stimulant to short-term memory. Caffeine binds to brain receptors, blocking the calming 

effect of the adenosine neurotransmitter, thus memory may be improved with the addition of caffeine. In 

this experiment, the mice given the dosage of caffeine completed the maze in steadily decreasing 

amounts of time (average of each run was 82.5, 47.5, 48, and 33.7 sec), while the mice given sugar water 

also completed the maze in a decreasing amount of time (average of each run was 140.3, 123.8, 106, 

and 85.3 sec),. This is because the mice became more familiar with the maze and the location of the 

cheese at the end of the maze. However, the mice given the caffeinated solution completed the maze in 

considerably less time than the mice given the control solution, as predicted. When the mice were given 

caffeine, they exhibited alertness within moments of receiving the caffeine dosage. The addition of 

caffeine prompted the mice to complete the maze almost immediately. 

15. THE EFFECT OF PH ON THE GROWTH OF BACTERIA (Escherichia coli). Nathaly Leal-Arteaga. 

Department of Biological Sciences, Saddleback College, Mission Viejo, 92692, California, USA 

Bacteria are unicellular microorganisms that can easily multiply through binary fission under the right 

conditions; temperature and pH levels are the two main factors that effect their growth. Most bacteria is 

not harmful to the human body and may benefit our immune system; however, there are pathogenic 

bacterium such as Escherichia coli (0157:H7) that causes illnesses. A counteraction to the growth of 

bacteria is acetic acid; most bacteria can not grow at pH levels below 4.6. In this experiment two different 

amounts of diluted E. coli was spread onto two groups of petri dishes consisting of solidified agar; half 

with a neutral pH and the other half with pH level three. After being placed in a 37°C incubator for two 

days bacteria colonies were present. In the 1/1,000 neutral solutions there were 11 bacterial colonies and 

in the 1/10,000 neutral solutions there was a total of 173 bacterial colonies. Consequently, both dilutions 

of E. coli that were placed in pH 3 petri dishes had no growth of bacteria. Overall, this study supports that 

pH 3 does prohibit the growth of E. coli bacteria. 

v 




16. THE EFFECTS OF OZONE ON ESCHERICHIA COLI ON SPINACH LEAVES. Aaron Echols and 

Crystine Gill*. Department of Biological Sciences, Saddleback College, Mission Viejo, California, 92692, 

USA 

Ozone treatment is an approved method of sanitation by the U.S. Food and Drug Administration. 

Using an ozone method of sanitation would yield greater benefits to both consumer and the environment. 

Although ozone is used as a disinfectant for various agricultural crops today, is not currently utilized to 

sanitize spinach. It was predicted that E. coli contamination on spinach would be significantly reduced by 

an aqueous ozone treatment. In this study, spinach was exposed to E. coli bacteria for twenty minutes. 

One portion of the spinach was washed in ozonated water (oxidation reduction potential 500), and the 

other portion was washed in untreated water, each for ten minutes. Samples of ozone-treated and waterwashed 

contaminated spinach were plated and incubated for two days. A mean count of 104 E. coli cfu 

per plate formed from the ozone-treated samples (n = 4, s.e. ± 5.7), and a mean count of 137 E. coli cfu 

formed on plates of water-washed samples of spinach (n = 4, s.e. ± 16.2). The quantity of E. coli on the 

spinach was reduced by the ozone treatment, although it was not found to be a significant reduction (one 

tail t-test 0.11; P = 2.13). 

17. THE EFFECT OF WAVELENGTH OF LIGHT ON THE DISCOLORATION OF WINE. 

Greg M. Fitzgerald and Michael B. Zilly. Department of Biological Sciences, Saddleback College, Mission 

Viejo, California, 92677, USA. 

Wines are usually corked in bottles varying in different colors. The different colors of bottles are due to 

help prevent light from entering the bottle, which discolors the wine. Since different wines are bottled in 

different color bottles, it was predicted that the effects of wavelengths of light on two different types of 

wine would discolor differently. A D’ Aquino Chianti and a La Loggia Barolo were obtained and 

experimented on. The two wines were diluted and placed into test tubes, some covered with different 

colors of cellophane. The test tubes were then placed in front of a full spectrum light for 14 days. Data 

was analyzed from the points given from a Beckman Coulter DU 730 spectrophotometer. Mean values of 

the two wines at blue light vs. mean values of the control showed p-values of p = 0.622 and p = 0.446 for 

a two tailed t-test assuming unequal variance for the Barolo and Chianti, respectively. In conclusion, for 

the time tested in this experiment, there is no significant difference between the discolorations of both 

wines under full light to their control samples. 

18. THE EFFECT OF APPLES ON THE RIPENING OF VALENCIA ORANGES (Citrus aurantium). 

Monica Mehran and Paris Aliyazdi. Department of Biological Sciences, Saddleback College, Mission 


Ethylene (C2H6) is a gaseous organic compound which can be thought of as a natural plant hormone, 

produced in small amounts by most fruits and vegetables. To assess the significance of ethylene on fruit 

ripening and maturation, we tested the effects of ethylene exposure on the Valencia orange (Citrus 

aurantium), by using ripening apples, commonly known for producing significant amounts of ethylene gas. 

The results of the experiment were consistent with the belief that the presence of ethylene accelerates 

the ripening, or what is frequently referred to as “degreening” in citrus fruits such as oranges and 

tangerines. There were very significant differences between the ripening time between the oranges that 

were exposed to apples and those that were allowed to ripen without the presence of apples. The 

ethylene released by the apples greatly reduces the time it takes for the orange to change color from 

green to yellow to orange. 

vi 




19. A COMPARISON OF THE WATER UPTAKE RESPONSES TO INTRAPERITONEAL BOLUS 

INJECTIONS OF ARGININE VASOTOCIN IN THE TOAD, BUFO AMERICANUS AND THE LEOPARD 

FROG, RANA PIPIENS. Ryan G. White and Michael Hadley. Department of Biological Sciences, 28000 

Marguerite Pkwy. Saddleback College, Mission Viejo, California, 92692. 

Arginine vasotocin (AVT) is a neuropeptide hormone found in most anurans, which is naturally 

produced and secreted into the plasma in response to dehydration. Within most anurans, natural 

response to AVT is conservation of water by the kidneys, enlargement of epithelial intercellular spaces, 

and increased permeability of the skin, along with additional neurological responses. Periodically 

catheterized specimens of the toad Bufo americanus and the Leopard Frog (Rana pipiens) were 

subjected to intraperinoneal bolus injections of AVT. Total body weight was monitored to quantify percent 

body weight gain of cutaneous water flux. Urine samples were examined for changes in osmolarity. 

Effects of single bolus injections of AVT dissipated after approximately 1hr. Total body weight gain 

(TBWG) percentage values indicated that there was a significant difference (P


22. EFFECT OF CAFFEINE ON BLOOD LACTATE IN EXERCISING HUMANS. John K. Davis and 

Jaclyn R. Kuluris. Department of Biological Sciences, Saddleback, 28000 Marguerite Parkway, Mission 


The purpose of this study was to observe the effects of caffeine on blood lactate during anaerobic 

activity in Homo sapiens. The study was done by taking a sample from three subjects. All three subjects 

were tested first by taking a blood lactate reading and then having them run 400 meters at full capacity. 

After completing the 400 meters another blood lactate reading was taken and compared to the initial 

reading. This procedure was repeated three more for a total of four test runs, two without caffeine and two 

with caffeine, in order to derive an average for both the control and the experimental runs. For the 

experimental runs, each subject was given a dose of 200 mg of caffeine one hour prior to the anaerobic 

exercise. To calculate the rise in blood lactate for each subject the following formula was used: Final 

reading-Initial reading = rise in lactate (mmol/l). The results of the study are that caffeine does have a 

significant change in blood lactate. One participant had the results that caffeine significantly (P


25. THE EFFECTS OF VITAMINS A, C, AND E ON THE GERMINATION OF RADISH SEEDS. Madina 

Ali and Rhonda Cheikh, Department of Biological Sciences, Saddleback College , Mission Viejo, 

California, USA 

To study the protective effects of antioxidant, Vitamin A, C, and E were added to Radish Seeds that 

were placed in a hostile environment containing hydrogen peroxide. To study vitamins protective effects 

as antioxidants, vitamin A, C, and E were added. The purpose of this project was to determine which type 

of vitamin is most effective for protecting plant cells against free radicals. Our results indicated that 

vitamin E was the vitamin that had the most germinating seeds in all three trails, but it wasn’t by much. 

The seed that had the least amount of germination was the one exposed to vitamin C. This experiment 

showed the effects of free radicals on seed germination, and the helpful effects of antioxidants. This 

experiment is relevant to our everyday lives because free radicals and antioxidants can affect us in 

carrying out a healthy lifestyle. 

26. THE EFFECTS OF ACID RAIN ON O 2 PRODUCTION IN ELODEA (Elodea camadensis). Nicole K. 

Baumgartner and Karl M. Neil. Department of Biological Sciences, Saddleback College, Mission Viejo, 

California, 92692, USA 

The majority of acid rain is the result of human emissions of sulfur and nitrogen compounds which react 

in the atmosphere to produce acids. Acidic rain harmfully affects aquatic life by lowering the pH of 

aquatic environments. It was predicted that decreasing the pH of the environment of elodea would 

decrease its oxygen production. Using a volumetric pipette and syringe as a manometer, individual 

samples of elodea were placed in solutions of differing pHs. The amount of time it took to produce ten 

milliliters of oxygen was measured using the constructed manometers, keeping temperature constant. At 

a pH of four, corrected mean time to produce 10mL of gas was 1.27 ± 0.17 min. At a pH of 7, corrected 

mean time to produce 10mL of gas was 0.14 ± 0.01 min. Time to produce 10mL of gas at a pH of four 

was significantly different (p = 7.088 x 10 -5 ) from a pH of seven. 

27. EFFECT OF NORMAL SALINE AND CONTACT LENS SOLUTION ON THE EPITHELIUM OF THE 

CORNEA. Larry T. Lam. Department of Biological Sciences, Saddleback College, Mission Viejo, 

California, 92692, USA. 

The tonicity of a solution depends on its concentration of solutes relative to the cell itself (Campbell 

and Reece, 2005). It has been suggested that normal saline solution (0.9% NaCl) can be used as an 

alternative to commercial contact solution. In the current study, the hypothesis that will be tested is 

normal saline will not be significantly different than Bausch and Lomb contact lens solution in lubrication 

of the eye and ocular irritation. Rabbit corneas were treated with both solutions, stained using a 

live/dead cell protocol, and imaged with a Zeiss confocal microscope. The number of dead cells was 

quantified per scan and the data for each treatment was compared. The mean for the normal saline 

treated tissues samples was 7.11 + 1.39 (+ standard error) dead cells per 350 m by 350 m. The 

mean for the Bausch and Lomb treated tissues samples was 4.44 + 0.74 (+ standard error) dead cells 

per 350 m by 350 m. In a two-tailed t-test, the p-value was 0.129 showing that there was no 

significant difference in using normal saline and commercial contact lens solution. 

28. THE EFFECT OF JADE PLANT ON ESCHERICHIA COLI. Kasra Abolhosseini and Harrison Pham, 


A common household plant, the jade plant, was used to determine its effectiveness in the prevention 

of bacteria growth of the bacterium, Escherichia coli. Leaves harvested from the jade plant placed in 

light and dark conditions were used in this study. Being that the jade plant is a CAM plant, it absorbs 

carbon dioxide at night, therefore leaves gathered from the dark conditioned plant were assumed to be 

more effective at killing bacteria than the leaves gathered from the day plant because of the acidity 

differences. 

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Volume 6, Spring 2008 - Saddleback College

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