Hixon 1 Concentrations and identities of fecal coliform bacteria from ...

Hixon 1 

Concentrations and identities of fecal coliform bacteria from tributaries 

of the Saint Johns River, Jacksonville Florida 

Benjamin Hixon and Anthony Ouellette 

Department of Biology and Marine Science, Jacksonville University 

October 23 rd , 2011


ABSTRACT 

Over the centuries humans have impacted many streams and rivers in Florida causing 

many of them to become polluted. Fecal coliform bacteria, which live in the guts of warm 

blooded animals, are common in polluted streams and rivers and are currently used as indicators 

of the presence of human pathogenic microorganisms. There are over 100 tributarites that flow 

into the Saint Johns River in Jacksonville Florida that are monitored for fecal coliform bacteria. 

According to the Florida Department of Environmental Protection (FDEP) if there are more than 

800 fecal coliform colony forming units (CFUs) per 100mL, the water is deemed not safe for 

human recreation. This paper focuses on descriptive and comparative research at six sites along 

the Saint Johns River near Jacksonville University (JU). The fecal coliform bacteria ranged from 

1 to 1067 CFUs/100mL, with the highest counts at Jacksonville University. There were no 

apparent relationships seen between fecal coliform bacteria CFUs and water temperature, 

dissolved oxygen, salinity, and pH. Common fecal coliform bacteria belonging to the genera 

Escherichia, Klebsiella, and Citrobacter were identified. 

INTRODUCTION 

Water quality is a serious issue that affects many U.S. streams and rivers, including the 

Saint Johns River in Jacksonville Florida. According to a report by Lui et al. (2010) that assessed 

39% of the United States streams and rivers, 19% were found to be impaired, of which 35% 

percent were polluted by fecal coliform bacteria. Fecal coliform bacteria are listed as the leading 

causes of water quality impairment in rivers and streams and the second leading cause in 

estuaries (Liu et al. 2010).


Fecal coliform bacteria are part of the gut flora of warm blooded animals and are 

commonly used as indicators of the presence of fecal material and sewage contamination 

(Toothman et. al. 2009). Water that contains contaminants from fecal material from warm 

blooded animals is likely to have human pathogenic microorganisms (Messyasz et al. 2010). 

Fecal coliform pathogens can become problematic if the water is unfiltered and used for 

recreational activities, shellfish harvesting, irrigation, and drinking water production (Servais et 

al. 2009). 

It has been proposed that fecal bacteria concentrations are enhanced by increased 

phosphorus concentrations. Burkholder et al. (2007) and Mallin et al. (2009) suggested that there 

is a positive correlation between fecal bacteria and nutrient concentrations due to either a 

common source or a random arrival of nutrients and fecal bacteria in one region. However, 

Sheheta et al. (1971) found that in areas with low nutrient concentrations fecal coliform bacteria, 

specifically E. coli, were dependent on phosphorus concentrations. Researchers from the 

University of North Carolina Wilmington continuously monitored water from the Bradley Creek 

watershed for 11 years; during a time of continuously high fecal coliform concentrations, urban 

storm water runoff was identified as the major contributor of fecal coliform bacteria to the 

watershed (Toothman et. al. 2009). 

Fecal coliform bacteria that are antibiotic resistant have also been isolated from river 

water near wastewater treatment plants. Microbial source tracking is typically used to track 

antibiotic resistance patterns of fecal bacteria to find the origin of the fecal pollution within 

specific aquatic environments. Servais et al. (2009) investigated antibiotic resistance of the fecal 

coliform bacteria Escherichia coli and intestinal enterococci isolated from the Seine River 

watershed using the disk diffusion method. It was found that 43% of the sampled E. coli and


83% of the intestinal enterococci were resistant to at least one antimicrobial agent (Servais et al. 

2009). In fact, most E. coli are resistant to multiple antimicrobial agents due to the pumps in their 

outer membrane (Musgrove et al. 2006). 

Fecal coliform bacteria are found in the Saint Johns River and its surrounding tributaries 

because of failing septic tanks, wastewater treatment plants, broken sewer lines, and animal 

waste (St. Johns River Keeper 2011). The Saint Johns River is a diverse ecosystem with slowly 

moving water that historically has had a lot of pollution problems; however, since the mid 1970’s 

and the creation of the clean water act of 1972, community efforts and initiatives addressing the 

bacterial sources have improved the overall quality of the river (Hollingsworth 2007). The major 

sources of pollutants, such as fecal coliform bacteria, are discharges from wastewater treatment 

plants and water runoff caused by rain (SJRWMD 2011). The root of the problem comes from 

nutrients and chemical contaminants that enter the river as either point-source or non-point 

source discharges. Point source discharges include domestic and industrial wastewater treatment 

plants, and usually enter the river through a man-made pipe or channel. The Saint Johns River is 

also affected by non-point source discharges which include septic tank leaching, ground water 

intrusion, runoff from roads and highways, pasture lands and forestry, and storm water collection 

systems which have no fixed entry point, making it difficult to find the main source of the 

pollution (Maher 2007). Algal blooms, which have detrimental effects on aquatic ecosystems, 

have the ability to form in the Saint Johns River due to the rivers warm water temperature and 

high level of excess nutrients, such as phosphorus and nitrogen, from both point source and nonpoint 

source discharges (Cadenhead 2007). 

The city of Jacksonville Florida (COJ) monitors over one-hundred tributaries belonging 

to the Saint Johns River on a quarterly basis. The tributaries are measured for dissolved oxygen


levels, fecal coliform bacteria concentrations, and other standards of water quality. According to 

Florida State standards, if a body of water contains 800 or more fecal coliform colony forming 

units (CFUs) per 100mL, then the water is deemed not safe for human recreational activities like 

fishing, and swimming (Tributary program 2011). 

This paper describes descriptive and comparative research on total bacteria and fecal 

coliform bacteria concentrations in tributaries that flow into the St. Johns River at or within a 

close vicinity of Jacksonville University. Fecal coliform bacteria CFUs were measured at four 

sites currently being measured by the city of Jacksonville (COJ) and at two other sites on the 

Jacksonville University Campus that were not previously monitored. Dissolved oxygen, pH, 

salinity, and water temperature were measured at all sites during both sample collections. The 

resulting data was compared to fecal coliform CFUs for both collections at all the sample sites in 

search of potential relationships. 

METHODS 

Six sampling locations at Jacksonville University in Jacksonville, FL or within a few 

miles of the university were monitored (figure1). All water samples were collected from either 

the Saint Johns River or surrounding tributaries. Four of the six samples (LB1, DR1, ALR7, 

ARL3) were tributary sites already being monitored quarterly by the city of Jacksonville and two 

(JU1, JU2) were not previously monitored and on the Jacksonville University campus. The order 

for both sampling collections was consistent and in the order: LB1, DR1, JU2, JU1, ARL7, 

ARL3. JU2 is the only sample site that is in the St. Johns River. For the sites that were 

previously monitored, data and results are available from the city of Jacksonville’s tributary 

monitoring website for February to March 2009 (Tributary program 2011).


NAME 

OF 

SAMPLE 

AREA 

LB1 

DR1 

JU2 

JU1 

ARL7 

ARL3 

LOCATION 

Long Branch at Wigmore Street 

Deer Creek at Talleyrand Avenue 

JU boathouse dock 

Creek/pipe runoff at JU dock inlet 

Unnamed Stream at Ferber Road 

Red Bay Branch at Lone Star Road 

Figure 1: Locations of Sample sites. COJ does not monitor JU1 and JU2. 

http://www.coj.net/NR/rdonlyres/ejld2jez3myhqoq37l2wi4c2rsf2paanfw5s4tqsa4zyg4ws2wkrhy 

q6w5bmj27lvb7aihpyupfxmve6kd3om3yslkc/Fecal+map+1st+quarter+2004.pdf 

At each sample site, an 800mL water sample was collected in a sterile plastic container 

just prior to low tide. Water temperature, salinity, and dissolved oxygen were measured in a 

small separate sample collected in a 50mL beaker using Exstik DO600 Dissolved Oxygen meter 

and Exstik pH/ Conductivity/ TDS/ Salinity/ Temperature meter. All samples were taken back to 

the Jacksonville University microbiology lab for testing no later than 4 hours after collection.


Testing began with using the membrane filtration technique: triplicates of 50mL of each sample 

was filtered on 0.45µm filters and then placed onto M-FC agar (Fisher Scientific Catalog 

Number DF0677173), which is selectable for fecal coliform bacterial growth, in 50x9mm Petri 

dishes. The incubation period for the 1/29/11 collection was 24 hours and 37 minutes and 18 

hours and 35 minutes for 3/12/11 collection at 46 ºC. Fecal coliform CFUs were visually counted 

using a Leica Quebec Darkfield Colony Counter and these numbers were multiplied by two to 

calculate fecal coliform CFUs per 100mL (table 1). Only blue colonies were counted, all brown 

or off white colonies were considered not to be fecal coliform bacteria CFUs. Water pH was 

measured using a Corning pH meter 125, which was calibrated prior to use. To calculate total 

bacterial counts, triplicates of serial dilutions using 0.85% NaCl in 100mL dilution containers 

were completed in tenfold increments from tenfold to one-thousand fold, followed by plating a 

0.1mL sample onto TSA and incubating for 42 hours and 5 minutes at 37 ºC prior to counting. 

IDENTIFICATION OF FECAL COLIFORM BACTERIA 

Morphological, physiological, and metabolic tests were used to identify and differentiate 

fecal coliform bacteria from each of the sampling locations. Three random and separate fecal 

coliform bacteria colonies, one from each of the three triplicate plates per sample site (totaling 18 

isolates), were chosen and successively quadrant streaked six times on m-endo agar (Fisher 

Scientific Catalog Number DF0677173), in order to promote only fecal coliform bacteria growth. 

Single colonies were used for testing and each sample was quadrant streaked on a new m-endo 

plate weekly in order to have living bacteria available for all tests. Before individual fecal 

coliform bacteria testing for differentiation among fecal coliform bacteria began, m-endo slants 

were streaked, incubated for 11 hours and 20 minutes, sealed tight, and store in a refrigerator 

with a temperature ranging from 0 to 5ºC for preservation. The methods for identification were


completed using the techniques described by Lammert et al. (2007), unless otherwise noted. All 

tests for differentiation were decided upon after researching various test result differences among 

the different fecal coliform bacteria genera Citrobacter, Enterobacter, Escherichia, and 

Klebsiella in Bergey’s Manual of Determinative Bacteriology. In order to confirm isolation of a 

single bacterium, gram stains were prepared with both a gram positive control Bacillus 

megaterium and a gram negative control Psuedomonas aeruginosa for comparison. Gram stains 

were also used to measure the length and width of individual bacterial cells and to note cell 

morphology using Motic Imaging Software and Swift Cam 5. Bacteria cell measurements were 

completed by measuring the lengths and widths of ten separate bacterial cells followed by 

calculating the average and the standard deviation. In order to confirm bacterial cell size and 

morphology a negative stain was completed. To test if the bacteria were able to produce capsules 

(Capsule stain) the bacteria were first grown on sucrose gelatin agar, whereas all other tests used 

bacteria samples grown on m-endo agar. The bacteria were tested for their ability to utilize 

citrate as a carbon source (Citrate test), to ferment acid by mixed acid fermentation (Methyl-red 

test), to conduct butanediol fermentation (Voges-proskauer test), and the ability to grow on 

media that inhibits the growth of Gram-positive bacteria and provides a color indicator 

distinguishing between organisms that ferment lactose and those that do not (EMB agar). The 

bacteria were also tested for motility and indole production (SIM test). 

RESULTS AND DISCUSSION 

FECAL COLIFORM CFU’s


All samples were collected around low tide in the Saint Johns River. The first collection 

of water samples were completed on January 29 th 2011 from 11:48 to 15:20 and the second 

collection of water samples were completed on March 12 th 2011 from 6:30 to 8:30. Low tide for 

the 1/29/11 collection was at 12:59 and 9:15 for the 3/12/11 collection. The outside air 

temperature for the 1/29/11 collection was 17.8ºC and 6.7ºC for the 3/12/11 collection. The 

average fecal coliform bacteria concentrations for the 1/29/11 collection ranged from 34 to 1067 

CFUs/100mL, with JU1 being the highest. For the 3/12/11 collection, the average fecal coliform 

bacteria concentrations was much less than 1/29/11 and ranged from 1 to 124 CFUs/100mL, with 

LB1 being the highest and JU1 being the second highest (99 CFUs/100mL). Site JU1 enters the 

Saint Johns River from a pipe that goes under the road leading to the Jacksonville University’s 

boat house. Based on numerous visual observations, the water coming out of this pipe is assumed 

to be constantly flowing. After tracking the stream it was found that it is short, going through the 

woods and eventually drying up upstream before approaching a road on main campus. It is 

believed the water in this stream is primarily from runoff from the densely wooded area the 

stream travels through. The root cause of the high fecal coliform CFUs has not been proven; but 

it is assumed that it is due to fecal material from animals running off into the stream. JU2 had the 

lowest fecal coliform CFUs at both collections, which was due to the site being the only sampled 

location that was in the St. Johns River and not a tributary; it is expected that the fecal coliform 

bacteria concentration is diluted in the river. Fecal coliform bacteria concentrations for the 

1/29/11 collection were more consistent with the City of Jacksonville (COJ) data than the 

3/12/11 collection. Comparing 1/29/11 and 3/12/11 fecal coliform bacteria CFUs data with COJ 

data from February 2009, the differences for the 1/29/11 collection ranged from 20 to 460 CFUs, 

with LB1 having the largest difference, and differences for the 3/12/11 collection ranged from

Location 

Date of Sample 

Data Source 

Water Temp (°C) 

pH 

Salinity (ppT) 

Dissolved Oxygen (mg/L) 

Fecal coliform CFUs per 100mL 

Bacteria CFUs per 100mL 


124 to 397 CFUs, with DR1 having the largest difference (table 1). The closest similarity among 

COJ data values and those of the 1/29/11 and 3/12/11 collections were seen at the ARL3 sample 

site, indicating there were no major flaws in experimental procedures giving reassurance that all 

collected data was accurate. For both sample collections, the highest fecal coliform bacteria 

concentrations were found at JU1 and LB1 with the exception of sample site ARL7 (table 1). All 

sample locations had lower fecal coliform CFUs at the 3/12/11 collection compared to COJ fecal 

coliform CFU data from February 2009 and data from the 1/29/11 collection (figure 2). Lower 

fecal coliform CFUs at all sites for the 3/12/11 collection could be due to all the water samples 

being collected before low tide causing them to be diluted while some of the 1/29/11 samples 

were collected past low tide, the colder air and cold water temperatures for all samples could of 

potentially not allowed the fecal coliform bacteria to replicate as efficiently (table 1), a 

difference in tidal heights potentially causing the samples be diluted, a shorter incubation period 

not allowing all the fecal coliform colonies to grow, or for some other reason not mentioned 

here. In order to accurately determine the cause or causes of this difference more research would 

need to be completed. All data for all sample collections and COJ data can be seen found in 

table 1.


573,300 

LB1 1/29/2011 TS 18.0 6.81 0.80 6.32 

941 ± 

81 

± 

197,300 

LB1 3/12/2011 TS 12.4 5.80 4.85 3.24 

124 ± 

143 - 

LB1 2/3/2009 COJ 13.9 - 0.65 6.54 480 - 

282 ± 

1,873,300 

± 

DR1 1/29/2011 TS 18.6 6.74 0.79 6.79 53 315,300 

DR1 3/12/2011 TS 11.6 6.30 0.97 2.26 43 ± 29 - 

DR1 2/3/2009 COJ 13.9 - 0.54 2.52 440 - 

140,000 

± 10,000 

JU2 1/29/2011 TS 15.9 7.60 7.84 9.05 34 ± 12 

10.0 

JU2 3/12/2011 TS 14.5 6.32 + 4.59 1 ± 2 - 

2,596,700 

± 

158,200 

JU1 1/29/2011 TS 17.8 6.67 0.21 7.29 

1,067 ± 

53 

JU1 3/12/2011 TS 12.1 6.34 0.22 3.83 99 ± 81 - 

ARL7 1/29/2011 TS 25.0 7.07 3.42 7.40 

724 ± 

71 

1,086,700 

± 75,700 

ARL7 3/12/2011 TS 12.9 6.19 4.81 3.63 4 ± 4 - 

ARL7 2/9/2011 COJ 17.2 - 0.15 8.12 380 - 

ARL3 1/29/2011 TS 19.1 6.76 0.28 6.58 

140 ± 

10 

195,000 

± 35,400 

ARL3 3/12/2011 TS 17.8 6.41 0.33 1.82 36 ± 47 - 

ARL3 2/9/2011 COJ 17.2 - 0.22 7.01 160 - 

KEY 

TS = Data from this study 

COJ= Data from the City of Jacksonville Tributary Monitoring Program 

- = Data is unknown or unavailable 

Table 1: Measured and COJ Fecal coliform CFUs, water temperature, Salinity, Dissolved 

oxygen, and bacterial CFUs for all water samples. COJ does not monitor JU1 and JU2 so no 

previous values are available.


Figure 2: Comparison of fecal coliform CFUs data from this study to COJ data. Comparing first 

collection fecal coliform CFUs per 100mL to second collection fecal coliform CFUs and City of 

Jacksonville Tributary Monitoring Program fecal coliform CFUs data from 2/2009 collection. 

According to the FDEP extreme levels of fecal coliform bacteria is 800+ displayed in red, 

moderate levels is 200 to 799 displayed in yellow, and low levels is 0 to 199 displayed in green. 

CHEMICAL AND PHYSICAL COMPONENTS 

Fecal coliform bacteria CFUs/100mL for each sample site were compared with the 

dissolved oxygen (DO), salinity, water temperature, and pH measurements to determine whether 

or not these measurements affect fecal coliform bacteria concentrations. Sample data from 

collection one (1/29/11) and collection two (3/12/11) were used for analysis along with COJ data 

and trends for comparison. After analysis of the data, no correlations between these factors and 

the fecal coliform bacteria CFUs per 100mL were found, as indicated by the low R 2 values for all 

of the graphs (figure 3). More sampling from each site would need to be completed in order to 

accurately determine if changes of pH, DO, salinity, or water temperature affect fecal coliform


bacteria concentrations. Also, each site would need to be analyzed separately because different 

sites naturally have different concentrations of fecal coliform bacteria to begin with, thus not 

allowing for an accurate comparison. 

Figure 3: Comparison of physical and chemical components and fecal coliform bacteria CFUs 

from data collected in this study on 1/29/11 and 3/12/11. A: Comparison of fecal coliform CFUs 

and water temperature. B: Comparison of fecal coliform CFUs and pH. C: Comparison of fecal 

coliform CFUs and salinity. D: Comparison of fecal coliform CFUs and dissolved oxygen levels. 

E: Comparison of fecal coliform CFUs and total bacterial CFUs. 

TOTAL BACTERIAL COUNTS 

When comparing fecal coliform bacteria CFUs per 100mL with the total bacterial CFUs per 

100mL there is no indication that samples with high concentrations of fecal coliform bacteria 

also had high concentrations of total bacteria (figure 3E). Fecal coliform CFUs are typically used


as indicators of the potential presence of disease causing organisms and counted for the purpose 

of alerting the person responsible for the water to take precautionary action 

(Total, Fecal & E. coli Bacteria in Groundwater 2007). 

IDENTIFICATION OF FECAL COLIFORM BACTERIA 

An attempt was made to identify three different fecal coliform bacteria from each sample 

site. Bergey’s Manual of Determinative Bacteriology was used to decide which tests to use to 

differentiate each genus of fecal coliform bacteria, and then to help identify individual species. 

The genera for sixteen of the eighteen bacteria were identified. Each bacteria’s test results are 

compared to the closest of the four (Citrobacter, Enterobacter, Escherichia, Klebsiella) fecal 

coliform bacteria genera (Table 2). A complete list of test results is available in the appendix. 

Citrobacter Escherichia Klebsiella Enterobacter 

Escherichia 

or 

Klebsiella 

LB1 X X X 

DR1 

X 

JU2 X X 

JU1 

X 

ARL7 X X X 

ARL3 X X 

Table 2: Genera of the bacteria found at each sample site.


Figure 4: Micrograph of Escherichia found in sample LB1. 

CONCLUSION 

Fecal coliform bacteria originate in the guts of warm blooded animals and are found in 

various tributaries for multiple reasons including failing septic tanks, wastewater treatment 

plants, broken sewer lines, and animal waste (St. Johns River Keeper 2011 2010). The highest 

fecal coliform CFUs were found during the 1/29/11 collection and at the site JU1 with the 

concentration of 1,067 CFUs/100mL. When analyzing the fecal coliform bacteria standards in 

regards to the state of Florida standards only samples LB1 and JU1from the 1/29/11 collection 

exceeded the state limit of 800 CFUs/100mL. More research needs to be done to distinguish 

whether fecal coliform bacteria concentrations are affected by pH, dissolved oxygen, salinity, or 

water temperature. Fecal coliform bacteria were identified that belong to the genera Escherichia, 

Klebsiella, and Citrobacter at all sites in this study.


APPENDIX 

SAMPLE / 

GENUS 

LB1 

BACTERI 

A 3 

0.95 +/- 

0.36 x 

0.36 +/- 

0.052 

JU2 

BACTERI 

A 3 

1.35 +/- 

0.29 x 

0.41 +/- 

0.07 

JU1 

BACTERI 

A 1 

0.91 +/- 

0.30 x 0.34 

+/- 0.052 

Coccobacill 

JU1 

BACTERI 

A 2 

0.79 +/- 

0.11 x 0.36 

+/- 0.052 

Coccobacill 

JU1 

BACTERI 

A 3 

0.84 +/- 

0.19 x 0.33 

+/- 0.048 

Coccobacill 

ARL7 

BACTERI 

A 1 

0.77 +/- 

0.16 x 0.35 

+/- 0.071 

Coccobacill 

Escherich 

ia 

6.0-2.0 x 

1.5-1.0 

SIZE (µm) 

MORPHOLO 

GY Bacillus Bacillus us 

us 

us 

us 

Bacillus 

GRAM Negative Negative Negative Negative Negative Negative Negative 

CAPSULES + + + + + + + 

EMB 

GROWTH + + + + + + UNK 

EMB COLOR 

OBSERVATI 

ON 23:30 

EMB COLOR 

OBSERVATI 

Dark 

Purple w/ 

GMS 

Dark 

Purple w/ 

Dark 

Purple w/ 

GMS 

Dark 

Purple w/ 

Dark Purple 

w/ GMS 

Dark Purple 

w/ GMS 

Dark Purple 

w/ GMS Purple N/A 

Dark Purple Dark Purple Dark Purple 

ON 49:30 GMS GMS w/ GMS w/ GMS w/ GMS Purple N/A 

MR w/ (T) + + + + + + + 

VP w/ (T) - - - - - - - 

Indole + + + + + + + 

Citrate - - - - - - - 

Motility + + + + + + +/- 

KEY 

+ = positive result 

- = negative result 

UNK = unknown result 

[-] = mostly all species within that genus are negative 

[+] = mostly all species within that genus are positive 

+/- = some species are positive and others are negative within that genus 

N/A = not applicable 

(T)= Triplicate 

GMS= Green Metallic Sheen 

Table A1: Isolated fecal coliform bacteria test results and comparison to the genus Escherichia 

test results.


SAMPLE / 

GENUS 

DR1 

BACTERI 

A 1 

2.23 +/- 

0.82 x 0.60 

+/- 0.067 

DR1 

BACTERI 

A 2 

2.62 +/- 

1.02 x 0.66 

+/- 0.07 

DR1 

BACTERI 

A 3 

1.68 +/- 

0.36 x 0.56 

+/- 0.053 

JU2 

BACTERI 

A 1 

2.80 +/- 

0.68 x 0.60 

+/- 0.16 

JU2 

BACTERI 

A 2 

1.99 +/- 

0.50 x 0.58 

+/- 0.063 

ARL3 

BACTERIA 

3 

0.74 +/- 

0.13 x 0.37 

+/- 0.048 

Coccobacill 

us 

Citrobact 

er 

6.0-2.0 x 

SIZE (µm) 

1.0 

MORPHOLO 

rodshaped 

GY Bacillus Bacillus Bacillus Bacillus Bacillus 

GRAM Negative Negative Negative Negative Negative Negative Negative 

CAPSULES - - - - - - - 

EMB 

GROWTH + + + + + + UNK 

EMB COLOR 

OBSERVATIO 

N 23:30 

EMB COLOR 

OBSERVATIO 

Dark 

Purple w/ 

GMS 

Dark 

Purple w/ 

Dark 

Purple 

Dark 

Purple w/ 

GMS 

Dark 

Purple w/ 

Purple 

Dark 

Purple w/ 

GMS 

Dark 

Purple w/ 

Pink and 

Raised 

Dark 

Light 


N 49:30 GMS Purple GMS Purple GMS Raised N/A 

MR w/ (T) + + + + + - + 

VP w/ (T) - - - - - + - 

Indole + + + + + + +/- 

Citrate - - - - - + [+] 

Motility - - - + - - [+] 

KEY 

+ = positive 

result 

- = negative 

result 






(T)= Triplicates 


Table A2: Isolated fecal coliform bacteria test results and comparison to the genus Citrobacter 

test results. 

N/A


SAMPLE / 

GENUS 

SIZE (µm) 

LB1 

BACTERIA 

4 

0.71 +/- 0.16 

x 0.41 +/- 

0.032 

ARL7 

BACTERIA 

2 

0.78 +/- 0.16 

x 0.36 +/- 

0.070 

ARL3 

BACTERIA 

1 

0.942 +/- 

0.33 x 0.38 

+/- 0.092 

ARL3 

BACTERIA 

2 Klebsiella 

0.79 +/- 0.17 

x 0.30 +/- 

0.00 

MORPHOLOGY Coccobacillus Coccobacillus Coccobacillus Coccobacillus 

6.0-0.6 x 

1.0-0.3 

rodshaped 

GRAM Negative Negative Negative Negative Negative 

CAPSULES + + + + + 

EMB GROWTH + + + + UNK 

EMB COLOR 

OBSERVATION 

23:30 Pink 

EMB COLOR 

OBSERVATION 

49:30 Pink Pink 

Pink, Purple 

outside 


Raised 


Raised 


Raised 


Raised 

MR w/ (T) - - - - +/- 

VP w/ (T) + - + + +/- 

Indole - - + - [-] 

Citrate + + + + [-] 

Motility - - - - - 

KEY 










Table A3: Isolated fecal coliform bacteria test results and comparison to the genus Klebsiella 

test results. 

N/A 

N/A


SAMPLE / 

GENUS 

SIZE (µm) 

LB1 

BACTERIA 

2 

1.062 +/- 

0.14 x 0.37 

+/- 0.048 

ARL7 

BACTERIA 

3 Escherichia Klebsiella 

0.87 +/- 0.22 

x 0.35 +/- 

0.053 

6.0-2.0 x 

1.5-1.0 

6.0-0.6 x 

1.0-0.3 

rodshaped 

MORPHOLOGY Bacillus Coccobacillus Bacillus 

GRAM Negative Negative Negative Negative 

CAPSULES + + + + 

EMB GROWTH + + UNK UNK 

EMB COLOR 

OBSERVATION 

23:30 Purple Purple N/A N/A 

EMB COLOR 

OBSERVATION 

49:30 

Dark Purple 

w/ GMS Purple N/A N/A 

MR w/ (T) + + + +/- 

VP w/ (T) - - - +/- 

Indole + + + [-] 

Citrate - - - [-] 

Motility - - +/- - 

KEY 










Table A4: Isolated fecal coliform bacteria test results and comparison to the genera Escherichia 

and Klebsiella test results.


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