Fecal Coliform - kmutt

Bacteriological
Sample Collection &
Analysis

Selection of sample containers: criteria or requirements
• Sterilizable glass or plastic bottles
– Heat resistant
– Permit proper washing
– Uncontaminated until examinations are completed

Types of Bottles
– Plastic or glass bottles
• Suitable size (hold @ least 120 ml)
• Wide-mouthed
• Non toxic
• Able to be sterilized repeatedly

Transportation/storage holding times for
collected samples:
• Optimum time from point of collection to start
of analysis - one hour.
• If the sample can not be processed within one
hour of collection, refrigerate or ice during
transportation

Transportation/storage holding times for
collected samples:
• The recommended time interval is six
hours.
• The maximum time elapsed between
collection and examination should never
exceed 24 hours.

Indicator Organisms
Analytical Techniques
Microbiological Analysis
•Total plate count
•Multiple fermentation tube or Most probable number
•Membrane filter

Bioindicator เปนตัวแทนบงชี ้วาน้ํานั้นมีการปนเป อน
จุลินทรียที่ทําใหเกิดโรค เกณฑในการเลือกจุลินทรียมีดังนี้
1.เปนจุลินทรียที่พบรวมกับจุลินทรียที่กอใหเกิดโรคอยูในน้ํา
และมีจํานวนมากกวา
2.ควรจะเปนชนิดที่เกี่ยวของกับแหลงกําเนิดของจุลินทรียที่
กอใหเกิดโรค
3.มีความทนทานตอกระบวนการบําบัดน้ําเสีย การฆาเชื้อโรค
และสภาวะแวดลอมไดดีกวาจุลินทรียที่กอใหเกิดโรค
4.สามารถทําการวิเคราะหไดเปนประจําบนอาหารเลี้ยงเชื้อที่
งายตอการแยก และสามารถแปรผลที่นาเชื่อถือได

1. Escherichia coli & coliform: G-, non-sporing rod,
facultative, ferment lactose with gas (Total
coliform/Faecal coliform)
2. Faecal Streptococci : G+ cocci, ferment lactose-no gas
3.FC/FS บอกการปนเปอน ถามากกวา 4 มาจากคน ถานอย
กวา 0.6 มาจากสัตว
4. Clostridium perfingens:G+, spore-forming rod,
anaerobic

5. Pseudomonas aeruginosa: aerobic, G- rod, non sporing, non
capsule
6. Staphylococcus aureus : G- cocci, non sporing, non capsule
7. Bacteriophages
Coliform/coliphage
87:1 --> raw sewage 4.2:1 --> lagoon eff. 0.15:1--> river water

Coliform Group
• All aerobic & facultative anaerobic
gram-neg non-spore-forming rod-shaped
bacteria which ferment lactose with gas
formation within 48 hrs @ 35 o C
• “Total coliform group”
• Indicates possible hazard exists

Sub-groups of Coliform Organisms
• Non-fecal
• FECAL

Non-Fecal Characteristics
• Example: Enterobacter aerogenes
• Primarily found in soil, grasses, and vegetation
• Survive only in temperatures up to 35 degrees Celsius
• Have longer life span than fecal coliforms
• If presence is greater than fecal coliform it can indicate
an older pollution incident or insufficient chlorination
of the drinking water

Coliform Subgroups - Fecal Coliform (FC)
• Examples:
– Escherichia coli (E. coli) - predominant coliform in
feces of warm-blooded animals; best measure of
drinking water contamination
• Best measure of water supply contamination

Fecal Characteristics
• Survive in temperatures up to 44.5 degrees
Celsius
• Short life span
– Presence in greater quantities than non-fecal
organisms can indicate recent pollution

Average bacteria / gram of feces
Animal Fecal coliform, 10 6 Fecal strep, 10 6 Ratio
Chicken 1.3 3.4 0.4
Cow 0.23 1.3 0.2
Duck 33.0 54.0 0.6
Human 13.0 3.0 4.4
Pig 3.3 84.0 0.04
Sheep 16.0 38.0 0.4
Turkey 0.29 2.8 0.1

Prepare Lab Bench
• Clear the lab bench
• Use Chlorox (bleach) solutions and paper towels to
disinfect
• Assemble all equipment and supplies
• Wash hands

Basic MF Procedure
• Sample volume: 100 ml
• Membrane filter: 47mm diameter, 0.45 micron pore
size
• After filtering water, place membrane filter in treated
petri dish
–Pad
– Media – M-endo

Basic MF Procedure
• Incubate for specified time in the appropriate
incubator
• Interpret results: any coliform indicates a positive
sample.

Media
• Total coliform: M-endo broth
• Fecal coliform: M-FC broth
• Fecal strep: KF streptococcus agar

Incubation
• Culture plates are incubated upside down so
moisture from condensation does not fall on
developing colonies and stress/kill them
• Total coliform:
– Dry air incubator, 35 o C +/- 0.5C
– 22-24 hours incubation time

Incubation
• Fecal coliform:
– Wet bath or aluminum block
incubator, 44.5 o C plus / minus 0.2 o C
– 24 hours incubation time

Incubation
• Fecal strep: dry air, 35 0 C +/- 0.5 o C, 48 hrs

Colony Identification (Positive Cultures)
• Characteristics of Positive Samples
– Total coliform: pink to dark red w/ gold/green
metallic sheen
– Fecal coliform: various shades of blue
– Fecal strep: pink to dark red with no sheen
– How does a negative sample look

Colony Identification
• Confluent growth:
– Continuous growth, colonies run together –
– No distinct colonies
• Too numerous to count (TNTC):
– Colony growth in excess of 200 colonies per 100 ml

Confirmation
• All positive sample results must be
confirmed
• Analyze and confirm to determine the
presence of fecal coliform or E. coli

Confirmation Procedure (Fecal)
• Media: EC media – specific for fecal coliform
– In tubes containing inner inverted fermentation tube
• Remove the membrane containing the total coliform
colonies from the petri dish with sterile forceps
• Carefully curl and insert into the tube of EC media
or…...
• Use sterile loop – “streak and swish”

Confirmation Procedure (Fecal)
• Incubate in water bath incubator at 44.5 o C +/-
0.2 o C for 24 hrs +/- 2 hrs
• Gas production in the inner tube of the EC
media indicates positive fecal coliform

Confirmation Procedure (E. coli)
• Media: EC media with MUG
– MUG = 4-methylumbelliferyl-beta-D-glucuronide
• Procedure and incubation same as for EC media
– Can use inverted tubes (fecal ID) or no tubes (E. coli)
• Positive for E. coli is shown by fluorescence under UV
lamp
– More on MUG later

Confirmation Procedure (E. coli)
• Media: nutrient agar with MUG
– In a petri dish
• Transfer positive membrane filter to nutrient agar plate with
sterile forceps
• Incubate in dry air incubator at 35 o C for 4 hours
• If fluorescence occurs under ultraviolet (UV) lamp, E. coli
is present

MPN Technique
1. Presumptive test : lactose or lauryl tryptose broth 24
hr,35 C ---> produced gas
2. Confirmed test : brilliant green lactose bile broth
48 hr,35 C ---> produced gas
3.Completed test : Endo or EMB plates, 24 hr 35 C ,
gram stain ----> G-, rod no spore

Principles of Enumerating Bacterial Population Densities Using the Multiple
Fermentation Tube Technique - II
• Within each dilution, an equal amount of sample is placed in 5 separate
fermentation tubes.
10 mL
Each
Tube
Sample Fermentation Tubes, #1- #5
• The process is repeated for three successive serial dilutions. 15 tubes total.

Principles of Enumerating Bacterial Population Densities Using the Multiple
Fermentation Tube Technique - IV
• As an example, consider the following possibility:
Set #1, Sample Set #2, 1:10 Dilution Set #3, 1:100 Dilution
4 Positive 1 Positive 2 Positive
• For this combination, the Poisson Distribution yields the following results:
– The most probable number (MPN) of bacteria is: 21 bacteria per 100 mL
– There is a 95% probability that the correct number is within the range: 9 - 55 bacteria
per 100 mL

ตารางที่ 9 เปรียบเทียบขอดี และขอเสียของการวิเคราะห coliform bacteria
โดยวิธี MPN และ membrane filtration (Hutton, 1983 in Gray, 1989)
Multiple tube MPN
Costs
Large quantities of culture media and glassware and large autoclave
Capital costs fairly high
Accuracy
Statistically based estimate Possibly large errors especially at low levels
Field use
Needs static base
Suspended matter
May be used for turbid samples
Convenience
Large amount of material to be prepared prior to analysis and disposed of after
incubation
Incubation times
Up to 48 h or 72 h

Positive Test for Total Coliform Organisms - Presumptive Test
• Initially sterile
• Lactose broth containing Lauryl Sulfate
{(C 12 H 25 O) 2 SO 2 }
• Inverted tube to capture CO 2 produced as part of
fermentation
• Inoculated with water sample
• Incubate 48 hours at 37°C (body temp.)
• If gas appears in inverted tube, test is positive
• Thus, an organism was introduced that ferments lactose
in the presence of lauryl sulfate at 37°C

Use of Confirmation and Presumptive Test for Total Coliform
• Each time a positive test is recorded, the population density of organisms
reported increases significantly.
• To make sure a positive test is indeed positive, organisms from a positive test
are transferred to a different medium.
• Although the medium is different, it relies on the same principle, fermentation
of lactose in the presence of bile salts.

Positive Test for Total Coliform Organisms - Confirmation Test
• Initially sterile
• Brilliant green bile broth, lactose and bile salts.
• Inverted tube to capture CO 2 produced as part of
fermentation
• Inoculated with loop from presumptive test.
• Incubate 24 hours at 37°C (body temp.)
• If gas appears in inverted tube, test is positive
• Thus, an organism was introduced that ferments lactose
in the presence of bile salts at 37°C

Positive Test for Fecal Coliform Organisms
• Initially sterile
• Lactose broth containing bile salts
• Inverted tube to capture CO 2 produced as part of
fermentation
• Inoculated with water sample
• Incubate 48 hours at 44.5°C (112.1°F)
• If gas appears in inverted tube, test is positive
• Thus, an organism was introduced that ferments lactose
in the presence of bile salts at 44.5°C

Positive Test for Fecal Streptococcus Organisms - Presumptive Test
• Initially sterile
• Glucose broth containing sodium azide (NaN 3 ).
Sodium azide is highly toxic, but serves to select for
streptococcus group.
• Inoculated with water sample
• Incubate 48 hours at 35°C (95°F)
• If contents of test tube become turbid, test is positive.
• Thus, an organism is present that is likely to be part of
the streptococcus group associated with fecal material.

Positive Test for Fecal Streptococcus Organisms - Confirmation Test
• Initially sterile
• Pfizer selective enterococcus (PSE) agar, complex
organic media (bacterial candy), bile salts, and sodium
azide.
• Streak with contents of positive presumptive test.
• Incubate 24 hours at 35°C (95°F)
• If brownish-black colonies with brown halos appear
the presumptive test was indeed positive.
• Thus, it is confirmed that an organism is present that is likely to be part of the
streptococcus group associated with fecal material.

Membrane filtration
Costs
Smaller quantities of media membrane expensive, Disposables (petridishes,
pipettes) expensive
Capital costs of proprietary equipment high
Accuracy
More accurate especially to low levels (less than 100 colonies per 100 ml)
Field use
May be operated in the field and in transport if portable incubator used.
Transport media can be used prior to incubation
Suspended matter
Not suitable for turbid waters due to membrane clogging
Convenience
Less manipulation and hence lower chance of contamination. Disposable,
presterilized equipjent can be purchased
Incubation times
24 h (or oven 7 h in some special cases)

Total & Faecal coliform
Inoculate into lactose broth
Gas
Inoculate into brilliant green
lactose bile broth
No Gas (negative)
Inoculate into EC medium
Gas
No Gas
Gas
No Gas
(positive)
(negative)
(positive)
(negative)

การคํานวณหา coliform bacteria โดย MPN
MPN/100 ml
= no. of positive tube x100
{(ml sample in neg.tube) x (ml sample in all tubes)} 1/2

Ex จงหาคา MPN จากการวิเคราะห coliform bacteria
จากขอมูลที่ตรวจนับไดดังนี้
ขนาดของตัวอยาง (มล.) จํานวนหลอดที่เกิดกาซ จํานวนหลอดที่ไมเกิดกาซ
10 4 1
1 4 1
0.1 2 3
0.01 0 5
40/100 ml

Solids

Settleable solids (ml/l, mg/l)
SVI = V30/MLSS (ml/mg)

. ขอมูลจากการบันทึกผลวิเคราะหของแข็ง มีดังนี้ (3 คะแนน)
น้ําหนักถวย 42.0512 กรัม
น้ําหนักถวยหลังอบ 103 o C 52.5310 กรัม
น้ําหนักถวยหลังเผา 550 o C 45.2458 กรัม
ปริมาตรตัวอยางน้ํา 100 มิลลิลิตร
2.1 จงหาปริมาณของแข็งที่เปนสารอินทรียในตัวอยาง
2.2 จากขอมูลขางตน ตัวอยางดังกลาวมีสารอนินทรียอยูรอยละเทาใด