How to Perform Fecal Coliform
Analytical Test for Drinking Water
Quality
Written by: Sheree Gossett-Johnson
www.WaterWorldCE.com
How to Perform Fecal Coliform
Analytical Test for Drinking Water
Quality
Educational Objectives
Upon completion of this course, the operator should understand:
•
How to prepare needed solutions and media
•
How to correctly interpret the test results with
respect to drinking water quality
•
How to use a table to determine sample volumes
from other water sources.
I.
Abstract
This operator education course reviews the chemical chlorine
to familiarize the operator with the disinfection portion of the
water treatment plant and explain in a step by step method how
to perform the Total Coliform analytical test, including quality
assurance and control measures. Upon completion of this Contact
Hour, the analyst should understand how to prepare needed solutions, media, count colonies and correctly interpret the test results
with respect to drinking water quality. In addition, a table outlining suggested volumes for nine water sources, including drinking
water is provided. Some sample calculations along with a question
and answer section is also provided to aid the analyst in understanding the material.
References: Standard Methods for Examination of
Water and Wastewater, 19th Edition.
II. Keywords and Definitions
Take a glance through this list and be sure to read carefully any
terms or abbreviations with which you are not familiar.
Autoclave: Provides uniform temperatures within a chamber (up to
and including the sterilizing temperature of 121° C.
Sterilization: To make free of living organisms.
Culture Dish: Shallow tight fitting plastic dishes.
Incubator type: Water bath or heat sink incubator.
Incubation: Providing an environment that is favorable for Coliforms to thrive.
Culture media: Dehydrated media (M-Endo medium) to provide
2
•
The water treatment plant chlorine disinfection
method as it relates to the Total Coliform analytical test.
•
How to calculate the Total Coliform density and
determine the statistical reliability of the results.
the appropriate nutrients for Coliform growth.
Quality Assurance: Procedure strictly followed to produce data of
known and defensible data.
Stainless Steel Filter: Specific Filter apparatus that consists of a
seamless funnel attached to a Unit base by a locking device.
Total Coliform: Facultative anaerobic, gram negative, non-sporeforming, rod-shaped bacteria that produce a red colony with a
metallic sheen within 24 hour incubation at 35° C on an Endo-type
media.
95% Confidence: Calculated confidence limits to determine the
statistical reliability of the results.
Contact Time (CT): The contact time is the product of disinfectant
residual and disinfectant contact time.
Disinfection: A treatment process used to destroy or inactivate
disease-causing (pathogenic) organisms.
III. Introduction
The purpose of a water treatment plant is to provide safe drinking
water. The two main types are ground water and surface water
treatment plants (WTP). The ground WTP water source comes
from one or more drilled wells that are pumped into the plant.
The surface WTP water source comes from a river or stream water
intake, which pumps the water from the river or stream into the
plant. The water pumped into either plant is processed to meet
the drinking water standards, which includes disinfect ion of the
water to produce safe drinking water. Disinfection is a treatment
process used to destroy or inactivate disease-causing (pathogenic)
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organisms.
Diseases caused by these pathogenic organisms in
water are called waterborne diseases. These diseases include Typhoid, Dysentery, Cholera, Infectious hepatitis,
Giardiasis, Cryptosporidiosis, and Legionallosis. Methods
of disinfection to destroy the disease causing pathogenic
include: Heat Treatment, Ultraviolet light, Chemical Treatment, and Chlorination.
The method of Heat Treatment was discovered many
years ago and simply involves boiling the water for at least
5 minutes to kill the pathogens. When there is contamination of a public water system a “Boil Order” is issued
to the public, stating that all water consumed by humans
must be boiled for 5 min. before consumption. Obviously
boiling is not well suited for large-scale use due to cost
and practicality. The second listed method is Radiation
Treatment, known as ultraviolet light treatment.
Ultraviolet light is very effective at destroying disease
causing pathogens. Specially designed lamps produce U.V.
light that is readily absorbed and scattered by turbidity in
the water. UV light does not remain in the water; all of the
killing action occurs during the exposure time. Due to the
lack of residual effects, UV light is most suitable for small
quantities of drinking water.
The Chemical Treatments include bromine, iodine,
ozone, potassium permanganate, chlorine dioxide, or
oxygen. The general effectiveness of these used oxidants
varies and causes the cost of water treatment to rise. In
addition, the equipment and expertise in using these
chemicals makes them less desirable.
Chlorination is a useful disinfectant for storage tanks,
pipes, oxidizing iron, manganese, and hydrogen sulfide as
well as for controlling taste, odor, algae, and slime. The
secondary benefits place chlorine as the most cost effective and therefore the most common in the United States.
Elemental chlorine is a poisonous, yellow-green gas
when kept at ordinary temperatures and pressure. The
gas is highly corrosive when wet, and is stored as a dry
liquid under pressure. The gas is highly irritating and its
penetrating odor is heavier than air by 2.5 times. Liquid
chlorine is clear amber in color, not flammable, and 1.5
times heavier than water. Therefore, for best results, in
large WTP, the operator will purchase the gas; in small
WTP, the operator will feed the aqueous solution. Chlorine gas disinfect ion requires appropriate housing, safety
controls, and dosing devices as does the liquid chlorine
disinfect ion method.
The gas chlorinator may be as large as one ton cylinders or as small as a 100 lb cylinder. Regardless of the
size, the cylinder must be on scales to allow the operator
the opportunity to replace the cylinder before running
out completely. Liquid chlorine can be measured by
the operator through calculating the demand needed in
advance. The room temperature of the chlorine gas feed
www.WaterWorldCE.com should be kept at 21 degrees Celsius. In addition, the room
should be enclosed with no sunlight provided. The liquid
chlorine should also be kept at 21 degrees Celsius, and not
exposed to sunlight. Temperature changes and sunlight
can cause the chlorine gas to be susceptible to re-liquification in the piping between the cylinder and fed device.
This phenomenon is accompanied by deposits of chlorine
impurities throughout the system, resulting in excessive
maintenance. Temperature changes and sunlight cause
liquid chlorine to breakdown and lose its effectiveness.
The Chlorinator room should be at or above ground
and equipped with an exhaust fan at floor level to remove
any “leaking” chlorine when changing cylinders or evacuate leaking chlorine from a faulty cylinder. The ventilation fan should have a manual “on” switch in addition to
“automatically” turning on when the chlorination door
opens. The chlorine room should also have an inspection
window as a means to view the equipment for deterioration before entering the room. The alarm system should be
highly audible and connected not only to the scales, which
indicate the absence of chlorine in the cylinder but also to
a chlorine leak detector capable of sensing atmospheric
chlorine concentrations less than 0.5 mg/L.
The water treatment plant operator can determine if
the chlorine dosage was effective by performing the analytical Total Coliform. A negative result indicates the water system is disinfected, while a positive result indicates
that the water is unhealthy for human consumption.
Coliform bacteria are present in the gut and feces of
warm blooded animals (including humans) so the significance of coliform bacteria in drinking water is that the
water may be contaminated with animal feces carrying
the pathogens. Waterborne pathogens include Salmonella species, Shigella species, and Giardia lamblia. The
Total Coliform test is used to determine if the drinking
water supply is an unsanitary water supply by a positive
(growth) or negative (no growth) result. Within this test,
all bacteria that are defined as in the coliform group are
comprised of facultative anaerobic, gram negative, nonspore-forming, rod-shaped bacteria that produce a red
colony with a metallic sheen within 24 hour incubation
at 35° C on an Endo-type media. As a rule of thumb, any
colony lacking sheen are considered non-coliforms by this
technique. A positive or negative is the acceptable reporting result; however, to determine the degree of pollutant
in the water sample, the analyst should “count” the number of colonies.
To “count” the number of coliform bacteria within
this test, the number of colonies with the metallic sheen
count must be within 20 - 80 and not more that 200. If
the counts yield more that 200 the reported count is Too
Numerous To Count (TNTC). If the counts yield several
numbers within the range, average the count per volume
and express as number per 100 mL, If the counts only yield
3
one that is within the limits than that one is expressed as
count per 100 mL. If there are no counts within the range
and the count is not above 200 it is considered “negative”,
if a number is required, add the counts and divide by volume and express in count per 100 mL.
The membrane filtration test is most appropriate for
water that has low turbidity and low “background” bacterial population. This technique is highly reproducible,
and is a fast method to determine the drinking water quality. Unfortunately, this technique may underestimate the
number of viable coliform bacteria, so using the assumption that the bacteria are distributed randomly, and follow
poison distribution, the analyst may employ a statistical
“check” referred to as the 95% confidence limit.
The Total Coliform test is generally used to determine
if there are Coliforms present in the drinking water or not.
Due to the importance of a positive or negative result, the
sample collection technique used is very important. All
water samples collected from sample spigot or distribution
tap must be sterilized through flaming the tap. Disinfecting the tap with a chlorine solution can be used; however,
if possible, the flaming of the tap opening is preferred. To
ensure that the source water for the drinking water system
is not contaminated, de-chlorination of the sample must
be performed. De-chlorination is achieved by adding a
tablet of Sodium Thiosulphate to the sample container.
U.S. EPA Total Coliform rule only requires that a
private or public water system report “positive” or “negative.” If the system has a “positive” result, the water from
that source is considered “unhealthy” for human consumption. Generally, if the water system disinfects with
chlorine, the follow up tests will be “negative” If positive
results continue to occur, the water system should be
evaluated to determine the source of contamination. In
some cases, this means the “positive” or contaminated
well or well field will be closed and another drilled.
IV. Procedure
Materials Needed
• Bunsen burner
• Stainless Steel Filter Unit
• Flasks
• Autoclave
• Pipette
• Smooth Edged Forceps
• Sample Bottles with Sodium Thiosulphate tablet
• Graduated Cylinders
• —Endo medium (Agar and Broth)
• Petri dish (60 mm)
• Water Bath
• 95% Ethanol
• Filters Membranes (0.45um pore size)
• Waterproofed Plastic Bag enclosures
4
•
•
Sterile Dilution Water
Absorbent pad (able to absorb 2 mL +/- 0.2 of M -Endo broth)
Preparation of Materials
Autoclave
Sterilize stainless steel filter unit, Petri dishes, sample
bottles (Sodium Thiosulphate tablet within), pipettes and
graduated cylinders, and 2 flasks of distilled water in the
autoclave, once it reaches 121°.C, set the timer for 15 minutes.
Note: Wrap the filter unit, Petri dishes, sample bottles
loosely capped with Sodium Thiosulphate tablet within,
and the openings of the pipettes and graduated cylinders,
in brown kraft paper before autoclaving.
Solutions
1. M-Endo medium: Follow manufacturers’ directions and incorporate the following:
•Agar preparation
•Rehydrate in a solution of 20 mL 95% Ethanol/800 mL
sterilized distilled water
2. Heat until close to boiling remove and cool to 45 - 50° C.
3. Measure between 5-7 mL of the dissolved agar into each Petri
dish.
4. Check pH of 7.2 +/- 0.2. Precipitation may be present.
5. Store in dark refrigerator at 4 to 8° C.
6. Discard after 2 weeks.
Note: Have on hand 95% Ethanol or Absolute Methyl Alcohol
to sterilize the forceps through flame burn between each filter.
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Table 1: Sample Volumes For Membrane Filter Total Coliform
Amount to Be Filtered mL
50
10
x
x
x
x
x
x
Water supply intakes
x
x
x
Bathing Beaches
x
x
x
River Water
x
x
x
x
Chlorinated Sewage
x
x
x
x
Raw Sewage
x
x
Water Source
100
Drinking Water
x
Swimming Pools
x
Wells, Springs
Lakes, reservoirs
1 0.1
0.01 0.001 0.0001
Sample Collection
Collect a minimum of 100 mL and maximum of 1000
mL grab sample as per Table 1 guideline, in a pre-sterilized sample bottle containing the Sodium Thiosulphate
tablet using the following guidelines:
Note: What is a grab test, and how is it different than
other tests?
Answer: Some test measurements may change rapidly,
so you must perform a grab test. A “composite test” is
automated and measures a sample set to act on a time basis or a flow pace basis. For example, collecting a sample
every half hour is a time basis test. Setting the test to be
taken every 100,000 gallons of water is a flow basis test.
Typically, as loads rise, then the organics increase. Flow
pace is usually better because it reflects the loadings.
Grab test: Collect a grab sample in a prepared sterilized sample bottle by holding the bottle near its base in the
hand and plunging it, neck downward, below the surface.
The bottle should then be turned until the neck faces the
outfall, and tilted upward. Lift straight out of the water,
capping immediately. Bacteria will use the dissolved oxygen so you must measure it right away, so grab a sample in
a bottle and read it right on the spot.
1. Water Plant Distribution sample: Within the service area, at a
metal, non-swivel faucet, remove any extra apparatus (filters
etc. including the screen).
• Flame the facade opening using a lighter.
• Turn on the cold water and allow it to run until the tap is
cool.
• Uncap the sample bottle; fill without overflowing the
bottle and recap
2. Swimming Pools: At sampling spigot, after filtration before
the water enters the pool.
• Flame the facade opening using a lighter.
• Turn on the cold water and allow it to run until the tap is
cool.
• Uncap the sample bottle; fill without overflowing the
bottle and recap.
3. Ground Water Well Source: At sampling spigot, after filtration before the water enters the pool.
• Flame the facade opening using a lighter.
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Turn on the cold water and allow it to run until the tap is
cool.
• Uncap the sample bottle; fill without overflowing the
bottle and recap.
4. Wells, Springs, Lakes, Bathing Beaches, and River Water:
Sodium Thiosulphate tablet is not needed.
• Holding the bottle near its base and plunge it, neck downward, below the surface. The bottle should then be turned
until the neck faces upward to the surface.
• Lift straight out of the water, capping immediately.
5. Water Supply Intakes (Surface Water Plants): Sodium Thiosulphate tablet is not needed.
• Holding the bottle near its base, plunge it, neck downward, below the surface.
• The bottle should then be turned until the neck faces
upward to the surface.
• Lift straight out of the water, capping immediately.
6. Raw and Chlorinated Sewage: Sodium Thiosulphate tablet is
not needed:
• Holding the bottle near its base, plunging it, neck downward, below the surface.
• The bottle should then be turned until the neck faces the
outfall, and tilted upward.
• Lift straight out of the water, capping immediately.
Samples may be preserved through immediate refrigeration or in cooler with ice (for field collection), then
refrigerate at 4 degrees Celsius for a holding time of 6 to
30 hours.
Test Procedure
1. Place pre-sterilized filter membrane on (0.45mm, grid marked)
porous plate of receptacle, with flame smooth-edged forceps.
2. Place filter holder into waste flask that is connected to a
vacuum. Place the filter receptacle onto the holder.
3. Shake sample bottle 10 seconds, hard, to loosen the colonies;
pour sample volume through filter membrane.
4. Rinse the interior of the filter 3 times with sterile water in 20 to
30 mL amounts.
5. Transfer filter membrane directly to Petri dish of M-Endo
Agar by rolling it to ensure that there is constant contact (no
bubbles between the filter and Agar). Re-roll the filter membrane to ensure constant contact.
6. Note: Flame forceps after each filter membrane transfer.
7. Invert Petri dish.
8. Place the inverted Petri dish in a waterproofed Plastic Bag
enclosure. Swirl and close the bag tightly. Place the bag so the
Petri dishes are bottom up on a rack in the hot water bath.
Water Bath
9. Incubate in the water bath at 35° C +/- .5° C for 22 - 24 hours.
5
10. After incubation count all colonies with 10 X 15 magnification
dissecting microscope. The colonies are pink to dark red color
with a metallic sheen.
11. If a plate doesn’t produce 20-80 colonies or more than 200,
throw out those results. If there is more than 1 plate with 20-80
colonies, average the results.
(10 + 12 + 0) divided by (50 + 5 + 1) X 100 = 39/100mL
If the plate counts are above 200 then report Too Numerous To Count (TNTC)
95% Confidence Limit
Upper limit = count + 2(square root of the count)
Lower limit = count - 2 (square root of the count)
Quality Assurance
• For each sample date a blank is analyzed.
• For each sample date duplicates are performed.
• Every other week (twice a month) a spiked sample is analyzed.
V. Calculation
Coliform Colonies and Volume Filtered
25 + 2(25 squared) = 35
25 - 2(25 squared) = 15
If all counts are between 20 - 80, then average the results. EXAMPLE:
In this example, the analytical result may be 10 more or 10 less than
what was counted. The analyst should tabulate or graph the results
to determine if there is a trend.
References: Standard Methods for Examination of Water and Wastewater
19th Edition
VI. Summary
(25 + 20 + 40) divided by (20 + 40 + 40) X 100 = 110/100 mL
If only one sample was between 20 and 80, the result is
expressed in Number / 100 mL. EXAMPLE:
40/10 X 100 = 400/100mL
If none of the plate counts are between 20 – 80, then add
them and a count was requested, divide by their sample
volume and report / 100 mL. EXAMPLE:
6
The purpose of a water treatment plant is to provide safe disinfected
drinking water. The Total Coliform analytical test can be used to
determine if the water has been adequately disinfected. Disinfection
is a treatment process used to destroy or inactivate disease-causing
(pathogenic) organisms. Diseases caused by these organisms in
water are called waterborne diseases, such as, Typhoid, Dysentery,
Cholera, Infectious hepatitis, Giardiasis, Cryptosporidiosis, and
Legionallosis. Some methods of disinfection include: Heat Treatment, Ultraviolet light, Chemical Treatment, and Chlorination.
Chlorination is the most common form of disinfection
in the United States. Chlorination is not only a useful disinfectant for storage tanks, pipes, but it also oxidizes iron,
manganese, and hydrogen sulfide as well as for controlling
taste, odor, algae, and slime. In addition, the low cost of
chlorine disinfection places this method as the most preferred.
If the operator has correctly fed the chlorine to effectively disinfect the drinking water, then the operator’s
Total Coliform results will be negative. If the results are
positive, then the test result is an indication for a high
likelihood that the water is contaminated by feces from
a warm-blooded animal. To correct the problem with
chlorine disinfection, the operator needs to inspect all
of the equipment and repair or replace damaged pumps,
switches, or piping. After the operator has determined that
the equipment is in working order, the operator should review the chlorine contact time his/her water system design.
The contact time is the product of disinfectant residual
and disinfectant contact time. Obviously, ensuring continuous disinfection feed for all water supplies is recomwww.WaterWorldCE.com
mended. The chlorine should be fed at the point that will
provide adequate contact time. Disinfection basins that
contain baffles will increase the contact time between the
chlorine and water minimizing the possibility of short
circuiting. From the end of the basin to the end of the
distribution system, the minimum free chlorine should be
0.2 to 0.5 milligrams per liter (mg/L). Chlorine residual
test equipment should be able to measure residuals to the
nearest 0.1mg/L.
Repair or replacing damaged pumps, switches, piping,
and/or adjusting the contact time may allow the operator
to get a negative total coliform test result, indicating the
water is not harmful to human health. Once the contact
time has been determined, maintaining clean disinfected
drinking water may be achieved through daily Total Coliform testing.
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Questions
1. When counting the colonies,
which ones do you include?
Choose the best response.
A. The colonies that exhibit sheen
B. All colonies are counted.
C. Only the blue or green colonies are
counted.
D. None of these
2. What should be done with the Petri
dish? Choose all that apply.
A. Inverted in a waterproofed plastic
bag enclosures
B. Closed in the bag tightly
C. Pre-sterilized
D. None of these
3. The correct microscope
used to count the colonies is:
Choose the best response.
A.
B.
C.
D.
Dissecting microscope
Compound microscope
Binocular microscope
None of these
4. U.S. EPA Total Coliform rule
only requires that a private or
public water system report:
Choose the best response.
A. a positive or negative.
B. the total count expressed in 100 mL.
C. the lowest count expressed in 100
mL.
D. the highest count expressed in 100
mL.
5. Coliform bacteria are found in:
Choose the best response.
A.
B.
C.
D.
warm blooded animals
all animals
only humans
none of these
6. If the plate counts are above 200,
then: Choose the best response.
A. report TNTC
B. add all of the results, then average
them and express per 100 mL
C. report the lowest count
D. report the highest count
7. Coliform bacteria are: Choose
the best response.
A. facultative anaerobic, gram
negative, non-spore-forming,
rod-shaped bacteria that produce
a red colony with a metallic sheen
within 24 hour incubation at
35° C on an Endo-type media
B. considered health pathogens
8
C. all aerobic and facultative
anaerobic bacteria that use
only human feces as food
D. none of these
8. After collection, the maximum holding
time is: Choose the best response.
A.
B.
C.
D.
30 hours
26 hours
20 hours
48 hours
9. Define “Sterilization”: Choose
the best response.
A.
B.
C.
D.
to make free of living organisms
to remove coliforms
to disinfect
none of these
10. The standard autoclave settings and
sterilization times for media used
in microbiological examination
are: Choose the best response.
A. 121° C for 15 min
B. 121° C for 18 min
C. 170° C for 15 min
11. What is the correct sample volume of
water for examining a drinking water
system? Choose the best response.
A.
B.
C.
D.
100 mL
1 mL
10 mL
50 mL
12. In the MF Total Coliform test, the analyst must maintain the water bath temperature at: Choose the best response.
A.
B.
C.
D.
35.0°C +/- 0.5°C
35.5°C +/- 0.2°C
44.5°C +/- 0.5°C
44.5°C +/- 0.2°C
13. When taking a water
sample from a drinking water
sample spigot, the sample spigot
must be sterilized. True or false?
A. True
B. False
14. When collecting a sample from
the drinking water distribution
system, which of the following must
be performed? Choose all that apply.
C. Flame the façade opening using a
lighter. Turn on the cold water and
allow it to run until the tap is cool.
D. None of these
15. Waterborne pathogens include:
Choose all that apply.
A.
B.
C.
D.
Giardia lamblia
Salmonella species
Shigella species
None of these
16. The 95% confidence limit assumes:
Choose the best response.
A. The bacteria are distributed
randomly and follow Poisson distribution.
B. The water sample is turbid.
C. The M.F. technique overestimates
the coliform density.
D. None of these
17. Calculate the 95% confidence for a
drinking water sample when the count
equals 25 and the volume filtered
equals 100. Choose the best response.
A.
B.
C.
D.
Upper limit = 35; Lower limit = 15
Upper limit = 45; Lower limit = 5
Upper limit = 25; Lower limit = 25
Upper limit = 20; Lower limit = 30
18. Sodium thiosulphate is
added to de-chlorinate the sample
thus allowing the viable coliforms
within the sample the ability to
produce colonies. True or false?
A. True
B. False
19. Can the Total Coliform count be
calculated from the following three
samples: 10 in 40 mL sample, 5 in
10 mL sample, 18 in 50 mL sample?
Choose the best response.
A. No
B. Yes
20. The Total Coliform test is used
to determine if the drinking
water supply is unsanitary by a
positive (growth) or negative (no
growth) result. True or false?
A. True
B. False
A. Sample from a metal, non-swivel
faucet, remove any extra apparatus
(filters etc. including the screen).
B. Uncap the sample bottle; fill without
overflowing the bottle and recap.
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How to Perform Fecal Coliform Analytical Test for Drinking Water Quality
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4. ❑ A ❑ B ❑ C ❑ D
5. ❑ A ❑ B ❑ C ❑ D
6. ❑ A ❑ B ❑ C ❑ D
7. ❑ A ❑ B ❑ C ❑ D
8. ❑ A ❑ B ❑ C ❑ D
9. ❑ A ❑ B ❑ C ❑ D
10. ❑ A ❑ B ❑ C ❑ D
11. ❑ A
12. ❑ A
13. ❑ A
14. ❑ A
15. ❑ A
16. ❑ A
17. ❑ A
18. ❑ A
19. ❑ A
20. ❑ A
❑B
❑B
❑B
❑B
❑B
❑B
❑B
❑B
❑B
❑B
❑C
❑C
❑C
❑C
❑C
❑C
❑C
❑C
❑C
❑C
❑D
❑D
❑D
❑D
❑D
❑D
❑D
❑D
❑D
❑D
COURSE EVALUATION and PARTICIPANT FEEDBACK
We encourage participant feedback pertaining to all courses. Please be sure to complete the survey included with the course and mail to:
[email protected].
RECORD KEEPING
PennWell maintains records of your successful completion of any exam. Please contact our offices for a copy of your professional
development hours report. This report, which will list all credits earned to date, will be generated and mailed to you within five business days
of receipt. © 2010 by the PennWell Corporation
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