Microbiology
Acute waterborne diseases such as cholera and typhoid fever were major epidemics in the late-
1800s and early-1900s. Methods to detect and remove these organisms were developed, and
water operators are responsible to ensure the water supply is safe.
Diseases caused by pathogenic bacteria, viruses, and protozoa can be transmitted through fecal
contamination to humans; and drinking water is just one of several carriers of these agents.
Pathogens are disease-producing organisms and the presence of these is often related to poor
sanitation practices. Microorganisms associated with recent waterborne outbreaks include the
protozoa, bacteria, and virus. The following lists some of the most common waterborne diseases
and their possible causes:
Waterborne
Pathogen Source of Pathogen Symptoms
Disease
Gastroenteritis Virus Animal or Human Diarrhea, Vomiting
Feces
Typhoid Fever Salmonella Typhosa Human Feces Inflamed Intestine,
Enlarged Spleen, High
Temperature
Dysentery Shigella species Human Feces Diarrhea
Cholera Vibrio Cholera Human Feces Vomiting, Severe
Diarrhea, Dehydration
Infectious Virus Human Feces, Shell Yellowed Skin,
Hepatitis Fish Abdominal Pain
Amebic Entamoeba Human Feces Mild Diarrhea
Dysentery Histolitca
Giardiasis Giardia Lamblia (a Animal or Human Diarrhea, Cramps, Nausea,
protozoa, one-celled Feces Weakness
animal)
Cryptosporidious Cryptosporiduim Animal or Human Diarrhea, Cramps, Nausea,
parrum Feces Weakness
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It is difficult, expensive and potentially hazardous to test for the presence of all types of
pathogens. Instead, the technician or operator tests for the presence of indicators, organisms that,
when present, indicate that pathogens may be present.
Coliforms have been chosen to be the bacterial group routinely tested to assess the
bacteriological safety of water. Presence of any of the coliform group of bacteria, i.e. total
coliforms, indicates general contamination, while the presence of fecal coliforms indicates
contamination of human or animal origin. An ideal indicator organism would have the following
characteristics:
• Indicator should always be absent in clean, uncontaminated water and present when
pathogens are present.
• Indicator should be present in large numbers in fecal matter.
• Indicator and pathogen should respond similarly to treatment processes.
• Indicator should be easy to isolate, identify, and count.
• Ratio of indicator to pathogen should be high.
• Indicator and pathogen should come from the same source.
While total coliform do not meet all the criteria in all cases, they are the best indicators available.
TOTAL COLIFORM
The total coliform group of bacteria has been used as indicators in water treatment since the early
1900s. This group of organisms is found both in soil and in the waste of warm-blooded animals.
It includes the Eschericia coli (E. coli), which is a common bacterium in the feces of warm-
blooded animals. USEPA has set microbiological standards for drinking water, and public water
suppliers that collect fewer than 40 samples per month are allowed one total coliform positive
per month. Systems that collect 40 or more samples per month are allowed 5 percent total
coliform positive results per month.
Fecal coliform provide stronger evidence of the possible presence of pathogens than do total
coliform. This group indicates the presence of fecal matter, which could be of human or animal
origin. One positive test does not prove that fecal contamination exists; and more tests must be
taken. Samples can be contaminated from external sources or there may have been other
problems such as unsterile bottles and laboratory error. The most common problem, however,
involve errors in sampling. The current regulations require that if a sample is positive (shows the
presence of coliforms), the water supplier must take four more samples, one at the same location
as the sample that was positive, one within five service connections on each side of the positive
location, and one from a representative site on the distribution system.
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HETEROTROPHIC PLATE COUNT
The total or heterotrophic plate count (HPC) test measures the numbers present of a large group
of bacteria, including both nonpathogens and pathogens. Because it does not isolate a specific
organism, the HPC cannot be correlated with the likelihood of waterborne-disease outbreak.
Water with a high HPC can contain many, few or no pathogens. The significance of using the
HPC test is that it indicates a generally poor biological water quality for PWSs using surface
water sources. Five-hundred colonies per milliliter have been suggested as an upper level, above
which corrective action should be taken. This is applicable only to surface water supplies.
The microbiological contaminant section of the Public Water Supply Regulations chapter shows
the frequency required.
SAMPLING AND SITE SELECTION
The number of samples required for the system is based on the size and number of people served
by the system. Routine sampling sites should be representative of the water system. The sites
should include some dead ends as well as areas where the flow is high. Sampling points may be
scattered across the system and can include such sites as park buildings and fire stations. It is
important that service lines are used and that the water being sampled comes directly from the
water system rather than from the indoor plumbing.
Sampling taps should be selected carefully, and taps that should be avoided, if possible, include:
• Outdoor faucets with a likelihood of contamination from the ground surface. Frost-free
hydrants should also be avoided since they can be contaminated by dust and snow.
• Mixing faucets where water from the hot side may not be representative of water from
the system.
• Faucets supplying dishwater in cafes, drug stores, or other sites that may contain higher
than normal bacterial contamination.
• Swing spouts because bacteria can grow where the faucet pivots.
• Leaky faucets or faucets that allow water to seep around the packing nut. A fixture in
poor condition can introduce contamination into the sample.
SAMPLE CONTAINER
Proper use of the sample container is important and the sample can be positive for total coliform
if the container becomes contaminated. Most laboratories supply sterilized sample bottles or bags
to be used when taking the samples in addition to approved mailing cartons and appropriate
forms. Sample containers may be made of glass or plastic.
• Bottles should have a wide mouth and a capacity of at least 125 milliliters. Only
containers that are sterile and contain sodium thiosulfate to neutralize the chlorine in the
water sample should be used. Most laboratories supply bottles that are ready for testing.
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• Caps used on the sample must be sterile. Sample bottles should be examined for possible
contamination. If the cap is loose, the bottle should not be used.
• Bottles that have been in storage for a long period of time have an increased likelihood of
contamination and should not be used.
• Bottles without a label may get misplaced at the laboratory.
SAMPLE COLLECTION
Technique used in taking the sample is important. The following steps should be followed:
• Remove the aerator, if there is one, from the faucet. Some utilities will allow the tap to
run for a short time and then flame the faucet with a torch. Flaming is not required but
will assure that the outside of the tap is sterile. Caution is required as plastic faucets may
melt if flamed. (The collector may also sterilize the tap with an alcohol pad.)
• Turn on the faucet and allow it to run for five to six minutes so that the sample drawn is
from the distribution system, not the interior plumbing of the building. If the service is
large, the tap should be run longer. The hot-water tap should not be used for sampling.
• After the lines have been flushed, open the container and hang on to the bottle cap, taking
care to not touch the inside of the cap. Do not rinse the container and do not overfill.
• Place the sample container in the flow from the faucet without contacting the tap. Fill the
bottle to the fill line, if provided. Leave space at the top of the container so that the
laboratory can mix the sample before testing it. If the bottle is overfilled, take a new
sample with a new container. The minimum sample required at the laboratory is 100 ml,
and it is important not to flush out the preservative in the bottle.
• Seal the container as soon as it is filled and removed from the flow. If a sample bag is
used, be sure it is sealed properly; these bags can leak easily.
Do the necessary paperwork, which includes filling in the PWS identification number, time, date,
collector name, and location taken on the sample’s label and on the reporting forms. The forms
may require other information, such as pH, chlorine residual, and iron concentration.
If additional tests are required, don’t use the same bottle used for total coliforms. Instead take a
different sample from the same water faucet. Different types of tests require different types of
bottles. Consult with laboratory prior to taking other types of samples to ensure that you use the
correct sample bottle.
TEST METHODS
All coliform tests done for compliance with the Safe Drinking Water Act must be performed by a
laboratory approved for such testing by the Minnesota Department of Health. Three basic test
methods are used to establish the presence of coliform bacteria. Selection of the test method is
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the responsibility of the laboratory and MDH. The laboratory must be certified by the state to
perform the method used. The technicians doing the tests must also be certified.
MULTIPLE TUBE FERMENTATION
Multiple tube fermentation is very rarely used. The
multiple tube fermentation or most probable number
(MPN) test progresses through two steps, the
presumptive and confirmed test. A final check may
be done by use of the completed test. The
presumptive test is not exclusive for coliform
bacteria; some other bacteria present in soil or water
may also produce a presumptive test, but the
confirmed test is specific for coliform bacteria. The
completed test, used for quality-control purposes,
definitely establishes the presence of coliform
bacteria. Bacteriological testing of most public
water supplies stops after the confirmed test. This is
the minimum test required of the positive samples.
The presumptive test is the first step of the MPN test. Samples are poured into each of five tubes
containing a culture media and an inverted vial. The samples are incubated at 37° C (98.6° F) for
24 hours, checked and then incubated for another 24 hours, then checked again. If coliform
bacteria are present, gas will be forming in the inverted vial within the 48-hour period. This
indicates a presumptive positive sample. If no gas forms, the sample is considered negative.
The confirmed test is more selective for coliform bacteria. Cultures from the positive samples in
the presumptive test are transferred to brilliant green lactose bile broth tubes, also containing
inverted vials, and incubated. If no gas is produced after 48 hours, the test is negative, meaning
no coliform bacteria are present. If gas is produced, the test is positive, indicating the presence of
coliform bacteria. From the number of positive samples found, the technician uses statistical
(MPN) tables to determine the number of coliforms present in the original sample.
If further confirmation is needed, positive samples may be transferred to the completed test
where the selection for coliform bacteria is even closer than for the confirmed test. The positive
sample is transferred to a plate containing a special growth media and incubated for up to 24
hours. A second portion is placed in a lauryl tryptose broth and incubated for the same 18 to 24
hours. The completed test is positive if gas is formed in the lauryl tryptose broth and coliform
bacteria are found on the plate. If no gas is formed, the test is considered negative.
MEMBRANE FILTER METHOD
Many private laboratories still use the membrane filter technique, but it is becoming less
common. The membrane technique was, until recently, the most common method used to isolate
coliform bacteria. A given size sample, generally 100 milliliters, is filtered through a membrane,
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small-pore filter, which is then incubated in contact with a selective culture agar at 37° C (98.6°
F). A coliform bacteria colony will develop at each point on the membrane where a viable
coliform was left on the membrane during filtration. After the incubation period of 24 hours, the
number of colonies per plate is counted. They represent the actual number of coliforms that were
present in the volume of samples filtered.
A typical coliform bacteria colony is pink to dark red with a distinctive green metallic sheen on
the surface. All organisms that produce such colonies within 24 hours are considered members of
the coliform group.
COLILERT TEST
The colilert test is probably the most widely used coliform detection method at this time and is a
method accepted by the U. S. EPA for coliform testing. The colilert test is used for simultaneous
detection and confirmation of both total coliforms and Eschericia coli (E. coli) fecal coliforms.
As the colilert test is a presence/absence test, it does not indicate the extent of contamination.
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The colilert test method is just as accurate as the membrane filtration method and many believe it
is more sensitive than the other methods.
The colilert reagent contains a formulation of salts,
nitrogen, and carbon sources that are specific to total
coliform. It contains specific indicator nutrients that
create a yellow color when total coliforms are present
and fluorescence when E. coil is present. The reagent is
added to a 100-milliliter water sample in a sterile, non-
fluorescent borosilicate glass container. The vessel is
capped and shaken vigorously by repeated inversion to
aid in mixing of the reagent. It is then incubated at 35° C
for 24 hours. (An 18-hour preliminary test may be
requested if there is reason to need test results more
quickly.) After 24 hours, the technician compares the
reaction vessels to the color in a comparator supplied
with the test kit. If the inoculated reagent has a yellow
color equal to or greater than the comparator, the
presence of total coliform bacteria is confirmed.
The technician tests each reaction vessel for
fluorescence by placing it five inches from an ultraviolet
light in a dark environment. If the
POSITIVE TOTAL COLIFORM RESULT
When total coliform are found in a routine sample, the
water supplier must collect at least four repeat samples
for each coliform positive sample found. One repeat
sample shall be from the site of the original positive, one
from within five service connections upstream, one from
within five service connections downstream, and one
from another representative site on distribution system.
Results of samples found to contain coliform bacteria and results of all repeat samples must be
reported to MDH within 24 hours. Any routine or repeat samples that are found to be positive for
total coliform must be analyzed for fecal coliform or E. coli. When maximum microbiological
contaminant levels are exceeded, the water supplier must use the proper notification process to
inform the affected consumers.
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Notes:
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