Examination of Water Quality Using Bacterial Indicators
The microbiologist is often required to estimate the number of microorganisms in a sample of water, food, beverage,
or other product. In addition to the number of microbes present, it may also be necessary to determine which types of
microbes are present to assess the quality of the product. For bacteria, many direct methods are available for their
enumeration. These methods include: (1) direct microscopic count, (2) electronic particle count, (3) DNA probes, (4)
most probable number, (5) dilution/spread plate, (6) dilution/pour plate, and (7) the membrane filtration technique.
The last four methods are commonly used for estimating the number of viable (living) bacteria and may require
sample dilution prior to plating/testing. Sometimes quantifying the number of bacteria is unnecessary. Instead, testing
is done to determine the presence or absence of specific bacteria using quality testing rather than quantity. Water
quality testing extends beyond detection of bacteria. Several other methods analyze water quality by the presence or
absence of specific viruses (mainly bacteriophage), “algae” and “protozoa”.
Heterotroph Counts
Heterotroph is the term used to describe organisms that use organic compounds for carbon and energy sources.
Determining heterotrophic counts in water is often used to show enrichment of water with agriculture or industrial
wastes, or to determine the quality and success of treating of drinking water. The majority of heterotrophs in aquatic
environments are saprophytic bacteria and fungi that decompose non-living materials such as leaf litter, fecal waste,
dead algae and dead animals. Population growth is directly proportional to the availability of organic matter such as
plant and animal detritus.
The process of determining heterotroph concentrations is done by the heterotroph plate count (HPC), whereby the
concentration of heterotrophic bacteria and fungi are determined. There is no “universal” HPC measurement. The
types of heterotrophs recovered is dependent on the growth media, temperature, atmosphere (aerobic or anaerobic),
and the length of incubation. Often, the test does not specify the specific bacteria or fungi that are detected, and only
a small portion of the population is actually culturable. Typically, microbes that are recovered through HPC include
those that are part of the natural microbiota of water; and in some instances, they may also include those derived
from diverse pollutant sources. To date, there are over 60 genera of bacteria recognized as normal constituents of
freshwater systems.
So how can HPC be applied to determining water quality? Within the heterotrophs are groups of bacteria that are
primarily associated with the gastrointestinal system of warm-blooded animals and not naturally occurring in aquatic
systems. These include the bacteria E. coli, Enterococcus, Clostridium perfringens, Bacteroides, Bifidobacterium.
Of these, E. coli and Enterococcus are the most widely used indicator organisms to determine water quality, although
several reports indicate the anaerobes Clostridium, Bacteroides, Bifidobacterium are better indicators. However,
culturing of anaerobic bacteria requires special handling and atmospheric conditions as they tend to be sensitive to
oxygen.
Fecal Coliform
Coliforms are a specific group of bacteria that are normal inhabitants of the gastrointestinal tract of animals and
found in the environment. Since 1885, it has been recognized that coliform bacteria were numerous and always
detected in animal feces and sewage. It was further discovered (1899) that these bacteria were detectable after
sewage had been diluted more than 10,000 fold when obvious signs of sewage pollution were not evident. The
concentration of E. coli in the human intestines is 1.5 x 109 cells/gram feces. Although this number may be
impressive, facultative anaerobic bacteria like E. coli are outnumbered by the anaerobic bacteria by 10 1 to 102 times.
Coliform bacteria are characterized as regular, rod-shaped Gram-negative bacteria that are able to ferment the sugar
lactose. Furthermore, these bacteria produce easily recognizable reactions when grown in/on specially formulated
laboratory media used to enhance their growth, but suppress the growth of other bacteria. Although the coliforms
may not cause disease (although some can), their presence indicates that other gastrointestinal pathogens such as
rotaviruses, hepatitis A virus, Salmonella, Shigella, Campylobacter, Giardia, Cryptosporidium may be present. Thus
coliforms are referred to as indicator organisms because their presence often indicates the presence of feces from
humans and other warm-blooded animals and concurrent existence of more serious pathogens.
1
Coliforms that normally reside in the intestines of warm-blooded animals have the abilities to grow at 44.5oC and
produce gas from the fermentation of lactose. These thermotolerant coliforms are referred to as fecal coliforms;
however, some coliforms (Klebsiella, Citrobacter, Enterobacter) that are associated with industrial effluents (i.e.
paper mill pulp wastes), decaying plant material or soil may also grow at elevated temperatures. Escherichia coli is a
thermotolerant, fecal coliform that inhabits the intestines of warm-blooded animals and reaches concentrations of
approximately one billion cells/ ounce feces. Although thermotolerant coliforms other than E. coli may grow under
the same testing conditions, the concentrations of fecal coliforms are, under most circumstances, directly related to
E. coli.
Water quality standards are set by each state to achieve the goals mandated by the Clean Water Act for “the
protection and propagation of fish, shellfish, and wildlife and provides for recreation in and on the water.” In
Georgia for the months of May through October, the quality limit for fecal coliforms in free-flowing freshwater
streams impacted by non-human sources is 500 bacteria/100 ml (geometric mean). For streams impacted primarily
by human sources, the limit is 200 bacteria/ 100 ml. For the months of November through April when recreational
contact with the water is less likely, the geometric mean limit is 1000/ 100 ml or 4000/ 100 ml for any individual
sample. Above this limit, the probability of illness through contact with the water increases as the counts increase.
The quality standard for drinking water is a strict 0 fecal coliforms/100 ml.. In drinking water, it is often enough to
test for the presence or absence of fecal coliforms or enterococci without the need to enumerate.
Although some strains of E. coli cause human disease, its primary uses in water quality analyses are to indicate the
fecal contamination and the potential presence of other gastrointestinal pathogens. The USEPA recommended
recreational water (swimming, fishing) quality standard for E. coli is based on two criteria: 1) a geometric mean of
126 organisms/100 ml based on several samples collected during dry weather conditions or 2) 235 organisms/100 ml
for any single water sample (USEPA, 1986). The current EPA water quality standard for E. coli corresponds to
approximately 8 gastrointestinal illnesses per 1000 swimmers. As the concentration of E. coli rises, so does the
number of illnesses per 1000 swimmers. GA DNR (2002) has recently proposed a change in the state water quality
standards using E. coli and a geometric mean of 548/100 ml based on a minimum 4 samples over 30 days (at least 24
hrs between samples). Water may not exceed a single sample of 1,783/100 ml from the months of May through
October. In the months of November to April, the proposed water quality limit is a geometric mean of 2,740/100 ml
or 8,915/100 ml on a single sample.
Enterococci Concentrations
Like coliforms, the fecal streptococci are bacteria that reside in the intestines of warm-blooded animals and are used
to indicate fecal contamination. The enterococci were once part of the fecal streptococci group, but are now
classified separately. These bacteria provide valuable supplementary data on the bacteriological quality of natural
water systems, especially because these bacteria rarely multiply in polluted water and are more persistent (survive
longer) than E. coli. High enterococci concentrations are usually associated with raw sewage discharge and
discharge/run-off from meat processing plants, dairy farms or feedlots and livestock range. Enterococcus faecalis
and Enterococcus faecium are the species found to dominate the human intestines (especially E. faecalis in the U.S.).
These two species also are found in other animals such as birds and cattle. Determining the species of enterococci or
fecal streptococci in a water sample may provide general information about the probable animal source. The quality
limit for recreational freshwaters is 33 enterococci/100 ml and 35 enterococci/100 ml for marine waters (geometric
mean). Traits used to identify the enterococci are spherical-shape (coccus), Gram-positive staining, resistant to
sodium azide, lack the enzyme catalase, utilize esculin (a carbohydrate) and can grow in broth containing 6.5% salt.
Whether testing for fecal coliforms or enterococci, it is generally accepted that these fecal indicator organisms
cannot grow in natural environments outside their host, because they are adapted to living in the gastrointestinal
tract. Temperatures exceeding growth tolerance, sunlight, competition from other bacteria, predation by protozoa
and other microorganisms, lack of food sources and inhibitory environmental chemicals are reported to limit the
survival time of intestinal bacteria from a few hours to days in water. The length of survival is extended for days to
possibly months in relatively protected (lack predators, toxins, sunlight and are cooler), moist, nutritive environments
such as sediments and deep soils (>40cm). Thus the concentration of bacteria is highly dependent on the timing of
the sampling, amount of fecal matter dose and dilution, and environmental conditions. In general, the closer a sample
is taken to the time and/or entry point of contamination, then the higher the concentrations of the bacteria. Inversely,
2
the longer and/or further away from the point of entry, the detectable concentrations of bacteria will be lower. It is
assumed that gastrointestinal pathogens in fecal matter will die at the same rate as the indicator bacteria; therefore,
high concentrations of fecal indicator bacteria indicate an increased likelihood of pathogens being present. It is
known that some disease-causing protozoa survive much longer than the indicator bacteria. E. coli and enterococci
are considered to have a higher degree of association with outbreaks of gastrointestinal diseases than fecal coliforms
and were recommended as the basis for bacterial water quality standards in the 1986 USEPA document Ambient
Water Quality Criteria for Bacteria. Quality standards are established on the basis of an acceptable risk model
generated from epidemiological studies. The acceptable risks model assumes that every activity, such as swimming
in a lake, carries a certain risk of illness and that most people are willing to risk becoming ill or sick from
participating in water-related activities if the probability of illness is low. For example, a concentration of 35
enterococci/ 100ml correlates to an expected illness rate of 19 diarrhea diseases per 1,000 swimmers, or a 1.9
percent chance of becoming ill. Thus, the probability of becoming ill from swimming in waters with enterococci
counts below 35/100 ml is low and acceptable.
Dilutions and Viable Plate Count
High numbers of bacteria are often present in samples. Some samples may contain up to 108 bacteria/ml. At this
concentration, a 0.1 ml volume plated onto a nutrient agar plate could potentially yield 10,000,000 colonies. Needless-tosay, counting one plate would be a tedious and somewhat impossible task. To overcome this problem, serial dilutions of
the sample prior to plating reduces the number of organisms such that when a small volume of the diluted sample is
plated, the colonies are countable. Remember that one colony arises from a single bacterium so the number of colonies
counted on the plate is representative of the number of viable bacteria, referred to as colony forming units (CFU), in the
sample. For statistical accuracy, only plates containing between 30 and 300 colonies for 100 mm Petri plates and
between 20 and 200 colonies for 50 mm plates are counted. Therefore, a suspension containing 10 8 bacteria/ml will
require a dilution of 10-6 (1/1,000,000) to achieve a countable number of 100 CFU.
Figure 1. Photographs of plates with countable colonies (left), too few colonies to count (middle) and too numerous
colonies to count (right). Colonies are the white circular forms and theoretically arise from a single cell called a
colony forming unit. (A Photographic Atlas for the Microbiology Laboratory. Michael J. Leboffe and Burton E.
Pierce. 2nd Edition. Pp 83-85, Morton Publishing Co, Englewood, CO).
Suppose flask A contained a bacterial suspension having a concentration of 10 6 bacteria/ml. If 1 ml of the suspension
from flask A (106 bacteria/ml x 1 ml= 106 bacteria) was transferred to flask B which contained 99 ml of a sterile,
isotonic saline solution (called a blank), then flask B would now have 10 6 bacteria/100 ml or 104 bacteria/ml. Notice
that the concentration in B is only 1/100th (104/106) of the original concentration in A, thus it represents a 1/100
dilution. Another way of determining dilutions is by using the following formula:
Dilution = Volume transferred/(Volume transferred + Volume of diluent)
Referring back to the example given above, since 1 ml was transferred into a diluent volume of 99 ml, then 1/(1 +
99)= 1/100. If 0.5 ml was transferred to a tube containing 4.5 ml of sterile saline, then the dilution would be 0.5/(0.5
+ 4.5) = 0.5/5.0 or 1/10. Since the number of cells present in a sample is unknown, it is necessary to make several
dilutions so that some dilution will contain a countable number of cells when plated. As a general rule the more
turbid the suspension, then more organisms are in the suspension. If a sample requires dilutions greater than 1/100,
3
then dilution is accomplished in series.
The serial dilution method is illustrated in the schematic diagram below. A calibrated volume of sample is
transferred from tube A to tube B in the first step. After tube B is mixed, a calibrated volume is transferred from tube
B to C, and then C to D, and so on throughout the series. Each tube represents a dilution. The overall dilution of the
sample is calculated by multiplying the dilutions of each individual tube. For example, the overall dilution in tube B
is 1/10 (1/1 + 9), in tube C is 1/100 (1/10 B x 1/10 C= 1/100), and in tube D it is 1/1000 (10 x 10 x 10). A 0.1 ml
volume from the last two or three dilutions is spread onto the surface of separate agar plates, the plates are incubated,
and the colonies are counted (plates with 30 to 300 colonies or 20 to 200 colonies).
Figure 2. Illustration showing serial dilutions for 1/2, 1/5 and 1/10 series.
To determine the concentration of bacteria in the original sample, the colonies are counted, the count is divided by
the volume plated, and then multiplied by the reciprocal of the total dilution factor:
CFU (bacteria)/ml = (Number of CFU/Volume plated in ml) X (1/Product of all dilution factors)
Suppose that a 0.1 ml sample was plated and after incubation, the plate had 50 colonies. The concentration of
bacteria in the tube from where the sample came can be interpreted as 50 CFU/0.1 ml or 500 CFU/ml. If this tube
represents a 10-6 (1/1,000,000) dilution of the original sample, then the original sample must contain 106 times as
many organisms, which can be written as 500 CFU/ml x 106 or 5.0 x 108 CFU/ml. To increase the accuracy of the
results, it is best to plate the samples in duplicate or triplicate and then determine the average count. Major sources
of error include inaccurate volumes of diluent and/or inaccurate transfer of volumes. Bacterial cells that adhere to
each other give rise to a single colony and produce low estimations of bacterial numbers.
4
Table 1. Deciding how far to dilute a sample depends on the predicted count. Suggested dilutions for different
samples are presented here:
Water source:
Iced tea
Pasteurized milk
Drinking water
Swimming pool
Wells, springs
Clear lakes, ponds
Stagnant lakes, ponds
River water
Toilet water (city)
Toilet water (rural)
Raw sewage effluent
0
+
+
+
+
+
10-1
+
+
+
+
+
+
+
+
+
10-2
+
+
+
+
+
+
+
+
+
+
10-3
+
+
+
+
+
10-4
10-5
10-6
10-7
10-8
+
+
+
+
+
The viable plate count and membrane filtration methods are often used to determine the concentration of specific
bacterial groups such as heterotrophs, coliforms, fecal coliforms and enterococci in water or fluid samples. However,
when the water has a high amount of suspended solids, it is often difficult to accurately pipette volumes due to
plugging of the pipette bore or clogging membrane filters with debris. Additionally, bacteria that adhere to
particulate matter may not be disbursed evenly in the sample. At best, these methods indicate the minimal
concentration of specific microbial groups in a sample. The application of these methods is best in fluids without
much suspended solids. The advantage of membrane filtration is that large volumes of clear fluid, mainly water, can
be filtered in order to detect the presence of bacteria or protozoa or algae that might be present in small numbers.
Most Probable Number
The most probable number test, also called the multiple tube fermentation assay, is an alternative to plate counts or
membrane techniques, especially for samples with higher turbidity. The MPN procedure is a tube-dilution method
using a nutrient-rich medium, which is less sensitive to toxicity and supports the growth of environmental-stressed
organisms. The MPN method detects and estimates the bacteria in water samples (and can be applied to foods and
soils) by the multiple fermentation tube technique. The number of bacteria per 100 ml of sample is estimated by the
use of probability tables. The method has two test stages: The Presumptive Test, and the Confirmed Test. The
presumptive tests are designed to grow the target bacteria. The media used in the confirmed tests are designed to
validate the growth of target bacteria in the presumptive test. Confirmed test conditions are usually more stringent
than presumptive conditions. Thus, the presumptive test provides a preliminary estimate of bacterial density based on
enrichment in minimally restrictive tube media. The results of this test are never used without further analysis; the
MPN must be carried through the Confirmed Test for valid results. The MPN per 100 ml is calculated from the MPN
table based upon the Confirmed Test results. This value is based on the number of positive and negative results
observed when five 10 ml, five 1.0 ml and five 0.1 ml volumes of sample are tested in confirmed/completed tests.
For example, if 3 of 5 tubes in the first series (10 ml) were positive, 2 of 5 were positive in the second series (1.0 ml)
and 1 of 5 positive in the third series (0.1 ml) for confirmed tests, then the pattern is read as 3-2-1. Referring to the
MPN index, 3-2-1 implies that the most probable number of bacteria is 17/100 ml. However, the actual range may be
between 7 and 40 (95% confidence).
The method is applicable to the examination of many bacterial groups including total coliforms and fecal coliforms
in chlorinated primary effluents and under other stressed conditions. The multiple-tube procedure is also better suited
for the examination of turbid samples, muds, sediments, or sludge because particulates do not interfere visibly with
the test.
The time required for the test may be as long as 96 hours for Confirmed Test results. The man-hour requirements to
prepare glassware and media and to perform the tests are also significant. The MPN tables (listed below) are
probability calculations, inherently having poor precision.
5
Table 2. Most probable number of bacteria per 100 ml of sample according to positive test results from 15 tubes at
three different dilutions.
Most Probable Number Index and 95 % Confidence Limits for Five Tube, Three Dilution Series
No. Of Tubes Giving Positive Reaction out of:
MPN Index per 95% Confidence Limits:
5 of 10 ml
5 of 1 ml
5 of 0.1 ml
100 ml:
Lower
Upper
0
0
0
<2
0
0
1
2
<0.5
7
0
1
0
2
<0.5
7
0
2
0
4
<0.5
11
1
0
0
2
<0.5
7
1
0
1
4
<0.5
11
1
1
0
4
<0.5
11
1
1
1
6
<0.5
15
1
2
0
6
<0.5
15
2
0
0
5
<0.5
13
2
0
1
7
1
17
2
1
0
7
1
17
2
1
1
9
2
21
2
2
0
9
2
21
2
3
0
12
3
28
3
0
0
8
3
24
3
0
1
11
4
29
3
1
0
11
4
29
3
1
1
14
6
35
3
2
0
14
6
35
3
2
1
17
7
40
4
0
0
13
5
38
4
0
1
17
7
45
4
1
0
17
7
46
4
1
1
21
9
55
4
1
2
26
12
63
4
2
0
22
9
56
4
2
1
26
9
78
4
3
0
27
9
80
4
3
1
33
11
93
4
4
0
34
12
93
5
0
0
23
7
70
5
0
1
31
11
89
5
0
2
43
15
110
5
1
0
33
11
93
5
1
1
46
16
120
5
1
2
63
21
150
5
2
0
49
17
130
5
2
1
70
23
170
5
2
2
94
28
220
5
3
0
79
25
190
5
3
1
110
31
250
5
3
2
140
37
340
5
3
3
180
44
500
5
4
0
130
35
300
5
4
1
170
43
490
5
4
2
220
57
700
5
4
3
280
90
850
5
4
4
350
120
1000
5
5
0
240
68
750
5
5
1
350
120
1000
5
5
2
540
180
1400
5
5
3
920
300
3200
5
5
4
1600
640
5800
5
5
5
1600
-
6
Membrane Filtration Technique
The membrane filter (MF) technique is highly reproducible, can be used to test relatively large volumes of sample,
and yields numerical results more rapidly than the multiple-tube procedure. The membrane filter technique is
extremely useful in monitoring drinking water and a variety of natural waters. In this technique, water samples are
drawn through a membrane filter with a particular pore size. Most filters used in this process have pore sizes of 0.22
or 0.45 microns (μm). The pores allow matter smaller than 0.22 or 0.45 um to pass through the filter, whereas
matter/cells that is larger gets caught on top of the filter. Therefore, microorganisms that are usually 1.0 um and
larger are caught on top of the filter. The filter is then removed and placed on top of a growth medium. The nutrients
freely diffuse through the membrane and the microbial cells are able to grow on top of the membrane.
The MF technique has limitations, particularly when testing waters with high turbidity or noncoliform (background)
bacteria. Water that contains a large amount of suspended solids usually results in filter clogging. Large numbers of
non-coliform bacteria may interfere with coliform growth or cause false-positive reactions. For such waters or when
the membrane filter technique has not been used previously, it is desirable to conduct parallel tests with the multipletube fermentation technique to demonstrate applicability and comparability.
The growth medium used in the MF technique often contains chromogenic substrates such that the growth of specific
groups of bacteria is detected by color change. These substrates are generally acted upon only by the enzymes
produced by a specific bacterial group. For example, the medium mEI has the substrate indoxyl-β-D-glucoside (as
well as ingredients to inhibit fungi and gram-negative bacteria). Enterococci produce the enzyme β-D-glucosidase.
When the enzyme acts upon the substrate, an insoluble indigo blue complex is formed, therefore colonies of
enterococci are seen as indigo blue.
Similar to detecting enterococci, media have been developed for the detection of E. coli. In the selective medium
mTEC, the chromogen 5-bromo-6- chloro-3-indolyl-β-D-glucoronide is acted upon by the β-D-glucoronidase
produced by E. coli. The result is glucoronic acid and an insoluble red/magenta colored compound. Therefore,
colonies of E. coli are detectable by their red to magenta coloring. Detegent compounds are also added to the
medium to inhibit growth of other gram-negative bacteria. Lactose is included as the primary carbohydrate source for
growth of E. coli.
Some members of the total coliform group may produce a dark red or nucleated colony without a metallic sheen.
When verified these are classified as atypical coliform colonies. When purified cultures of coliform bacteria are
tested they produce a negative cytochrome oxidase (CO) and positive Beta-galactosidase (ONPG) reaction.
Generally, all red, pink, blue, white, or colorless colonies lacking sheen are considered non-coliforms by this
technique.
Objectives
In this lab, students will use the spread plate, most probable number and membrane filtration techniques to determine
the concentration of heterotrophs, fecal coliforms and enterococci in water samples. Detection and enumeration of
specific indicator groups will be based on culture methods such as lactose fermentation (E. coli) and esculin
hydrolysis (enterococci), presence of turbidity, and/or characteristic colors or fluorescence. New testing methods
use enzyme-substrate reactions that result in specific color changes or fluorescence to detect indicator bacteria. For
example, the chromogenic substrate indoxyl-ß-D-glucuronide or the fluorogenic substrate 4-methylumbelliferyl-ß-Dglucuronide (MUG) are designed to react with specific enzymes (generally, ß-glucuronidase in E. coli and ßglucosidase in enterococci) to produce color changes or fluorescence to detect the target organism. This testing
methodology will be used in the multiple tube fermentation and membrane filtration assays in this lab exercise to
determine the concentration of E. coli and enterococci in water samples.
7
Sampling
Obtain four sterile water sample bottles. Label the bottles with the date, group number, and sample source. Fill out
the chain of custody form. Fill the bottles with water samples from a lake or other natural body of water. Be sure to
collect water that has not been subjected to sediment disturbance as a result of walking in the sediment by the
sampler. Transport back to lab. Water samples should be processed within 1 hour of sampling. If longer, place on ice
during transport.
Spread Plate for HPC, Coliform count and Enterococci Count
1. Make 1/10 dilutions of your sample according to table 1. Use a micropipetter to transfer 1.0 ml (1000 ul) from the
source to a tube containing 9.0 ml diluent. Mix. This is a 1/10 (10-1) dilution. Transfer 1.0 ml from the 1/10 dilution
tube to a second tube containing 9.0 ml diluent. Mix. From tube 1 to 2, another 1/10 dilution was made. However,
the dilution from the original sample was 1/10 x 1/10 = 1/100 (10 -2) dilution.
2. Once your dilutions are made, set a micropipetter to 100 ul (0.1 ml). You will need two each of tryptic soy agar
(TSA), eosin methylene blue agar (EMB) and bile esculin agar (BEA) plates for each dilution you are plating. Label,
the bottom of the plates with your group, agar type, date and dilution. Beginning with the highest dilution, transfer
0.1 ml onto each plate corresponding to the dilution
3. Sterilize a glass L-shaped rod by dipping in alcohol and passing through a flame. Once the alcohol has burned off,
use the rod to spread the sample across the surface of the plate. Sterilize the glass rod between plates.
4. Incubate the EMB plate at 44.5oC and the Bile esculin plate at 41.0oC and TSA at 30oC.
5. The next lab period, examine the EMB plates. All colonies that are dark purple, and dark purple with a green
sheen are fecal coliforms. Record the data. Calculate the concentration of fecal coliforms/100 ml.
6. Examine the BE plates. All colonies exhibiting a brown reaction on the agar are counted as enterococci. Record
the data. Calculate the concentration of fecal coliforms/100 ml.
Multiple Tube Fermentation:
1. Each group will get two sets of 15 tubes. One set will contain lauryl tryptose broth (LTB) for determining fecal
coliform concentration. The second set will contain azide dextrose broth (ADB) for determining enterococci
concentration.
2. To the first row containing double strength broth, use a 10 ml pipette to aseptically transfer 10 ml of your
undiluted water sample to each tube.
3. To the second row containing single strength broth, use a micropipetter to aseptically transfer 1.0 ml of your
undiluted water sample to each tube.
4. To the third row containing single strength broth, use a micropipetter to aseptically transfer 0.1 ml of your
undiluted water sample to each tube.
5. Incubate the lauryl tryptose broth tubes in a 35oC water bath for 24-48 hours.
6. Incubate the azide dextrose broth tubes in a 41 oC water bath for 24-48 hours.
7. Fecal Coloforms: The next lab period, examine the tubes of lauryl tryptose for growth (turbidity) and gas. Record
the number of positive tubes for each row. Refer to table to for the MPN. This is a total coliform count.
8. For each positive tube, transfer a loopful of broth to a tube of sterile EC medium with MUG. Remember to label
which row the sample came from (10 ml, 1 ml or 0.1 ml). Inoculate a positive control tube with Escherichia coli and
a negative control tube with Proteus vulgaris. Place your tubes in a 44.5 oC water bath for 24-48 hours. Examine the
tubes for growth (turbidity). All tubes showing growth will be examined for fluorescence under long wave UV light.
Place all tubes with growth, the positive control, negative control and a tube with no growth under long wave UV
light. Record the number of tubes that fluoresce.
9. Refer to table 2 for the MPN based on the pattern of positive results. This will be your confirmed fecal coliform
concentration.
10. Enterococci: In the second lab period, examine the tubes of azide dextrose broth for growth (turbidity). Record
the number of positive tubes for each row. Refer to table to for the MPN. This is your presumptive enterococci
count.
11. For each positive tube, transfer a loopful of broth to sterile brain heart infusion broth supplemented with 6.5%
salt. At the same time, inoculate a sector on bile esculin agar. Incubate the tubes and plates at 37 oC.
12. After 24 hours incubation, tubes positive for growth (turbid) and brown reaction on bile esculin agar are
confirmed positive results for enterococci.
13. Refer to table 2 for the MPN based on the pattern of positive results. This will be your confirmed enterococci
8
concentration.
Membrane Filtration
1. The instructor will demonstrate how to operate the vacuum manifold for membrane filtration.
2. Aseptically place a sterile 0.45 um membrane filter onto the reservoir apparatus.
3. If using drinking water, tap water or toilet water, add 100 ml to the reservoir, If using pond water, add 50 ml
sterile water to the reservoir, then add 10-50 ml of pond water. The more turbid the water is, then use a smaller the
volume of sample. If possible, fill to 100 ml mark with more sterile water. If using raw sewage water, fill reservoir to
100 ml with sterile water. Use a micropipetter to transfer 0.01 ml (10 ul) of waste water to the reservoir.
4. Turn on vacuum pump and open the channels. When the entire sample has been filtered through the membrane,
turn off the pump.
5. Aseptically remove the filter and place it on top of mEI agar. Try to avoid creating any air bubbles between the
agar and the bottom of the membrane. Repeat for a second mEI plate. This agar is used for enterococci counts. Place
these membranes in a 41oC incubator.
6. Repeat the procedure, but place the next two filters onto mTEC agar plates. This agar is used for E. coli counts.
Place plates in 35oC incubator for 2 hrs, then transfer to 44.5 oC incubator for 22 hours.
7. Enterococci. The next lab period, examine the mEI plates. Any colony exhibiting a blue halo regardless of colony
color is counted as Enterococcus. Record the data and calculate the number of enteroccocci per 100 ml.
8. E. coli. Examine the mTEC plates. Any colony exhibiting red or magenta appearance is counted as E. coli.
Record the data and calculate the number of E. coli per 100 ml.
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Examination of Water Quality Using Bacterial Indicators
1A. Spread Plate: Fecal Coliforms
Water Source
Avg. CFU
Dilution
*CFU/ml
**CFU/100 ml
*CFU/ml
**CFU/100 ml
*CFU/ml
**CFU/100 ml
2. A. Multiple Tube Fermentation: Fecal Coliforms
Water Source
Presumptive
Confirmed Positive
CFU/100 ml
Tubes
Confirmed
CFU/100 ml
Range
2B. Multiple Tube Fermentation: Enterococci
Water Source
Presumptive
Confirmed Positive
CFU/100 ml
Tubes
Confirmed
CFU/100 ml
Range
Volume Used
*CFU/ml
**CFU/100 ml
Volume Used
*CFU/ml
**CFU/100 ml
*CFU/ml = (CFU/0.01ml ) x (1/dilution factor)
**CFU/100 ml = (CFU/0.01ml ) x (1/dilution factor) x (100/100)
1B. Spread Plate: Enterococci
Water Source
Avg. CFU
Dilution
*CFU/ml = (CFU/0.01ml ) x (1/dilution factor)
**CFU/100 ml = (CFU/0.01ml ) x (1/dilution factor) x (100/100)
1C. Spread Plate: Heterotrophs
Water Source
Avg. CFU
Dilution
*CFU/ml = (CFU/0.01ml ) x (1/dilution factor)
**CFU/100 ml = (CFU/0.01ml ) x (1/dilution factor) x (100/100)
3A. Membrane Filtration: E. coli
Water Source
Avg. CFU
*CFU/ml = CFU/volume used
**CFU/100 ml = (CFU/volume used ) x (100/100)
3B. Membrane Filtration: enterococci
Water Source
Avg. CFU
*CFU/ml = CFU/volume used
**CFU/100 ml = (CFU/volume used ) x (100/100)
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4A. Comparison of fecal coliform/E.coli indicator bacteria concentrations according to 3 different techniques.
Water Source
Spread Plate
CFU/100 ml
MPN
CFU/100 ml
Membrane Filter
CFU/100 ml
1. Do the results from the three different tests tend to agree? Explain.
2. The standard for drinking water is 0 fecal coliforms per 100 ml. Do any of the water sources meet the drinking
water standard? Were the results surprising? Explain.
3. The EPA has suggested a limit of 126-235 E. coli/ 100 ml for recreational water. Which water sources would meet
this standard assuming that fecal coliform and E. coli are the same?
4A. Comparison of enterococci indicator bacteria concentrations according to 3 different techniques.
Water Source
Spread Plate
CFU/100 ml
MPN
CFU/100 ml
Membrane Filter
CFU/100 ml
1. Do the results from the three different tests tend to agree? Explain.
2. The EPA has suggested a limit of 35-33 enterococci/100 ml be the limit for recreational water. Which of the
natural water sources tested would be safe for human recreation based on this standard?
3. If you were performing 100 to 200 water quality tests per day, which of the test procedures would you choose and
why?
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ANALYSIS REPORT
Using the background information and data write a summary on the use of bacteria as indicators of water quality, and
what they indicate about the conditions of the lake that was tested. Be sure to use data in your statements concerning
water quality. Also, include discussion on what you would do to examine the water quality of this lake over time and
what you may expect to find. What frequency of sampling would you use and what method?
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