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Detection of Total and Fecal Coliform Bacteria and Escherichia coli by Broth Culture,
Quantal (Most Probable Number) Methods: Multiple Fermentation Tube (MFT) and
Defined Substrate Methods
Fecal Coliforms
Although the total coliform group as presently operationally defined includes bacteria
usually found in the feces of humans and other warm-blooded animals, some of the
bacteria detected by these procedures are sometimes also found in soil (Citrobacter,
Enterobacter and Klebsiella), on various plants, including grains and trees (Serratia,
Klebsiella and Enterobacter) and in certain industrial wastes. Furthermore, some
coliform bacteria can also be pathogenic for humans and animals when present either in
the gut (e.g., various pathogenic E. coli) or in other parts of the body (e.g., Klebsiella
pneumonia in the respiratory tract). In addition, organisms that are not members of the
family Enterobacteriaceae and not exclusively of intestinal origin, such as members of
the genus Aeromonas, may be detected in the total coliform test. For these reasons,
another group, the "fecal coliforms" or thermotolerant coliforms has been established
in an attempt to exclude those total coliforms that are non-fecal and detect only those of
fecal origin. The basis for this exclusion and selection is a higher incubation temperature
of 44.5OC, at which presumably coliforms of only fecal origin will grow because they are
adapted to the higher temperature of the mammalian intestinal tract of about 36-37oC.
Coliforms from non-fecal, environmental sources are generally incapable of
growing at this elevated temperature. Thus, fecal or thermotolerant coliforms can be
defined as gram-negative, non-sporeforming, rod-shaped bacteria which ferment lactose
with the production of gas at 44.5oC within 24 hr. The fecal coliform test is now widely
applied to surface and ground water, sewage treatment systems, biosolids (treated
sludges) and general monitoring of natural waters for sanitary quality, including
recreational and shellfish waters, and water quality standards have been developed for it.
However, the coliform test is still used in the examination of potable waters in the USA
and some other countries, because coliform bacteria of any kind are considered
undesirable and are not tolerated in a finished drinking water. Today, both the total and
fecal coliform tests are used in the examination of water, even though E. coli has been
identified as the most feces-specific member of the coliform group.
There are actually several approved Multiple Tube Fermentation Techniques for
fecal coliforms and two main ones are described here: a single-step and a two-step
procedure. The latter is the traditional test that has been widely used for some time, and
the former is a more recent test approved for some applications. In the single-step
procedure dilutions of the sample are inoculated into fermentation tubes of A-1 medium.
The tubes are first incubated for 3 hr at 35oC and then transferred to a 44.5oC water bath
for an additional 21 hr of incubation. Tubes showing growth plus gas are considered
positive for fecal coliforms. For the two-step Multiple Tube Fermentation Technique for
fecal coliforms, positive tubes from the Presumptive (total) coliform test are inoculated
into fermentation tubes of EC medium and incubated at 44.5oC for 24 hr. Tubes showing
growth with gas production are considered confirmed positives.
Escherichia coli.
Because both the total and fecal (thermotolerant) coliform tests often still detect
bacteria of non-fecal origin, test have been devised to detect E. coli exclusively as the
coliform bacterium most specific for fecal contamination. An early approach to this
effort was the use of four biochemical tests to separate E. coli from other
lactose-fermenting Enterobacteriaceae:
Indole test - detects indole production from tryptophane.
E. coli is positive (+); many other coliforms are negative.
Methyl Red test - detects acid production in the medium; intended to distinguish between
type of fermentation (mixed acid versus butylene glycol).
E. coli is (+) and some other coliforms are (-).
Voges-Proskauer test - detects acetoin, an intermediate in the butylene glycol pathway.
Acetoin is oxidized to diacetyl under alkaline conditions in the presence of air, and when
reacted with creatine, it forms a pink color.
E. coli is (-) and some other coliforms are (+).
Citrate utilization as sole carbon source.
E. coli is (-) and many other coliforms are (+).
Therefore, in this series of four tests, called the IMViC tests, E. coli is typically +
+-- and Enterobacter aerogenes is typically --++. Other coliforms give different reaction
patterns and are designated "intermediates". However, the IMViC reactions have been
found to be imperfect in speciating E. coli, and subsequently, other approaches to E. coli
and coliform speciation have been developed.
Additional or alternative approaches to detecting and speciating E. coli are worth
noting here. One additional approach to coliform speciation is the use of rapid,
commercial, biochemical test kits designed to carry out several biochemical tests
simultaneously in incubation periods of 4 to 24 hours. The results of each test are scored
as (+) or (-) and assigned a number based on the relative reliability of the test. These
results constitute a "code" which is compared against a data base of accumulated reaction
codes for nearly all of the medically important Enterobacteriaceae. The code that is
identical or closest to the organism tested is taken as its identity at the genus and species
level.
An alternative approach to the simultaneous detection and quantitation of
coliforms and E. coli in water and other samples is the use of a single property or
biochemical test in addition to lactose fermentation to definitively identify E. coli among
other coliforms. One such property is the presence of the enzyme beta-glucuronidase,
which is found only in E. coli and some Salmonella and Shigella. Tests for this enzyme
have been developed in which a beta-glucuronidase substrate, a hydrolysable beta-
glucuronide compound, such as MUG (4-methylumbelliferyl-beta-D-glucoronide) is
incorporated into a coliform medium. If E. coli is present, the MUG is hydrolyzed to
yield a fluorogenic product (4-methylumbelliferone) whose blue fluorescence can be
readily seen by shining a long wavelength UV light onto the culture. The MUG substrate
can be added to coliform and fecal coliform media so that when E. coli is present and
grows in the culture medium broth, the medium fluoresces blue under long wavelength
UV light. Other tests for coliforms and E. coli have been devised in which the Beta-
galactoside substrates (for the beta-galactosidase enzyme of any coliform) and the beta-
glucuronide substrates (for the beta-glucuronidase activity of E. coli) can be detected
because the hydrolysis products have a unique and distinguishable color. That color
appears in the broth culture medium or in the bacterial colonies if the bacteria are
cultured on a solid medium (one containing agar as a gelling agent, for example).
Some commercially available media and test systems for E. coli and coliforms use
defined substrates for beta-galactosidase and beta-glucuronidase (e.g., MUG) in a mineral
salts solution to simultaneously detect and quantify coliforms and E. coli, respectively, by
characteristic color changes resulting from hydrolysis of the defined substrates. These
substrates are present as the only food supply for the bacteria, resulting in tests with high
specificity in detecting the target bacteria, coliforms or specifically E. coli. The
commercial liquid or broth culture tests are available in several formats, including
multiple tubes or multiple wells. One widely is known brand of this test is Colilert"
(IDEXX Corp.). It is now widely used in bacteriological analysis of drinking water,
although similar commercial tests have been developed as well, such as Colitag (CPI
International). Similar tests based on defined chromogenic substrates detect the target
bacteria as colonies on a solid medium (containing agar or pectin) or on a membrane
filter, on which the colonies have a distinctive color resulting from hydrolysis of a
chromogenic beta-galactoside (total or fecal coliform) or beta-glucuronide (E. coli)
substrate that yields a distinctively colored hydrolysis product. One such test is Easygel,
which uses pectin as the gelling agent and in which coliform bacteria grow as pink-
magenta colonies while E. coli grow as purple-blue colonies.
To estimate the total and fecal coliform and E. coli concentrations in samples of surface
water or wastewater by broth culture quantal techniques using traditional Multiple-Tube
Fermentation Techniques and Defined Substrate techniques.
III. PROCEDURE:
A. Aseptic Technique: Use it for all operations. Use pipets and pipet aids.
Dilute the sample you select serially 10-fold as directed by the instructor.
Mix the sample by shaking 25 times before diluting.
To make a 10-fold dilution, pipet 11 ml of sample into a 99-ml dilution blank. Seal the
top of the dilution container and mix vigorously 25 times.
Repeat these steps in the 10-fold dilution to make a subsequent 10-fold dilution (100-fold
overall); repeat as required for further dilutions.
C. Two-Step-Tube Fermentation Tube Technique for Total and Fecal Coliforms and E.
coli (5 tubes per dilution):
Place 25 fermentation tubes of lauryl tryptose or lactose broth in a test tube rack as
groups of 5 dilutions, and mark the tubes as to their dilutions. Using a 5- or 10-ml pipet,
inoculate 1-ml volumes of each sample dilution to be tested into the 5 replicate tubes.
Incubate the tubes in a 35oC air incubator.
2. After 24 and 48 hr: Reading of presumptive positive tubes and inoculation of tubes
for Confirmed Total and Fecal Coliform Tests and for E. coli.
Gently shake the rack of tubes back and forth several times to release gas in positive
tubes.
Examine all tubes for the presence of growth (turbidity or cloudiness) and gas (look in
the small inverted tube), and score tubes showing both growth and gas as presumptive
positive. Submit all lactose broth fermentation tubes that are Presumptive positive at
about 24 hr and all additional Presumptive positive tubes at 48 +/- 3 hr to the confirmed
tests.
Confirmed tests: inoculation of material from presumptive positive tubes into tubes of
conformation media
Insert a sterile wood applicator into the broth of a positive tube to a depth of >1 inch to
wet the end. Transfer the organisms on the wetted end of the applicator to a fermentation
tube of brilliant green lactose bile (BGLB) broth (coliform confirmatory broth) by briefly
immersing the wet end of the applicator into the BGLB broth. Use the same applicator
and same procedure to transfer material from the same Presumptive positive tube to a
fermentation tube of EC medium containing MUG (fecal coliform-E. coli confirmatory
broth). Discard the applicator. For each positive Presumptive tube, use a fresh applicator
stick and repeat these steps above.
Incubate the BGLB broth tubes in a 35oC incubator and the EC-MUG tubes in a 44.5oC
water bath.
After 24 (+/- 4) and 48 (+/- 4) hours of incubation: Examine BGLB tubes for growth plus
gas. Tubes with growth plus gas are confirmed positive. If a tube is negative after 24
hours, continue incubation to 48 hours and read again for positivity (growth plus gas).
Record
Examine the EC tubes at 24 +/- 2 hr for growth plus gas. Tubes showing both are scored
as Confirmed positive for fecal coliforms. Examine the positive EC tubes under a long
wavelength UV light. Tubes showing bluish fluorescence under long wavelength UV
light (in addition to growth plus gas) are scored as positives for E. coli.
Record the results of all BGLB (total coliform) and EC-MUG (fecal coliform and E. coli)
tubes as confirmed positive or as negative.
Calculate the total and fecal coliform and E. coli densities of your sample from the
number of positive and negative tubes of three sample dilutions according to the
procedures described in Standard Methods for The Examination of Water and Wastewater
and below. Bring these results to the next scheduled laboratory period.
A. If your sample is waste water, dilute the sample serially 10-fold according to
directions from the instructor.
If your sample is treated sewage effluent or Morgan Creek water is does not need to be
diluted.
1. Briefly, dispense a 100 ml sample into a sterile bottle and aseptically add the
powdered contents of a Colilert packet to the sample. Close the sample container and
swirl to dissolve the medium in the sample.
2. Open a Quantitray by peeling back a portion of the seal as per manufactures
instructions. Pour the 100 ml sample containing dissolved Colilert medium into the
Quantitray and re-seal it according to the manufacturers instruction (using the special
sealing device).
3. Incubate the Quantitry at 35oC for the specified time period and then remove to score
for positive results in each well.
4. Examine each well and score as positive for total coliforms if it has a bright yellow
color indicative of ONPG hydrolysis.
5. Shine a long wavelength UV light on the Quantitray and score each well as positive
for E. coli if it fluoresces with a blue color indicative of MUG hydrolysis.
6. Record these results for positive and negative wells for total coliforms (yellow color)
and E. coli (blue fluorescence) and bring to the next lab class to compute the MPN
concentration using the manufacturers MPN table.
Compute and record the Most Probable Number of total and fecal coliforms and E. coli
per 100 ml using the data from the confirmed tubes (BGLB and EC-MUG). Also record
the upper and lower 95% confidence limits for these MPN values.
The MPNs and confidence limits are found in the table for three dilutions and five tubes
per dilution in Standard Methods for the Examination of Water and Wastewater.
Note that the MPN table in Standard Methods is set up for a dilution series of 10, 1 and
0.1 ml sample volumes. The sample volumes examined in your samples are smaller( 1,
0.1 and 0.01 ml or even less), and therefore, the MPN values in the table have to be
multiplied by factors of 10 (or more for some samples) to correct for the difference in
sample volumes analyzed.
1. For each method (multiple fermentation tube and Colilert) how well do the results for
the replicate analyses of each sample agree? Specifically, are they identical? If not, does
the MPN value for one replicate fall within the 95% confidence limit of the other
replicate?
2. For each sample, compare the traditional multiple fermentation tube MPN
concentrations of total and fecal coliform and E. coli. Which indicator concentration is
highest? Which is lowest? Are these results consistent with what you would expect,
based upon the definitions of each indicator and the expected relationships among them?
3. For each sample, compare the Colilert MPN concentrations of total coliforms and E.
coli. Which indicator concentration is highest? Which is lowest? Are these results
consistent with what you would expect, based upon the definitions of each indicator and
the expected relationships among them?
4. For the corresponding samples, compare the MPN concentrations of total coliforms
and also E. coli based on traditional multiple fermentation tube MPN results and Colilert
Quantitray MPN results. How well do the results of these two methods agree? Are they
identical? Does the MPN value of one method fall within the 95% confidence interval of
the other method?
5. Recall that the four samples analyzed were: raw sewage, treated sewage effluent,
Morgan Creek (the receiving water) upstream of the sewage effluent discharge, and
Morgan Creek downstream of the sewage effluent discharge.
6. Compare the levels of the three indicators in the four samples. Which sample had the
highest levels of indicators? Which the lowest?
7. Considering that the raw sewage was treated and then discharged to Morgan Creek,
what is the impact of the sewage effluent discharge on the concentrations of indicator
bacteria in Morgan Creek?
8. How effective was the sewage treatment plant in reducing the concentrations of
indicator bacteria in raw sewage. (Sewage treatment at this plant consists of (i) primary
settling, (ii) secondary (biological) treatment and (iii) chlorine disinfection of the
secondary-treated effluent.).
Specifically, calculate the % (or log10) reductions of total and fecal coliforms and E. coli
by dividing the concentrations of these bacteria in the treated effluent by the
concentration in the raw sewage, then subtracting from 1 and multiplying by 100.
9. Also, compute the log10 concentrations of each group of indicator bacteria in the raw
sewage and treated effluent, and subtract the latter from the former to compute the log10
reduction by sewage treatment.
Log10 concentration in raw sewage - log10 concentration in treated effluent = log10 reduction.