Coliform bacteria are good indicator organisms for the presence of pathogenic
bacteria due to their realtionship with these pathogenic bacteria, their
relative ease of determination by simple methods, and by their occurrence in
large quantities in human feces. The MPN method used in this experiment is one
of the prescribed techniques for the determination of these coliform bacteria
from the Standard Methods for the Examination of Water and Wastewater as
prescribed by the EPA. It consists of three stages, each of which necessitates a
positive result for the previous stage. The first stage (presumptive test)
determines the gas-producing coliform characteristic during
lactose-fermentation. The second stage (confirmed test), determines the
gram-reaction and also the lactose fermentation abilities of the organism, while
the last stage (completed test) determines the endospore presence to determine
if the organisms in the sample indeed are coliforms. The number of coliforms or
bacteria present is readily seen with the use of a special table and then the
statistically estimated numbers are determined. The samples, however, did not
produce positive results for the presence of coliforms. Enventhough there was a
large MPN value for one of the samples, about 1100 MPN per 100 ml, the sample
still tested negative in the last stage. It is therefore suffice to say that the
samples did not present any health risks for humans. Future researchers should,
however, device or perform other more specific procedures due to the fact that
there might have been still coliforms present but these may have been negated by
possible endospore-forming relatives. Introduction Human health has always been
a hard condition to preserve and the detection and control of pathogens in the
environment have been the very key to the success of the human race. Although
microbial pathogens are relatively few in comparison to the total number of
microorganisms, their detection have been made easy with the use of indicator
organisms. Indicator organisms give researchers the benefit of making good
assumptions on the presence of pathogens before the pathogens multiply in
distressing numbers. For a microbe to be accepted as an indicator organism, it
must be present in human feces in large amounts so much so that the presence of
these bacteria in a given sample would already point to human fecal
contamination. It was reasoned that the largest amount of pathogens was present
in human feces, and thus, the indication of the entry of large amounts of human
waste, from healthy persons or not, already indicate a great risk (NCSU). Also,
indicator organisms must be present wherever and whenever the pathogen organisms
are present. More importantly, these indicator organisms must be easily
detectable in samples and tests for the measurement of their numbers must be
simple enough (Tortora et al. 1995). Coliform bacteria fit all the requirements
and are even safe to handle in the laboratory. Coliform bacteria are
gram-negative and non-spore/endospore forming bacteria, which include aerobes
and facultative anaerobes, and when incubated at 35ºC with lactose in the
media, will evolve gas (CO2) within 48 hrs, like Escherichia, Klebsiella,
Citrobacter and Enterobacter (NCSU). They are also prevalent in the colon and
intestinal tract (but not all groups are present) of warm-blooded mammals,
including man (Anderson et al. 1998). They are also related to pathogenic
bacteria in that a large number of these coliform bacteria usually imply the
presence of some pathogenic bacteria (Frank). These characteristics of coliform
bacteria already suffice the conditions outlined for these organisms to be
classified as indicator organisms. They occur in large amounts in human feces,
in fact, humans excrete billions of these coliforms (called fecal coliforms).
They are present whenever and wherever the pathogen organisms are present. More
importantly, their presence is easily detected as their characteristics are
easily tested with the use of simple procedures like gram-staining, endospore-staining
and lactose fermentation tests. These principles and procedures now form the
basis and the rationale for the methods by which this experiment was conducted.
Actually, the use of coliform bacteria as indicators of the presence of
pathogenic bacteria is not new already. It as been established since 1880, and
because of their reliability as indicator organisms, the procedures have not
changed much and have only geared on specifically measuring the amount of fecal
coliforms by use of special growth media and techniques. Today, the basis of the
Standard Methods for the Examination of Water and Wastewater that are being used
(also in this experiment) have been specified by the Environmental Protection
Agency (EPA) (NCSU). There are several methods prescribed by the EPA and
although the Most Probable Number (MPN) method is not the most frequently used,
it still provides adequate proof for the presence of coliform bacteria. Better
and more simple methods are being used, like the Colilert methods that is done
by just adding special powdered media to a sample water and then observing color
changes within 24 hrs after incubation at 35ºC (yellow = coliform, and if the
yellow-colored solution fluoresces under UV light, the fecal coliforms are
present) (Frank). The MPN method operates on a somewhat deductive manner,
providing stages by which each step builds up or confirms for the manifestation
of the coliform characteristics and thus, would readily separate coliform from
non-coliform bacteria based on cytological (gram reaction and endospore
formation) and lactose fermentation reactions. Thus, one can expect sterile
water to already be given a negative result on the first stage while sewage
water would be expected to test positive for all stages. The number of the
coliforms are determined by the use of a special table if coliforms are indeed
present, based on the last stage. In this experiment, all mentioned coliform
cytological characteristics as well as the ability to produce gas during lactose
fermentation are done in stages by which, the colonies left at the end (if any)
have coliform characteristics. Methodology The procedures were grouped into
three stages, each of which necessitates a positive result from the previous
stage, otherwise, the process is stopped at the particular stage and the sample
gets a negative result on the presence of coliform bacteria. The samples tested
in this experiment were from drinking water, tap water, AS pond, and from the UP
lagoon but this paper concentrates more on the sample obtained from the AS pond.
Presumptive Test 10-ml portions of the water samples were inoculated into three
large test tubes containing 10ml lactose broth and an inverted Durhan tube each,
per sample (note that the Durham tubes must be rid of air inside before
inoculation). Then, 1-ml portions were inoculated into three test tubes
containing each an inverted Durham tube and 10ml lactose broth. Afterwards,
0.1-ml portions were inoculated into three test tubes containing 10ml lactose
broth and an inverted Durham tube, each. These were inoculated for 24 hrs then
the presence of air in each of the Durham tubes was observed. For the test tubes
with gas inside the Durham tubes, these were called the positive presumptive
test and were then subjected to the confirmed test. The other test tubes were
then incubated for another 24 hrs and after which, were also observed for the
presence of gas inside the Durham tubes. If gas were present, these were then
called the doubtful test and were subjected to the confirmed test. The other
test tubes with no gas inside the Durham tubes were then set aside and labeled
negative tests. Confirmed Test All test tubes that were either positive
presumptive or doubtful tests from the first part were subjected to this test.
The test tube/s with the largest dilution from these test tubes was then chosen
for the next processes (priority = 0.1-ml sample test tubes*1-ml sample test
tubes*10-ml sample test tubes). Two each of pre-poured EMB and MacConkey agar
plates were then inoculated, using streak plating technique for isolation, with
samples from the test tube chosen. These plates were then incubated for 48 hrs
at 37ºC. For the EMB plates, the presence of colonies with green-metallic
shades or colonies that were dark purple were detected. For the MacConkey agar
plates, the presence of red colonies was observed. These colonies were possible
coliform bacteria and were subjected to the last stage, the completed test.
Completed Test Portions were picked up and inoculated onto a lactose broth and a
nutrient agar slant, individually, from the possible coliform bacterial colonies
from the previous stage. These were then incubated for 48 hrs at 37ºC. The
lactose broth tubes were observed for gas production from lactose fermentation
while the colonies inside the nutrient agar tubes were subjected to the
gram-staining and endospore staining procedures (see Appendix). Results
Fortunately or unfortunately, there were no coliform bacteria observed from the
samples. The samples from tap and drinking water already did not give positive
results in the confirmed test (no green-metallic or purple colored colonies in
the EMB plates nor red colonies on the MacConkey agar plates). The samples from
the other sources did go through all the stages but did not give positive
results for the last stage. Table 1 gives us a summary of the results for each
stage of each sample. Stage AS Pond UP Lagoon Tap Water Drinking Water
Presumptive Gas present in all tubes Gas present in some tubes Gas present in
some tubes Gas present in some tubes Confirmed Reddish colonies found on a
MacConkey plate Purple colonies found on an EMB plate No possible coliform
bacterial colonies No possible coliform bacterial colonies Completed
Gram-negative, endospore-forming, small rods and lactose fermenting bacteria
Gram-negative, endospore-forming, small rods and lactose fermenting bacteria N/A
N/A Table 1. Results from the stages for each sample tested. Coliform bacteria
are gram-negative, non-endospore forming and lactose fermenting small rods. As
seen, none of the results from the samples gave positive indication for the
presence of coliform bacteria. This is surprising due to the fact that there are
a number of marine organisms (hence more wastes and coliform bacteria) in both
the AS pond and the UP lagoon. It is not surprising and even convenient however,
to know that there are no coliform bacteria in both tap water and drinking
water. If we compare this to the number of bacteria present, we would now have a
notion of the relative amount of bacteria that are not coliform living on the
sample. Using an MPN table (see Table 2), we now determine that there are about
1100 bacteria per 100ml of the sample taken from the AS pond. This is about the
largest MPN for bacteria in the MPN table and it is really surprising that not
even one of these bacteria is a coliform bacterium. Number of tubes Giving
positive Reaction out of MPN index per 100ml 95% Confidence Limits 3 of 10ml
each 3 of 1ml each 3 of 0.1ml each Lower Upper 0 0 1 3 *0.5 9 0 1 0 3 *0.5 13 1
0 0 4 *0.5 20 1 0 1 7 1 21 1 1 0 7 1 23 1 1 1 11 3 36 1 2 0 11 3 36 2 0 0 9 1 36
2 0 1 14 3 37 2 1 0 15 3 44 2 1 1 20 7 89 2 2 0 21 4 47 2 2 1 28 10 150 3 0 0 23
4 120 3 0 1 39 7 130 3 0 2 64 15 380 3 1 0 43 7 210 3 1 1 75 14 230 3 1 2 120 30
380 3 2 0 93 15 380 3 2 1 150 30 440 3 2 2 210 35 470 3 3 0 240 36 1300 3 3 1
460 71 2400 3 3 2 1100 150 4800 Table 2. MPN values from multiple tube tests.
(source: Standard Methods for the Examination of Water and Wastewater, 14th ed.
American Public Health Association, American Water Works Association, Water
Pollution Federation, Washington, D.C., 1975.) Errors were minimal and if there
were contamination, there would be coliform bacteria in the results. Possible
reasons why there where no coliform in the AS pond and the lagoon would be that
they were eaten by large amounts or protozoans, etc. or that bacteriophages were
present and killed all of them, or that the samples were taken where the water
was cleanest (shallow parts). Discussion The tests made were done by stages in
order to narrow down the possibilities in the determination of the presence of
these coliform bacteria. The presumptive test selects out the
gas-producing-lactose-fermenting bacteria, which is one of the characteristics
of coliform bacteria. Characteristically, coliform bacteria produce CO2 under
anaerobic conditions and the gas production was manifested as the presence of
air inside the Durham tubes (Lindquist 1998). This narrows it down to a few
groups of bacteria that ferment lactose. The confirmed test further narrows the
coliform bacterial characteristics by growing the positive presumptive tests in
selective and differentiating media, EMB and MacConkey agar. EMB is a selective
medium, due to the fact that it inhibits the growth of gram-positive bacteria.
This is because EMB contains crystal violet, which characteristically is the
component that inhibits the growth of gram-positive bacteria. MacConkey agar
also contains crystal violet and thus, is also a selective medium. However it
also contains lactose by which, lactose-fermenting bacteria (red/pink colonies
on the MacConkey agar) may be differentiated from non-lactose-fermenting
bacteria (colorless colonies on the MacConkey agar) (Tortora et al. 1995). Thus,
in the confirmed test, we were looking for red/pink colonies in the MacConkey
agar plates, which are gram-negative and lactose fermenting bacteria, and
green-metallic or purple colonies on the EMB plates (although all bacteria in
the EMB are gram-negative, coliform bacteria exhibit the said colors). The
bacteria that “passed” the confirmed test (bacteria sought for in the
confirmed test) were then subjected to a last and final test, the completed
test. In this test the bacteria left are screened using again, lactose broths,
for the final assurance of gas-production in lactose fermentation, gram
staining, also for final assurance that the bacteria that passed are really
gram-negative, and endospore staining, which will separate the non-coliforms
from the coliforms. In this case, since coliform bacteria are non-endospore-forming
bacteria, the presence of endospores would mean that they are not coliforms and
are just very close relatives with the coliform bacteria. Since the results
showed that there were no coliform bacteria on any of the samples, we could then
say that the bodies of water these samples were in are relatively safe (but not
necessarily safe for drinking). The presence of 1100 MPN non-coliform bacteria
per 100ml should not be taken as a health hazard. On the contrary, based on
Philippine standards, the maximum tolerable level of coliform bacteria is at
1000 MPN coliform bacteria per 100ml (Infortech 1998). Thus, the 1100 MPN per
100ml free of coliform is an indication that the water sample from the AS pond
taken is very safe, and more safe are the other samples with lower MPNs and
negative for coliforms. However, if we analyze, the procedures, there might
still be coliforms in the sample. This is due to the fact that there are other
gram-negative, lactose fermenting bacteria but produce endospores. Thus, they
might have tested positive for the endospore stain but if there were coliforms
present with these endospore-forming realtives of coliforms, the presence of the
coliforms would not be detected and the sample would be given a negative on the
presence of coliforms. Better and more specific tests should thus be made by
future researchers to make more accurate and definitive conclusions on the
presence of coliforms in bodies of water. Appendix General Staining Procedures
used in the Experiment: I. Gram Staining This staining method required at least
18-24 hr. cultures of the organism in the nutrient agar slant that were fixed on
a slide. The stains used were crystal violet, iodine solution, 2% safranin O,
and 95% ethanol. A microscope, staining rack and forceps were also used for this
staining procedure. The smear, on a staining rack, was flooded with crystal
violet. The flooded smear was allowed to stand for a minute. It was then rinsed
with tap water (excess water was drained off). The smear was next stained with
iodine solution for a minute, rinsed with tap water then drained. 95% ethanol
was then dropped on the slide until no more crystal violet was washed off.
Afterwards, the slide was rinsed then drained. Safranin was then dropped on the
slide, and after a minute, the slide was rinsed with tap water. After the
staining was done, excess moisture was blotted off with tissue paper. The slide
was then air-dried. The slide was next studied under OIO (immersion oil was
used) of the microscope (the slide was placed under LPO first, where a good area
to examine was located). Gram-positive will retain the violet color,
gram-negative bacteria will be stained red. II. Endospore Staining This process
required at least 36-hr. cultures of the organisms in the NA slant enumerated
earlier that were fixed on a slide (like the smears on Gram staining). 5%
malachite green and 0.5% safranin (see Appendix) were the stains used for this
staining method. A disposable plastic, forceps, a microscope and an alcohol
burner were used in this method. First, the working area was covered with the
plastic because the stains might splatter out. Then the slide was flooded with
malachite green. This was passed over low flame several times for five minutes,
allowing the stain to steam but not to boil. The stain was replenished from time
to time and after five minutes, the slide was rinsed. The slide was then stained
with safranin and was allowed to stand for a minute. The slide was then rinsed
with tap water and air-dried. The dried slide was then examined under LPO, to
locate a good area, then placed under OIO (immersion oil as used) for a more
detailed study. The presence of green bodies the presence of endospores.

Bibliography
Anderson, J., Liukkonen, B., and Bergsrund, F. “Indicators of Health
Risks.” 1998. http://www.mes.umn.edu_Documents_D_D_Othoer_0814-04.html (2 Oct
1999) Frank, K. “Northern Testing Laboratories, Inc. Water Quality Fact Sheet:
Coliform Bacteria.” http://www.ptialaska.net_~ntl_Coliform.html (2 Oct 1999)
Infortech. “Eco-problems in Boracay.” 1998. http://www.sinfornia.or.jp_~infortec_hotspots_boracay_infopol.html
(2 Oct 1999) Lindquist, J. “Differential Media: Glucose Fermentation Broth and
O/F Medium.” 1998. http://www.bact.wisc.edu_bact102_dfglocosenf.html (2 Oct
1999) NCSU. “Bacteria.” http://h2osparc.wq.ncsu.edu_info_bacteria.html (2
Oct 1999) Tortora, G., Funke, R. and Case, C. 1995. Microbiology An
Introduction. US: The Benjamin/Cummings Publishing Company, Inc.153, 678-679.