FREE ESSAY ON THE DETERMINATION OF THE PRESENCE OF COLIFORM BACTERIA FROM SELECTED SOURCES BY MEANS OF THE MOST PROBABLE NUMBER (MPN) METHOD

THE DETERMINATION OF THE PRESENCE OF COLIFORM BACTERIA FROM SELECTED SOURCES BY MEANS OF THE MOST PROBABLE NUMBER (MPN) METHOD

Abstract
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 35?C 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 35?C (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
37?C. 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 37?C. 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.
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