Coliform Bacteria
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 35ēC 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 35ēC (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 37ēC.
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 37ēC. 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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