Coliform Bacteria Essay, Research Paper

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.

684

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.