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.
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.
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.
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.
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.
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).
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.
StageAS PondUP LagoonTap WaterDrinking Water
PresumptiveGas present in all tubesGas present in some tubesGas present in some tubesGas present in some tubes
ConfirmedReddish colonies found on a MacConkey platePurple colonies found on an EMB plateNo possible coliform bacterial coloniesNo possible coliform bacterial colonies
CompletedGram-negative, endospore-forming, small rods and lactose fermenting bacteriaGram-negative, endospore-forming, small rods and lactose fermenting bacteriaN/AN/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.
3 of 10ml each3 of 1ml each3 of 0.1ml eachLowerUpper
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).
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.
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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Infortech. ?Eco-problems in Boracay.? 1998. http://www.sinfornia.or.jp_~infortec_hotspots_boracay_infopol.html (2 Oct 1999)
NCSU. ?Bacteria.? http://h2osparc.wq.ncsu.edu_info_bacteria.html (2 Oct 1999)