.. 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.
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