Microbiology of Water

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					Microbiology of Water ABSTRACT The human body can survive without food, but not without water. Drinking water is important to both humans and animals. To determine if water is safe to drink, a series of three tests (presumptive, confirmed, and completed) are done. To filter water samples and quantitatively analyze them, the membrane filter method is used. INTRODUCTION This laboratory experiment focused on the types of microorganisms present in water and the methods used to determine the potability of water using standard qualitative and quantitative procedures. With increasing industrialization, water sources have been polluted by industrial, human, and animal wastes. Water is a nutritional vector for microbial and bacterial growth. It is important that water is potable and free of fecal contamination for the public health. There are three basic tests to detect coliform bacteria in water are presumptive, confirmed, and completed. To quantitatively analyze water, the membrane filter method is used. Membrane filters are capable of retaining microorganisms larger than 0.45 micrometers. They are advantageous in that the results are available in a little amount of time, larger volumes can be processed, and the results are readily reproducible. A disadvantage lies within processing turbid specimens with large amount of suspended materials. MATERIALS/METHODS (Experiment 50) Materials: Part A: Presumptive Test 5 double-strength lactose fermentation broth (LBX2) 10 single-strength lactose fermentation broth (LBX1) 1 10-ml serological pipette 2 1-ml serological pipettes 1 water sample (sewage, pond, or tapwater) Part B: Confirmed Test

24 hour-old positive lactose broth culture from one of the three series from the presumptive test 1 eosin-methylene blue agar plates Part C: Completed Test 1 24-hour old coliform-positive EMB agar culture form one of the three series of confirmed test 1 nutrient agar slants 1 lactose fermentation broths Crystal violet Gram’s iodine 95% ethyl alcohol safranin Methods: For the presumptive test, samples from the sewage, pond water, and tap water is quantitatively analyzed for bacteria. The tubes are inoculated with the specified water source and volume sample. Following incubation, the results are recorded as positive if 10% or more of gas appears in a tube in 24 hours, doubtful is gas develops in a tube after 48 hours, and negative if there is no gas in a tube after 48 hours. For the confirmed test, confirm the presence of coli-form bacteria in a water sample for which the presumptive test was positive. The media is streaked with the positive presumptive test. If E. coli is present, dark centers and a green metallic sheen will appear on the EMB. The completed test confirms the presence of coli-form bacteria in a water sample or any suspicious or doubtful result from the presumptive and confirmed test. The isolated colony from the confirmed test is used to inoculate the tube of lactose broth and streaked on a nutrient agar slant. Following incubation, a gram stain of the culture is conducted for the presence of gram-negative short bacilli, which is indicates E. coli. (Experiment 51) Materials: 1 influent, post-settlement, or effluent (choose one) 1 sterile membrane filtration apparatus per group 4 90-ml water blanks per group 2 10-ml serological pipettes per group 5 KF agar plates per group 5 small petri dishes per group 5 sterile absorbent pad per group 5 E. coli Blue media per group

Methods: The samples consist of influent, post-settlement, and effluent. A water sample is passed through a sterile membrane filter that is attached to an apparatus contained in a suction flask. The filter that contains the trapped microorganisms is aseptically transferred to a sterile Petri dish containing the absorbent pad saturated with the blue media. The cultures are incubated. Following incubation, the colonies present on the filter are counted with the aid of a microscope. RESULTS Experiment 50: Part A: Presumptive Test
Water Sample 1 Sewage Pond Tap + + + LB2X-10 Tube 2 + + + 3 + + + 4 + + + 5 + + + 1 + + Gas LB1X-1 Tube 2 + + + 3 + 4 + + 5 + + + 1 + LB1X-0.1 Tube 2 + 3 + + + 4 + 5 + + Reading 5-5-5 5-4-2 5-2-1 MPN ≥2400 220 70 Range 95% Probability 700 more than 4600 70-440 22-170

Part B: Confirmed Test
Water Sample Sewage Pond Tap Coli-form EMB Plate + Potability Potable Nonpotable √ √ √

Part C: Completed Test
Water Source Sewage Pond Tap Lactose Broth A/G (+) or (-) + + Gram Stain Reaction/ Morphology Gram(-)/ bacilli Gram(-)/rod Gram(-)/ rod Potability Potable Nonpotable √

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Experiment 51:
Dilution Undiluted 10-1 10-2 10-3 10-4 Influent TCC FSC TNTC TNTC TNTC TNTC TNTC TNTC TFTC(10) TNTC TFTC(3) 32 Post-Settlement TCC FSC TNTC TNTC 107 58 TFTC TFTC TFTC TFTC TFTC TFTC Effluent TCC FSC TNTC TNTC TNTC TNTC TNTC TNTC 294 237 0 10

DISCUSSIONS In the sewage water sample, using the presumptive test, the most probable number (MPN) is more than 2400 organisms per 100ml of sample with a95% probability that there are

more than 4600 microorganisms present and no less than 700 microorganisms. The confirmed test shows that E. coli is present because the growth on the EMB plate has a metallic green sheen on it. Presence of E. coli means that it is not potable. The completed test shows that the sewage water is not potable. The gram-stain indicates the presence of gram-negative microorganisms that are bacilli-shaped, meaning E. coli is present and that the water has fecal contaminants. The presumptive test for pond water shows that its MPN is about 220 microorganisms per 100ml of sample. There is a 95% probability that 70-440 microorganisms are present. This goes to show that drinking straight from a pond water, is inviting bacteria and foreign microbes into your body. Using the confirmed test, it seems that pond water is potable, because there is no metallic green sheen on the bacterial growth on the EMB agar. Using the completed test, pond water had presence of 10% gas in the lactose broth. When a gram staining was done, it showed a gram-negative microorganism that was rod-shaped. Still, pond water was rendered non-potable. For tap water, the presumptive test shows that it has 70 organisms per 100mL of water. There is a 95% probability that 22-170 microorganisms are present. The confirmed test shows that the sample is potable because it does not have any coliform according to the EMB test that was done. The completed test also shows that tap water is potable because it did not produce any gas during lactose fermentation and the gram staining reveals that the microorganisms present are gram-negative and rod-shaped. Thus, tap water is the cleanest water sample of the three and is potable. For the undiluted effluent, influent, and post-settlement, their fecal streptococcal count (FSC) and total coliform count (TCC) are all too numerous to count. For the dilution factor of 10-1, the post-settlement had a 107 total coliform count and 58 fecal streptococcal count. The effluent and influent still had too numerous microorganisms to count. In the 10-2 dilutions, the post-settlement had too few total coliform and fecal streptococcal to count while both effluent and influent were too numerous to count. For the 10-3 dilutions, effluent had 294 TCC and 237 FSC. Influent had about 10 total coliform count and too numerous fecal streptococcal count. For the last dilution, influent has about 3 total coliform count and 32 fecal streptococcal count. The effluent has no total coliform count and 10 fecal streptococcal count. Post-settlement still had too few total coliform and fecal streptococcal to count.

REFERENCES

Cappuccino, Sherman, “Microbiology: A Laboratory Manual”. 8th Edition By Ana-Alicia Alvarado

 Manual”. 8th Edition


				
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Description: The human body can survive without food, but not without water. Drinking water is important to both humans and animals. To determine if water is safe to drink, a series of three tests (presumptive, confirmed, and completed) are done. To filter water samples and quantitatively analyze them, the membrane filter method is used.