lab_24_water_quality - Miramar College Biology 205...

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Lab Exercise 24: ® Test Kit Lab Page 1 of 5 Miramar College Biology 205 Microbiology Lab Exercise 24: ® Test Kit Lab Background Most Probable Number ( MPN ) is a procedure used to estimate bacterial populations in the environment (including bacterial contamination of water). This procedure is based on the application of the Theory of Probability with the following given assumptions: 1. The organisms are randomly and evenly distributed throughout the sample; 2. The organisms exist as single entities, not as chains, pairs or clusters and they do not repel one another; 3. The proper growth medium, temperature and incubation conditions have been selected to allow even a single viable cell in an inoculum to produce detectable growth; 4. The population does not contain viable, sub-lethally injured organisms that are incapable of growth in the culture medium used. Environmental samples are diluted prior to use in order to reduce the number of positive tubes to a manageable number. The number of tubes prepared is generally based on the expected population contained within the sample. Reliable results occur when all tubes at the lower dilution are positive and all tubes at the higher dilution are negative for growth, as the dilution scheme has accurately “bracketed” the population, much as the case in dilution plating. Typically, this results in serial dilutions for MPN analysis in sets of 3, 5 or 10 MPN tube series, with more tubes increasing accuracy. MPN values are an indirect measurement of the number of viable organism in a given sample. However, MPN counts are useful for their technical ease and are particularly useful when low concentrations of organisms (<100/ml) are encountered in such materials as milk, food, soil and water where a low concentration of organisms and/or particulate matter of the matrix may interfere with accurate colony counts. Once dilutions have been performed, samples that are positive for microbial growth are indicated by gas production and/or visible turbidity. In this lab, you will be inoculating a series of 3 MPN tubes with the sample serial diluted ( i.e ., undiluted; a 10 -1 dilution; and a 10 -2 dilution). You will be looking for the presence of aerobic or facultatively anaerobic, Gram negative, non- endospore forming rods that ferment lactose and produce gas within 24 hours at 35°C. Organisms having these characteristics are typically considered indicator organisms for fecal contamination of water. However, there are many organisms that fit this description but are not indicative of fecal contamination. For this reason, positive MPN results are a putative result for fecal contamination. Confirmation of this must be done by further biochemical and physiological analysis of the cultured microorganisms. More sophisticated testing can be directly on water samples through the use of test kits which examine the bacteria’s ability to
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lab_24_water_quality - Miramar College Biology 205...

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