TOPIC A

1. TOPIC A Applied and Environmental Microbiology

2. What is a TMDL TMDL = Total Maximum Daily Load TMDL is the maximum quantity of a specific pollutant that a body of water can have to meet water quality standards Standards are set by City, County or State Varies depending on type of water TMDL levels are lower for potable water than for recreational water Varies depending on type of pollutant TMDL for E. coli is <126cells /100ml TMDL for Enterococci is <33cells/ 100ml

3. What is an Indicator Organism An indicator organism is an organism whose presence or absence can indicate the contamination of an environment Ex: of fecal coliform indicator organisms E. coli, Citrobacter freundii, Klebsiella pneumoniae Ex: of non-coliform fecal indicator organisms Streptococcus bovis (cow) Streptococcus equinis (horse) Streptococcus faecalis and S. faecium (all organisms)

4. Culture Based Methods for detecting Fecal Contamination MPN (Most Probable Number) Method For testing for fecal coliforms, 15 tubes containing Lauryl Tryptose medium are inoculated with 10ml of the sample for each of 3 series dilutions (10,100,1000) and there are 5 tubes per dilution. Once inoculated they are incubated for 24 hours at 35� C. If after inoculated there is gas production in the media the samples are then inoculated into EC medium and incubated for another 24 hours at 44.5�C. If there is gas production at this stage it is a positive result for fecal coliforms. The tubes that test positive are then compared to a chart that will show the MPN of microbes for a specific dilution. From this chart the number of coliforms per ml can be determined and the values can be compared to the TMDL values to determine if there is contamination. Another test for fecal coliforms involves placing 10ml of the sample from each of the 3 dilutions into 15 tubes containing A-1 medium and incubating the tubes for 3 hours at 35� C. The tubes then are incubated for 21 hours at 44.5�C. If gas is present in the media after the second incubation this a positive result for fecal coliforms. The tubes that test positive are then compared to a chart that will show the MPN of microbes for a specific dilution. From this chart the number of coliforms per ml can be determined and the values can be compared to the TMDL values to determine if there is contamination.For testing for fecal coliforms, 15 tubes containing Lauryl Tryptose medium are inoculated with 10ml of the sample for each of 3 series dilutions (10,100,1000) and there are 5 tubes per dilution. Once inoculated they are incubated for 24 hours at 35� C. If after inoculated there is gas production in the media the samples are then inoculated into EC medium and incubated for another 24 hours at 44.5�C. If there is gas production at this stage it is a positive result for fecal coliforms. The tubes that test positive are then compared to a chart that will show the MPN of microbes for a specific dilution. From this chart the number of coliforms per ml can be determined and the values can be compared to the TMDL values to determine if there is contamination. Another test for fecal coliforms involves placing 10ml of the sample from each of the 3 dilutions into 15 tubes containing A-1 medium and incubating the tubes for 3 hours at 35� C. The tubes then are incubated for 21 hours at 44.5�C. If gas is present in the media after the second incubation this a positive result for fecal coliforms. The tubes that test positive are then compared to a chart that will show the MPN of microbes for a specific dilution. From this chart the number of coliforms per ml can be determined and the values can be compared to the TMDL values to determine if there is contamination.

5. Culture Based Methods for detecting Fecal Contamination Membrane Filtration Filters sample through a membrane w/ small pore size�captures bacteria Filter is placed on FC agar containing aniline blue. Fecal coliforms produce acid that reacts w/ analine blue causing the colonies to appear blue Absence/presence test Place sample into tube containing: Bromcresol purple Lactose and lauryl tryptose Durham tube If yellow color and gas production result: Positive for presence of bacteria

6. Non-Culture Based Method and Viral Method Non-culture based Extract DNA, PCR, sequence, identify Viral Look for the presence of specific viruses that are correlated/found in the fecal material of animals