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OTCO Water Laboratory Analyst Workshop May 17, 2012 Microbiological Laboratory Update Drinking Water Micro 101.1 Todd W. Bidlack Laboratory Evaluation Officer Ohio EPA/DES/Lab Certification Section The human body contains hundreds of species of microorganisms in a living, evolving, ecosystem that make up our normal flora. The normal flora of the enteric or digestive system contain many beneficial microorganisms. • Help digest food • Help regulate our metabolic process • Help fend off ‘unfriendly’ microorganisms that have been ingested Microbial pathogens that can cause disease when ingested will pass through the digestive system and exit with feces. • Specific potential pathogens are too numerous to isolate individually, occur at relatively low concentrations, and are much more difficult to test for in water. Total coliform & E. coli are bacterial indicators of microbial contamination in drinking water. • Total coliform & E. coli occur normally and in great numbers in the enteric system of humans and other warm-blooded animals. • The microorganisms are relatively easy and inexpensive to test for. • When these indicators occur at specific levels, the risk is great enough the water should not be consumed. In the realm of microbiology & microorganisms there are always many exceptions. • The normal flora of our digestive system contains E. coli & we could not survive without it. • But good bacteria by genus & species can develop into bad or pathogenic strains that may cause disease in some individuals. There are also opportunistic pathogens. • These microbes may have no effect on most healthy individuals. • But, infants, elderly, and/or individuals with a compromised immune system due to medical condition, and/or treatment may become ill. When we look at drinking water we are dealing with living dynamic microorganisms that may have been away from the host for a period of time and may be attenuated and not produce a typical laboratory reaction. • This can lead to alternative metabolic pathways evolving in some microorganisms to survive and maintain their existence in the environment. • This is why environmental microorganisms are much more difficult to test for. • Clinical specimens are taken directly from the human host, typically occur in large numbers, & have a strong analytical test response. • ATCC pure cultures grown in ideal laboratory conditions and delivered into an ideal sterile matrix of laboratory pure water have well defined analytical test reactions. So what are total coliform bacteria? • Group of several genera belonging to the family Enterobacteriaceae. • Traditionally include members from: Escherichia, Citrobacter, Klebsiella, & Enterobacter. • But, may also include some Serratia, Hafnia, etc. Since the total coliform group is rather broad based from a taxonomical approach it has often been based or defined by more specific analytical reactions. • Historical definition based on fermentation technique has been: all gram-negative, facultative anaerobic, non-spore forming, rod shaped bacteria, that ferment lactose with acid & gas production within 48 hours at 35 degrees C. • With the advent of MMO-MUG the total coliform group is now defined as all bacteria possessing the enzyme β-D-galactosidase which cleaves the chromogenic substrate resulting in chromogen release. So… What does MMO-MUG stand for? Minimal Medium ONPG-MUG test • ONPG – ortho-nitrophenyl-βgalactopyranoside test reagent used to determine β-galactosidase activity of the family Enterobacteriaceae • MUG – 4-methylumbelliferyl-β-Dglucuronide Drinking Water Laboratory Certification Website http://www.epa.ohio.gov/ddagw/labs.aspx