ENVR 421 Laboratory #2: detection of bacteria in water Introduction The purpose of this laboratory exercise is to familiarize you with the most common assays for the detection of bacteria in water. We will be using two types of assays: membrane filtration, which, and the Most Probable Number (MPN) assay. We will simultaneously detect three groups of organisms: total coliforms, fecal coliforms, and E. coli. The Most Probable Number Assay Purpose In this lab, you will use the MPN assay to measure the numbers of total coliforms, fecal coliforms and E. coli in water from Morgan Creek. Principle Indicator bacteria Traditionally, indicator bacteria have been used to determine the possible presence and to estimate the amount of fecal contamination in water, foods and other samples. The detection of indicator bacteria is preferred over direct pathogen detection because the former are considered to be normal, non- pathogenic intestinal inhabitants that are present in feces, wastewater and other fecal wastes in much higher numbers than pathogenic microorganisms and because they are technically easier to detect and quantitate than pathogens. Present criteria and standards for the sanitary quality of water, foods and other materials with respect to fecal contamination are expressed in terms of concentrations of indicator bacteria. The most widely-used indicator bacteria in the U.S. and worldwide are the so-called total and fecal coliforms and specifically Escherichia coli, the bacterium in these groups that is most feces-specific. The word "coliform" has been used historically to represent various genera of the family Enterobacteriaceae that ferment lactose. The definition of a coliform has now been updated to include those Gram-negative bacteria that have the ability to metabolize Beta-galatosides as substrates (because they possess the enzyme Beta-galactosidase). The ability to ferment lactose depends upon the possession of the enzyme beta- galactosidase as well as a galactoside permease which facilitates lactose entry into the cell. It should be emphasized that "coliforms" are actually defined in operational or functional terms based upon the media, biochemical reactions and incubation conditions used for theirisolation and quantitation. None of these operational definitions detects all lactose-fermenting (or Beta-galactosidase-producing) members of the family Enterobacteriaceae and some include members of other families. The ability to ferment lactose (and produce Beta-galactosidase) is thus a characteristic of considerable diagnostic importance in distinguishing among the various groups of enteric bacteria. Fecal Coliforms Some of the bacteria detected by coliform procedures are sometimes also found in soil (Citrobacter, Enterobacter and Klebsiella), on various plants, including grains and trees (Serratia, Klebsiella and Enterobacter) and in certain industrial wastes. For these reasons, another group, the "fecal coliforms" or “thermotolerant coliforms” has been established in an attempt to exclude those total coliforms that are non-fecal and detect only those of fecal origin. The basis for this exclusion and selection is a higher incubation temperature of 44.5OC, at which presumably coliforms of only fecal origin will grow because they are adapted to the higher temperature of the mammalian intestinal tract of about 36-37oC. Coliforms from non-fecal, environmental sources are generally incapable of growing at this elevated temperature. Thus, fecal or thermotolerant coliforms can be defined as gram-negative, non-sporeforming, rod-shaped bacteria which ferment lactose with the production of gas at 44.5oC within 24 hr. The fecal coliform test is now widely applied to surface and ground water, sewage treatment systems, biosolids (treated sludges) and general monitoring of natural waters for sanitary quality, including recreational and shellfish waters, and water quality standards have been developed for it. However, the coliform test is still used in the examination of potable waters in the USA and some other countries, because coliform bacteria of any kind are considered undesirable and are not tolerated in a finished drinking water. Escherichia coli Because both the total and fecal (thermotolerant) coliform tests often still detect bacteria of non-fecal origin, test have been devised to detect E. coli exclusively as the coliform bacterium most specific for fecal contamination. Today, both the total and fecal coliform tests are used in the examination of water, even though E. coli has been identified as the most feces-specific member of the coliform group.Methods The MPN method uses liquid media in a multi-step assay that detects total coliforms, fecal coliforms, and E. coli all in one method. The assay for total and fecal coliforms actually consists of three successive steps or tests: Presumptive, Confirmed and Completed. For the Presumptive Test, dilutions of the sample are inoculated into fermentation tubes of lactose or lauryl tryptose broth and incubated at 35°C for 24 hr. At the end of the first 24 hours, results for total coliforms are read. For the Confirmed Test, organisms from all positive fermentation tubes (those with growth plus gas) of the Presumptive Test are transferred to fermentation tubes of brilliant green lactose bile broth or EC broth with MUG (the broth we will use in this lab) and incubated at 44.5°C for 24 hr. Tubes showing both growth and gas are considered positive Confirmed tubes. At this point, results for fecal coliforms and E. coli are read. For the Completed Test, organisms from positive Confirmed tubes are isolated in pure culture on agar plates of differential/selective media (Endo or eosin methylene blue agar) and then tested for: (1) growth and gas production in fermentation tubes of lactose or lauryl tryptose broth incubated at 35°C for 48 hr.; and a negative reaction in the Gram stain. For a positive Completed Test, the organisms must show growth plus gas production in the fermentation tubes and be Gram negative. The Completed test is rarely done in practice due to its complexity and