ENVR 421 - Environmental Health Microbiology - Spring, 2008Total and Fecal Coliform and E. coli Analyses by Membrane Filter MethodsINTRODUCTIONAs previously noted, E. coli and other coliform bacteria can be analyzed in water and other environmental samples by membrane filter (MF) as well as other methods. MF methods for totaland fecal coliform analyses are well-known historically and are described in standard reference works (Section 9222, Standard Methods for the Analysis of Water and Wastewater). MF methods for E. coli were developed more recently (Mates and Shaffer, 1989) and have been standardized by the US EPA for routine use (Fed. Reg, 1991; US EPA, 2000; 2002; USEPA). Initial E. coli MF methods required a confirmatory step in which membranes with presumptive E. coli colonies were transferred to a urea substrate to test for urease activity (E. coli is urease-negative). Newer developments in MF methods for E. coli are the transfer of TC and/or FC membrane filters with their colonies to nutrient agar containing the glucuronide substrate MUG (4-methylumbelliferyl -D-glucuronide). After 4 hours of incubation at 35oC, the colonies are examined under long wavelength UV light, and those colonies fluorescing blue are considered E.coli (Mates and Shaffer, 1989; Fed. Reg., 1991). More recently, glucuronide substrates for β-glucuronidase activity by E. coli, either fluorogenic ones like MUG or chromogenic ones, have been included in the primary culture medium. Hence, as bacteria grow and form colonies on the membrane, they produce the β-glucuronide hydrolysis product that either fluoresces under long wavelength UV light (as from MUG) or has a distinctive color (red or green, for example) (Grand and Baumgartner, 1996; Moini et al., 1996).In this lab exercise several types of membrane filter methods will be used to detect total and fecal coliforms and E. coli in water and wastewater samples and the results from the different methods will be compared.PURPOSETo use the following membrane filter methods for analyses of total and fecal coliforms and E. coli in selected samples of water and wastewater.Total coliforms on mEndo LES medium followed by blue fluorescence detection of E. coli on nutrient agar-MUGFecal coliforms on mFC medium followed by fluorescence detection of E. coli on nutrient agar- MUGTotal coliforms and E. coli on medium containing a TC chromogen and an E. coli fluorogen.Total coliforms and E. coli on medium containing a TC chromogen and E. coli chromogenMATERIALSWater and wastewater samples - assigned by instructorMembrane filter apparatus - consists of membrane filter holder, manifold assembly or vacuum flask and vacuum sourceMembrane filters - mixed cellulose esters, 47 mm diameter, ~0.45 um pore size, "up" side has a grid pattern.Flat blade forceps - for holding and transferring filters50 and 60 mm diameter petri dishes of media:mEndo LES for total coliforms mFC for fecal coliforms Nutrient agar containing 100 μg/ml MUG (to identify which fecal coliforms are E. coli based on appearance of blue fluorescing colonies TC and E. coli medium (TC chromogen and E. coli fluorogen) (Based on Manafi and Kneifel, 1991.). Bio-Rad TC and E. coli agar (TC and E. coli chromogens) (Based on Grand and Baumgartner, 1996; Moini et al., 1996).Diluent – Standard Methods buffer OR peptone water; in milk dilution bottles OR polypropyleneround bottles, dispense 99 ml per bottlePipets - 10 mlWashing (“squirt”) bottles - containing Standard Methods buffer OT peptone water; to rinse interior of filter funnel after filtering samplesWhirl-pac or zip-lock leak-proof plastic bags - for mFC platesWater baths - set at 44.5oCAir incubator - set at 35oC for mEndo, fluorogen-chromogen and chromogen-chromogen platesHand-held long wavelength UV light to detect fluorescence (from fluorogenic substrate hydrolysis product)NOTE: Before making dilutions, make sure the diluent volumes in dilution bottles is 99. Sample Dilution:-mix the sample 25 times by shaking. -then dilute the sample serially 10-fold as directed by the instructor. To make each dilution, pipetexactly 11 ml of sample (or previous dilution) into 99 ml of dilution water in a dilution bottle. Replace bottle cap and mix 25 times before making the next dilution.Sample Filtration, Plating and Incubation1. Set up a vacuum assembly and connect the lower section (base) of a filter unit to it. Be sure tohandle the base using aseptic technique (avoid touching the top surface of the base on which the membranes are placed).2. Use a sterile forceps to put (aseptically) a membrane filter, grid side up, on the surface of the filter base. Align and attach the upper, funnel section of the filter unit to the base.3. Starting with the highest sample dilution to be tested, shake the sample 25 times, remove the bottle cap and pipet exactly 20 ml into the funnel. Then, turn on the vacuum to filter the 20 ml through the membrane. As soon as the sample has filtered, squirt sterile diluent water onto the inside of the filter funnel and allow it to filter through the membrane. Then, turn off the vacuum.Aseptically remove the filter funnel from the filter base and set aside (turn upside down on a sterile surface).4. Using a pair of flat-blade forceps, lift the membrane filter from the base by its outermost edge and place it grid side up onto the surface of a plate of mEndo LES. The filter is applied to the medium by "rolling" it onto the surface. Avoid trapping air bubbles under the filter.Repeat the procedures 2, 3 and 4 above a total of three (3) more times, each with 20-ml volumesof the same sample dilution. Place each membrane on:a plate of mFC medium for fecal coliformsa plate of fluorogen-chromogen medium for TC and E. colia plate of chromogen-chromogen medium for TC and E. coliRepeat these membrane filtration procedures for the next lowest sample dilutions in succession. Check with instructor for which dilutions of water, treated sewage effluent and raw sewage are tobe filtered.Incubate all plates inverted in a 35oC air incubator. After 2 hours of incubation remove fecal coliform (mFC) plates from the incubator, place them in watertight plastic bags and incubate submerged in a 44.5oC water bath.Reading Results (after 22 +/- 2 hours of incubation, total).Remove the plates from the water bath or the air incubator. Count the total coliform colonies on mEndo LES and the fecal coliform colonies on mFC. You may have to carefully