Lab 17 Microbiology of Water

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Feb 20, 2024

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Microbiology of Water I/II OBJECTIVES: Students should be able to 1. perform the three steps used to determine water quality 2. define coliform and explain why they are good indicator organisms of water quality 3. explain how the MPN test is used in measuring water quality 4. discuss the selective and differential nature of EMB agar INTRODUCTION Water can be a vehicle to spread diseases such as typhoid fever ( Salmonella typhi ), cholera ( Vibrio cholerae ), and amoebic dysentery ( Entamoeba histolytica ). These diseases may be caused by a low number of organisms and so analyzing for specific pathogens is extremely difficult. It is therefore easier to screen for indicator organisms that suggest the potential presence of pathogens. Sewage water that is to be released into the environment as well as drinkable (potable) water is subject to such tests in order to ensure the safety of the water supply and to reduce the risk of disease. Indicator organisms are ones that indicate the presence of human wastes; these are the group known as coliforms . Coliforms are defined as Gram negative non-spore-forming, facultative rods that ferment lactose. Especially indicative of human or animal waste is the coliform E. coli . The microbiological analysis of water is intended to detect these organisms and give an estimate of their concentration as the more indicator organisms present, the more likely that pathogens will also be present. The test consists of three parts: the Presumptive test, the Confirmed test, and the Completed test. 1. Presumptive Test The presumptive test uses a statistical method to estimate the number of coliforms in a water sample. The test used here is called the MPN (Most Probable Number). This test shows whether lactose fermenting organisms may be present in the water. You can view a video of this process described below at https://www.micro.iastate.edu/video/microbiology-014-most-probable-number 2. Confirmed Test The confirmed test is used to verify the presence of lactose fermenting Gram negative rods by inoculating Eosin Methylene Blue (EMB) agar plates. 3. Completed Test The completed test is used to verify the presence of the coliforms, which can be identified by a Gram stain on an isolated colony on EMB and inoculating an IMViC series of tests to determine the potential pathogenicity of the isolate. (See Water analysis III)

Part 1: Presumptive Test MATERIALS : We will be using 4 sources of water: tap water, distilled water, toilet water and pond water Before starting your experiment: Record your hypothesis about which water(s) will be positive and explain your reasoning. The medium often used is lactose broth . There is a Durham vial in the broth. Ingredients per liter of water: Peptone 190 (Pancreatic Digest of Gelatin) 5.0 g Beef Extract 3.0 g Lactose 5.0 g pH 6.7 + 0.2 at 25°C METHODS 1. Inoculate three tubes of single strength lactose broth (SSLB) with 0.1 ml of your water into each tube. 2. Inoculate three tubes of single strength lactose broth (SSLB) with 1 ml of your water into each tube. 3. Inoculate three tubes of double strength lactose broth (DSLB) with 10 ml of your water into each tube. DSLB is made using half the volume of liquid (500 ml rather than 1 liter) in making the media. 4. Incubate the tubes at 37 o C.

RESULTS Record how many tubes show signs of lactose fermentation (gas in the Durham vial). Use the accompanying table to determine what the Most Probable Number (MPN) of organisms is per 100 ml of the water. Tap Distilled Toilet Pond #tubes positive 10 ml #tubes positive 1 ml #tubes positive 0.1 ml MPN Note that the MPN for all negative tubes is not 0. Remember that MPN is a test of probability of organism concentration in 100 ml of water sample and note how much of that 100 ml has been tested. Questions for Presumptive Test: 1. Is this a selective medium? A differential medium? If lactose is fermented, what specific chemical is present in the Durham vial? 2. After you add 10 ml of water to 10 ml of DSLB, what will the final LB concentration be in the tube now containing 20 ml of liquid? 3. After all inoculations, what is the total volume of water tested from each sample? 4. Why is the MPN for all tubes positive listed as >1400 rather than a specific number?

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Part 2: Confirmed Test MATERIALS: EMB agar METHODS : 1. Inoculate samples from positive presumptive tests onto eosin-methylene blue agar (EMB) . The dyes in the medium can readily enter and kill Gram positive cells by diffusing through the peptidoglycan layer to the cell membrane. The outer membrane of the Gram negative cell prevents access through the cell wall making this a selective medium for Gram negative bacteria. EMB is also a differential medium . It contains lactose and organisms that can ferment lactose will reduce the pH resulting in precipitation of the dyes. Colonies of lactose fermenting bacteria have a blue-black appearance often with a green sheen ( E. coli below left). Non-lactose fermenters grow as colorless or translucent colonies ( Proteus vulgaris , below right). Questions for Confirmed Test: 1. What would growth of each of the above organisms look like on MacConkey agar? Part 3: Completed Test MATERIALS: IMViC tests (Indole test, Methyl Red test, Voges-Proskauer test, Citrate test) METHODS: Using an isolated colony from the EMB plate that shows lactose fermentation, inoculate a series of IMViC tubes, incubate and read the results to determine whether the organism is a truly indicative of fecal contamination ( E. coli ) or an environmental organism ( Enterobacter aerogenes ).

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