Enumeration of Food Borne Bacteria - Master's Research and Thesis | FOR 500, Exams of Forestry

Download Enumeration of Food Borne Bacteria - Master's Research and Thesis | FOR 500 and more Exams Forestry in PDF only on Docsity! 1 Experiment 2: Enumeration of Food-borne Bacteria Objective: The goal of this experiment is to become familiar with methods of preparation of food homogenates, dilution schemes for liquid and solid samples and enumeration of aerobic mesophilic organisms, coliforms and Staphylococcus aureus present in foods. Experiment: Part A: Preparation and Dilution of the Food Homogenate A. Brief Background: Methods of isolation and enumeration of the microorganisms present in non-liquid foods requires treatment of the food samples to release the microorganisms into a fluid medium. The homogenate produced can be use to prepare appropriate dilutions as called for in enumerative procedures. In these procedures care must be exercised since excessive blending may injure the microbial cell; heat generated by the procedure may be detrimental; and aerosols created by mechanical blenders, may endanger the microbiologist, especially when pathogens could be in the food sample. B. Materials a. Liquid food sample, 25 ml per team b. Solid raw food sample, 25 g per team c. Solid processed food sample, 25 g per team d. Balance e. Sterile 100-ml beakers, 2 beakers per team f. Sterile spatulas, 2 spatulas per team g. Sterile 0.1% peptone water diluent, two 225-ml flask per team h. Sterile 0.1% peptone water diluent, Nine 99-ml dilution bottles per team i. Stomacher j. Stomacher bags k. Sterile 1-ml pipettes C. Procedure a. Liquid food sample i. Measure 1 ml of liquid food sample into a 99-ml dilution blank. Shake this and all subsequent dilutions vigorously 25 times in a one-foot arc. This procedure will yield a 10-2 (1 ml) and 10-3 (0.1 ml) dilution of the original sample. ii. Measure 1 ml of the 10-2 dilution of the homogenate into a 99- ml dilution blank. Shake this and all subsequent dilutions 2 vigorously 25 times in a one-foot arc. This procedure will yield a 10-4 (1 ml) and 10-5 (0.1 ml) dilution of the original sample. iii. Measure 1 ml of the 10-4 dilution of the homogenate into a 99- ml dilution blank. Shake this and all subsequent dilutions vigorously 25 times in a one-foot arc. This procedure will yield a 10-6 (1 ml) and 10-7 (0.1 ml) dilution of the original sample. iv. The procedure outlined above is summarized in a diagram appearing on a following page of this manual. The diluted samples, prepared by this procedure, will be used in the procedures outlined below. b. Solid food samples (raw or processed) i. Aseptically weigh a 25 g sample of food (use a sterile beaker and a sterile spatula). ii. Place this amount in a stomacher bag. iii. Add 225 ml of 0.1% peptone water diluent to the bag. This will yield a dilution of 10-1 (1 ml) and 10-2 (0.1 ml). iv. Blend the food and diluent using a stomacher then allow the homogenate to stand for a few minutes. During this time the foam will settle and microorganisms contacting the peptone solution will tend to resuscitated. v. Measure 1 ml of the 10-1 dilution of the homogenate into a 99- ml dilution blank. Shake this and all subsequent dilutions vigorously 25 times in a one-foot arc. This procedure will yield a 10-3 (1 ml) and 10-4 (0.1 ml) dilution of the original sample. vi. Measure 1 ml of the 10-3 dilution of the homogenate into a 99- ml dilution blank. Shake this and all subsequent dilutions vigorously 25 times in a one-foot arc. This procedure will yield a 10-5 (1 ml) and 10-6 (0.1 ml) dilution of the original sample. vii. Measure 1 ml of the 10-5 dilution of the homogenate into a 99- ml dilution blank. Shake this and all subsequent dilutions vigorously 25 times in a one-foot arc. This procedure will yield a 10-7 (1 ml) and 10-8 (0.1 ml) dilution of the original sample. viii. The procedure outlined above is summarized in a diagram appearing on a following page of this manual. The diluted samples, prepared by this procedure, will be used in the procedures outlined below. Part B: Total Aerobic Plate Count A. Brief Background: The number of aerobic mesophilic microorganism found in food has been one of more commonly used microbiological indicators of the quality of foods. It has value as an indicator of the adequacy of sanitation and temperature control during processing, transport and storage. The standard pour plate technique is well established as the procedure choice in conducting a total plate count. B. Materials 5 f. After the incubation period, count those plates showing 15 to 150 colonies. On un-crowded plates, coliform produce purplish-red subsurface colonies at least 0.5 mm in diameter. Further, the colonies may be surrounded by a reddish halo of precipitated bile. Since some non-coliforms, in particular cocci, may produce pinpoint red colonies in this medium, caution must be exercised in counting coliforms. g. Calculate and record the number of coliforms per g or ml of specimen. Part D: Enumeration of Staphylococcus aureus A. Brief Background: The growth of Staphylococcus aureus in foods presents a potential public health hazard since strains of this organism produce an enterotoxin. When such food is ingested, this toxin contributes to the nausea, vomiting, and general gastrointestinal upset. In processed foods, the presence of S. aureus usually indicates contamination from skin, mouth, or nasal passages of the food handler. This contamination may be introduced directly into the food by process line workers or by contacting line equipment previously contaminated. In raw foods, the presence of S. aureus is common and may not be related to human contamination. The significance of the presence of S. aureus in foods should be made with caution. The presence of large numbers of organisms, without identification of specific enterotoxin, is not sufficient cause to incriminate a food as the vehicle of food poisoning. Nor is the absence or presence of low numbers of S. aureus complete assurance that the food is safe. There are number of methods available for the enumeration of staphylococci, their difference resting with the type of selective agent used. The most common agents include sodium chloride, lithium chloride, and potassium tellurite. Media containing egg yolk offer additional differential qualities. B. Materials a. Baird Parker Agar Medium plates, 14 plates per team b. Sterile 1-ml pipettes c. Hockey sticks d. Incubator (35oC) e. Colony counter and tally, 1 each colony counter and tally per team f. 3% Hydrogen peroxide C. Procedure a. Use the dilutions of processed food sample homogenate for this procedure. b. You will use 14 plates (2 plates x 7 dilutions = 14 plates). Label petri plates with the names of the food product (e.g. potato salad), medium (e.g. BPA), dilution (e.g. 10-1), and Set number (e.g. Set I or Set II). c. To duplicate sets of Baird Parker Agar plates, pipet 0.1-ml aliquots from 10-1 to 10-7. d. With a sterile “hockey stick”, spread the inoculum over the surface of the agar until the surface appears dry. 6 e. Invert the plates and incubate them at 35oC for 24 to 48 hours and examine. After 24 hours staphylococcal colonies are 0.1 to 1.5 mm in diameter and appear jet black or dark grey in color. Additionally they exhibit one or more of three types of egg yolk reactions: i. discrete zones of precipitated egg yolk around and beneath the colony; ii. a clear zone of halo often occurring together with a zone of egg yolk precipitation beneath the colony; and iii. absence of either a precipitation zone or halo but visible precipitation beneath the colony f. Count all typical colonies on plates possessing between 25 and 250 colonies. Record the number of staphylococci per gram of sample. Remember to account for the 1:10 dilution produced by using an inoculum of 0.1 ml. g. Test for catalase production by flooding a Baird Parker Agar plate with 3% hydrogen peroxide. S. aureus is catalase-positive, indicated by bubble formation. h. Normally a series of typical colonies are tested for coagulase activity. 7 Total Aerobic Plate Counts on Standard Methods Agar: Liquid sample: ---------- Dilution 10-0 10-1 10-2 10-3 10-4 10-5 10-6 Plate #1 Plate#2 Average Solid raw food sample: ---------- Dilution 10-1 10-2 10-3 10-4 10-5 10-6 10-7 Plate #1 Plate#2 Average Total Aerobic Plate Counts on Standard Methods Agar containing TTC: Liquid sample: ---------- Dilution 10-0 10-1 10-2 10-3 10-4 10-5 10-6 Plate #1 Plate#2 Average Solid raw food sample: ---------- Dilution 10-1 10-2 10-3 10-4 10-5 10-6 10-7 Plate #1 Plate#2 Average