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1 Microbial Survival Introduction The main objectives of this practical are: Verify, or otherwise, the Department of Health recommendation that food be cooked to 70 o C for 2 minutes or an equivalent time-temperature regime. The reasoning behind this has already been covered in lectures. Assess the production of sublethally injured cells following heat treatment. Determine the persistence of micro-organisms on chopping boards. Determine the effectiveness of anti-microbial chopping boards Practical details: You are provided with overnight cultures of hazard group 2 organisms and therefore you must handle accordingly. The Pseudomonas is the only hazard group 1 organism provided. You will have a lot of agar plates to incubate at the end of this practical. It is VERY IMPORTANT that you: (1) Take your time (2) Pre-dry the plates if necessary (2) Label the plates with your bench letter and your name (3) Label the plates with the particular experiment. You will also have a large number of saline bottles to use on a time schedule, therefore label them before you start the experiment. Next week: (1) You must calculate the number of colony forming units per ml of original sample. If you do not know understand the model calculation given at the end of the schedule then ask now. You will subsequently need to calculate the % survival after each heat treatment. (2) A strong suggestion is to bring in a semi-completed table for you to write your results on next week. (3) Include in your Introduction :Thermal sensitivity of Gram-negative and Grampositive organisms, protection factors (extrinsic parameters). The reason for the Department of Health time and temperature regimes.

2 Experiment 1 is carried out by only 3 groups: Group 1 Death of Pseudomonas spp. on a Microban cutting board Group 2 Death of Salmonella spp. on a wooden cutting board Group 3 Death of E. coli O157:H7 on a Microban cutting board Group 4 Death of Bacillus cereus on a Microban cutting board Group 5 Death of Clostridium perfringens on a Microban cutting board. Note you will need to incubate you plates anaerobically Experiment 2 is carried out by all groups: Groups 1-5 Temperature sensitivity of Salmonella spp. Groups 6-11 Temperature sensitivity of E. coli O157:H7 Experiment 3 is carried out by 6 groups : Group 6 Bacillus cereus Group 7 Listeria spp. Group 8 Clostridium perfringens. Note you will need to incubate you plates anaerobically Group 9 Salmonella Group 10 E coli O157 Group 11 Staphylococcus aureus Experiment 1: Antimicrobial activity of chopping boards Using the green tape provided mark the chopping boards into 8 sectors. These will be referred to as : A1 A2 A3 A4 B1 B2 B3 B4 At a known time inoculate sectors A1-4 and B1-4 with 20µl of your organism. Using a dampened cottonwool swab sample A1, and streak on PCA. Using an ATP swab sample B1 and determine the RLU. At a known time place the chopping board in a warm oven. After 30 minutes sample A2 (Cottonwool swab, PCA plate) and B2 (ATP swab, RLU). After 1 hour sample A2 (Cottonwool swab, PCA plate) and B2 (ATP swab, RLU). At the end of the practical sample A2 (Cottonwool swab, PCA plate) and B2 (ATP swab, RLU).

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4 Experiment 2: Thermal death of E. coli O157:H7 and Salmonella Part 1 preparation of materials (1) You will be assigned a waterbath to use. (2) Place the two Universal bottles `Saline and `Meat extract in the assigned water bath C) to equilibrate for 5 minutes. Ensure the water level covers the tube contents, otherwise the experiment will not be valid. (3) Label four 9ml salines bottles `Heat treated saline sample and either 30, 60, 90 or 120 sec. These are for step 7. (4) Label four 9ml saline bottles `Heat treated meat extract sample dilution and either 30, 60, 90 or 120 sec. These are for step 7. Part 2 Preparation of inoculum (5) Dilute the overnight culture by aseptically pipetting 1ml into 9ml saline (=10-1 dilution), and repeat the sequence to (6) Use the spreadplate method to put 0.1ml volumes of dilutions 10-8, 10-7, 10-6, 10-5 in duplicate on to Tryptone soya agar (TSA) and MacConkey agar. Spread the sample using a sterile glass rod. This must be done very carefully as these results are crucial for your calculations of survival. Part 3 Temperature treatment (7) At a known time pipette 1ml of the original overnight culture into the two waterbath Universal bottles. (8) After 30, 60, 90 & 120 second intervals remove 1ml from each bottle into 9ml of appropriately labelled saline. (9) Spreadplate by pipetting 0.1ml of each sample on to two TSA and two MacConkey agar plates and then spreading with a glass rod. (10) Incubate plates at 37 o C.

5 Experiment 3 : Factors affecting bacterial temperature sensitivity The standard time and temperature recommended by the DoH for killing vegetative bacteria is 70 C for 2 minutes (and equivalents), but what is the affect of the suspending medium? Prepare the following tubes. Tube % Salt (ml) % Nitrite (ml) Dw (ml) Tube CMB (ml) ph (10ml) ph5 ph7 ph9 Dw (ml) Place tubes in the 70 C waterbath for 10 minutes to equilabrate. At known timed intervals; inoculate with 1ml of your organism and leave for exactly 2 minutes. Enumerate the number of survivors by pipetting 0.1ml onto the surface of a BHI plate and spread over the surface using a sterile glass rod. Dilute your organism 10-1 to 10-8 in sterile saline and inoculate five BHI plates with 0.1ml of the 10-8 to 10-3 (in this order to save pipette usage) and surface spread with glass rod (also 10-8 to 10-3 sequence). Determine the survival of the organism after the heat treatment and the number of decimal reductions in viability.

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