9.2: Procedures

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Note

You will be using sterile spreaders when you inoculate plates in Procedures A and B of this lab. Each table will have a tinfoil packet of sterile spreaders. Please be careful when you open the packet (unfold the narrow end first) so you do not contaminate the spreaders. There will be a beaker of vesphene specifically for these spreaders at each table. Place the spreaders in this beaker. DO NOT throw the spreaders in the garbage— they will be autoclaved and reused for later lab sections. DO NOT use spreaders from the disinfectant beakers in your experiment.

A. The Microbiology of Milk

In this exercise, you will compare the amount of bacteria found in regular pasteurized milk with the amount found in UHT pasteurized milk. You will also examine the effects of leaving the two different types of milk at room temperature.

Per pair of students: 2 TSA plates

Per pair of students:

Either: 2 tubes of pasteurized milk: one refrigerated, one left out at room temperature for 1 week, or 2 tubes of UHT pasteurized milk: one refrigerated, one left out at room temperature for 1 week

1. Each pair of students will choose either the regular pasteurized milk or the UHT pasteurized milk.

2. Using the 20-200 µl pipettor (also called the P200), transfer 200 µl of the refrigerated milk on a TSA plate. (Appendix III provides instructions on how to use micropipettors.)

3. Use a sterile spreader to spread the 200 µl of the milk across the plate.

4. Using a different sterile spreader, repeat Steps 2-3 using the room temperature milk.

5. Incubate the plates until the next lab.

Type of milk chosen: ____________________________________

Per Table, you should have the following plates:

Screenshot (146).png

What type of bacteria will grow on the TSA plates? ________________________________________

Predict which type of milk will have the most bacteria. Explain the rationale for your prediction. ___________________________________________________________________________________

B. The Microbiology of Poultry

In this exercise you will determine the number of bacteria found on chicken wings using serial dilutions followed by a standard plate count.

There will be 1 resealable plastic bag containing 9 ml of sterile water and a chicken wing at the instructor’s table.

Per table: 3 TSA plates ,4 tubes, each containing 9 ml of sterile H₂O,1 tube of chicken wing wash fluid (given to you by your instructor)

1. The instructor will rinse the chicken wing in the water, massaging the chicken wing to remove any bacteria that are adhering to the skin. Using a 200 - 1000 µl pipettor (also called the P1000), the instructor will transfer 1 ml of the wash fluid into 99 ml of H₂O, and will mix thoroughly. This will be a dilution of 1/100 (10^-2). The instructor will transfer 2-3 ml of this 1/100 dilution into 4 sterile tubes (1/table).

2. Label one of the tubes of sterile water 10^-3(1/1,000)—this will be Tube 1. Label one of the tubes of sterile water 10^-4 (1/10,000)—this will be Tube 2. Label the next tube of sterile water 10^-5 (1/100,000)—this will be Tube 3. Label the last tube of sterile water 10^-6 (1/1,000,000)—this will be Tube 4.

3. Using the P1000, remove 1 ml of the 1/100 wash fluid from the tube given to you by the instructor, and transfer it to Tube 1. Mix the contents of the tube by carefully by flicking the tube. Using the same pipettor, transfer 1 ml from Tube 1 into Tube 2; transfer 1 ml from Tube 2 to Tube 3, and transfer 1 ml from Tube 3 to Tube 4. After each transfer, flick to mix thoroughly. The figure below shows you how to make the transfers.

Instructions for plating

Using the P200, transfer 100 µl from Tube 2, Tube 3 and Tube 4 to 3 different TSA plates. Make sure to label your plates. The figure below indicates how the transfers should be done.

Screenshot (147).png — Figure 9.2.1: *Serial dilutions and transfers to plates*

You will be using 1 sterile spreader for all of your plates, so you must be careful to work from the most dilute sample to the least dilute sample. Make sure that all of the fluid has been absorbed into the plate before you invert the plate for incubation.

C. Microbiology of Food

Per table: 1 TSA plate

Per table: Choose 1 of the following four food materials: ground beef, mushroom, bread or lettuce.

Chosen food material: _________________________________________

1. Flame sterilize forceps. (Note: follow the recommendations for forcep sterilization described in Lab 9.)

2. Pick up a portion of your chosen food product, gently rub it over the surface of the TSA plate, then discard the food.

3. Incubate the plate until the next lab. Of the four food materials, which do you think will contain the most bacteria? __________________