# 7.2: Procedures

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## A. The Autoclave: (Optional Field Trip)

Each table: 1 broth culture of Geobacillus stearothermophilus

Each table: 1 TSA plate

1. Divide TSA plate into 2 areas, labeled “before” and “after”.

2. Make a streak of Geobacillus stearothermophilus on the area labeled “before”.

3. Place the culture tube in a wire rack for autoclaving. Your instructor will take you to the Micro Prep room, where our technician will discuss the use of the autoclave.

4. After the culture has been autoclaved, make another streak on the side of the plate labeled “after”.

5. Incubate the plate at 55° C until the next lab period.

## B. Effect of Temperature

Each student: broth cultures of Micrococcus roseus, Geobacillus stearothermophilus, Escherichia coli, and Serratia marcescens

Each student: 1 TSA plate

1. Divide your agar plate into 4 areas, and inoculate with each of the following bacteria (use a small streak or spot inoculation): Micrococcus roseus, Geobacillus stearothermophilus, Escherichia coli, and Serratia marcescens

Your instructor will collect all 4 plates from your table and place them at 4 different incubation temperatures: 4˚C (refrigerator), room temperature (about 25˚C), 37˚C, and 55˚C. After incubation, you will determine which temperatures are appropriate for the growth of these species.

## C. Effect of Moisture

Each student: 1 empty sterile tube, sterile cotton swab and liquid culture of one of the following: Proteus vulgaris, Staphylococcus aureus, Mycobacterium phlei, or Bacillus subtilus

Instructions

Week 1: Each student at the table should choose one of the bacteria listed above.

1. Make sure your bacteria are suspended in the broth by gently flicking the tube.

2. Dip a sterile cotton swab into your liquid culture to pick up some bacteria.

3. Press the swab against the inside of the culture tube to make sure you are not transferring too much moisture.

4. Use the swab to introduce bacteria into the bottom of your empty sterile tube.

5. Discard the swab in the disinfectant beaker, and incubate the tube until the following week.

Week 2: tubes of nutrient broth, 5 ml pipets, pipet pumps

1. Observe any changes in the appearance of your tube.

2. Use a sterile 5 ml pipet and pipet pump to add 2 ml of nutrient broth to your tube.

3. Incubate this tube until the following lab period.

Week 3: record growth (a turbid culture indicates that the bacteria survived dry conditions for one week and were able to grow after nutrient broth was added)

## D. Demo Experiment: Determination of the TDT

There will be 8 plates at the instructor’s table which you will use to determine the TDT at 65˚C and at 80˚C for the following organisms: M. roseus, E. coli, S. aureus, and G. stearothermophilus. Each of these organisms was heated at 65˚C and at 80˚C for 15 min., 20 min. and 30 min. 0 min. is the control. They were then streaked on the plates. Record the results as instructed, and then calculate the TDT for each organism at each temperature.

## E. Effect of UV radiation

Per table: 12 TSA plates (6/pair of students), liquid cultures of Serratia marcescens and Bacillus subtilis, sterile cotton swabs, UV lamp, visible light source

Note

Serratia marcescens produces a red pigment known as prodigiosin especially when it is grown at room temperature. However, cells that have mutations in the genes responsible for producing this pigment may lose their ability to make it.

1. Each pair of students will work with one type of bacteria (Serratia marcescens or Bacillus subtilis). Label your plates with the name of the organism and the experimental conditions as follows:

1. UV lid off 5 sec

2. UV lid off 10 sec

3. UV lid off 1 min

4. UV lid off 10 min

5. UV lid on 10 min (control)

6. Visible light (lid off) 10 min (control)

2. Use the sterile cotton swab to inoculate a lawn of bacteria (cover the whole surface of the plate) for all 6 plates.

3. Your instructor will demonstrate how to use the UV lamp (be extremely careful to minimize your exposure). Expose each plate to the UV light source with the lid off for the specified amount of time. Be sure to time the exposure carefully!

a. Visible light control: this plate should be placed under the small lamp with the lid off for the maximum exposure time.

b. UV lid on control: this plate should be placed under the UV lamp for the maximum exposure time used but with the lid in place.

4. Incubate all plates until the next lab period.

Note

If you are working with Serratia, your plates will need to be wrapped in foil before incubating them. This is to present any photoreactivation (a DNA repair mechanism) from occurring.

7.2: Procedures is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Joan Petersen & Susan McLaughlin.