8.2: Procedures

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A. Disinfectants

For this exercise, you will perform a filter paper disk diffusion assay to determine the effectiveness of 5 different types of disinfectants.

Materials: 1 TSA plate/student, liquid cultures of Staphylococcus aureus and Escherichia coli, sterile cotton swabs, filter paper disks, 5 different disinfectants, forceps, and beakers with 70% ethanol

Go up to the instructor’s table and write down the names of the 5 disinfectants you will test in the chart below.

Disinfectant #	Name	Concentration	Active ingredient/class of disinfectant





6	Sterile distilled water (control)

1. Choose one of the bacterial species listed above. Use a cotton swab to inoculate a lawn of bacteria on your TSA plate. Discard swabs in the beaker provided at your table.

2. Label your TSA plate with the name of the microbe you are using, and numbers 1-6 evenly spread out on the plate (see demo plate done by your instructor). The numbers (and corresponding disks) should not be too close to the edges of the plate.

3. Bring your inoculated plate up to the front table, where you will be adding filter paper disks soaked in disinfectants.

4. Before each use, forceps should be flame sterilized as follows:

a) Remove forceps from beaker of alcohol. Keep the tips angled down at all times.

b) Put the forceps in the Bunsen burner flame just to ignite the alcohol.

Note

You are not heating the forceps, just igniting the alcohol.

c) Keep a careful eye on the forceps (hold them steady in one location) until all the alcohol has burned off (this will be very quick). To avoid fires, do not hold them over any beakers of alcohol!

d) Once the alcohol has burned off, use the forceps to pick up one filter paper disk from the glass Petri dish.

5. Dip the disk into disinfectant #1.

Note

Just touch the surface of the liquid—the disinfectant will soak into the disk by capillary action. It is important to not have the disks too wet when you place them on your TSA plate.

6. Place disinfectant #1 on the appropriate area of the agar plate. Tap it down gently with the forceps to ensure that it adheres to the surface of the agar.

7. Repeat this step for all disinfectants.

Note

If you want to test some other product, just omit one of the disinfectants in the front of the room.

8. Add a filter paper disk that has been dipped in sterile water to the area of the plate labeled “#6”- this will serve as your negative control.

9. After each use, place the forceps back into the beaker with 70% ethanol. DO NOT heat them after use before returning them to the beakers.

10. Incubate plates (inverted, as usual) until the next lab period.

B. Antibiotic susceptibility testing

Each table: 3 large (150 mm) Mueller-Hinton agar plates, liquid cultures of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, sterile cotton swabs, antibiotic disk dispensers.

1. Work as a table for this exercise. Use a sterile cotton swab to inoculate the Mueller-Hinton agar plates with a lawn of bacteria for each species listed (one species/plate). Be sure to cover the entire area of the plate. This can best be accomplished by swabbing the entire surface of the plate three times, rotating the plate approximately 60º each time, and finishing the swabbing by going around the entire outer surface of the plate.

2. After all plates are inoculated, your instructor will show you how to use the disk dispenser to introduce antibiotic disks onto the plates. Be careful to always keep the dispensers in the upright position.

a. You will notice that each disk that is introduced to the plate is stamped with a letter and a number—these indicate the type of antibiotic as well as the concentration. For example, P10 is an abbreviation for 10 units of penicillin; PIP-100 is an abbreviation for 100 mcg (micrograms) of piperacillin.

3. Return to your lab table, and use your inoculation loop or needle to gently push down on the disks to ensure that they are adhered to the agar surface.

Note

Flame your loop in between plates to avoid cross-contamination

4. Incubate the Mueller-Hinton agar plates (inverted) until the next lab period.

C. Demonstrating antibiotic production with Streptomyces

Materials: Soy Flour Mannitol (SFM) plates containing cultures of Streptomyces coelicolor and two additional Streptomyces cultures, liquid cultures of Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis, TSA agar plates (1/pair of students), sterile cotton swabs, sterile 1000 µl pipet tips.

Before beginning this experiment, write down the names of the organisms you will be testing here, and provide a brief description of their appearance.

Strain #	Strain name	Description	Additional information

1. Divide your plate into 4 areas (labeled 1, 2, 3 and – for negative control).

2. Choose one bacterial liquid culture from the list above, and use a sterile cotton swab to inoculate a lawn of bacteria. Discard swab in the beaker at your table.

3. Using the large round end of a sterile pipet tip, punch a hole out of the center of each of the 4 quadrants you created on your TSA Plate. Discard the tip in the disinfectant beaker.

4. Use another sterile tip to cut out a plug of agar from the lawn of the corresponding SFM plate (your instructor may provide these for you).

5. Transfer this plug of agar into the well you created in your agar plate.

6. Repeat for the other two Streptomyces strains, using a fresh sterile tip each time.

7. Place a plug of uninoculated SFM agar on your plate in the negative control quadrant.

8. Incubate the plates at 28˚C until the next lab period.