Lab #5 Differential Staining

Name and Course Section: 

Teressa Guerrero, Neveta James

MICROBIOLOGY 216-705

Title and Number: 

LAB#6 Methyl Red Voges-Proskauer Test

Purpose:

Procedure:

• We started this experiment by setting the tubes of MR-VP broth aside to warm to room temperature.
• We disinfected our work area using a 10% bleach cleaning solution.
• The tubes were labeled E. coli and S. epidermidis.
• We inoculated each tube with the appropriate organism, and passed the top of the tube thru the flame of the candle, both before and after to ensure other organisms would not transfer with our specimen.
• The tubes were incubated for 48 hours.
• After the 48 hours, the tubes were set aside to cool to room temperature.
• We used 4 sterile test tubes and labeled 2 E. coli, and 2 S. epidermis.
• We transferred ½ of each of the MR-VP broth tubes that we incubated, into each of the 4 sterile tubes.
• We used 1 E. coli tube, and 1 S. epidermis tube and added about 7 drops of  

Methyl Red reagent to each of the tubes.

• We then used the other E.coli and S.epidermis  and added 12 drops of Barrit’s A and gently mixed as indicated.
• We then added 4 drops of Barritt’s B to both tubes and shook the tubes gently for 30 seconds as indicated.
• We opened the caps to allow exposure to oxygen and let the tubes sit for 30 minutes.
• After recording our results we disinfected our work are and allowed the tubes to soak for an hour.

Procedure

1. Disinfect the work area.

2. Label two microscope slides E. coli and S. epidermidis.

3. Use a plastic inoculation loop to transfer a sample of each organism to the corresponding

slide.

4. Add a drop of hydrogen peroxide to the smear. Mix with the plastic loop if needed. Do not use

a metal loop when using hydrogen peroxide as hydrogen peroxide will give a false positive and

degrade the metal.

5. Interpret the results.

a. A positive result is the evolution of oxygen gas evidenced by bubbling or foaming.

b. A negative result is evidenced by no bubbles or only a few scattered bubbles.

Figure 7: Catalase Test Results

6. Record the results.

7. Return the E. coli culture and the S. epidermidis stock culture to the refrigerator for use in

future experiments.

8. Clean and disinfect the slides and work area.

Observations