Lysozyme Activity

1. Prepare a fluid extract from a plant.

2. Prepare a 1:10 dilution of the plant extract by adding 0.5 ml plant extract to 4.5 ml lysozyme buffer. What is the purpose of the buffer?

3. Mix 2.5 ml of the plant extract preparation with a 2.5 ml Micrococcus luteus suspension in a spectrophotometer tube. Carefully pipette the contents of the tube up and down three times to mix.

4. Record the absorbance at 540 nm at 30 seconds, 60 seconds, 120 seconds, 180 seconds, 240 seconds, 5 minutes, and at 5-minute intervals for the next 15 minutes.

5. Repeat steps 3 and 4 using egg-white lysozyme.

6. Plot your data. Place the Petri plate, dilution tube, and spectrophotometer tube in the appropriate To Be Autoclaved container.

Bacteriocins Figure

A. What happens is you treat the extract with proteinase?

B. What happens if you heat the extract?

1. Aseptically prepare 1:10², 1:10⁴, and 1:10⁶ dilutions of a bacterial culture (S. aureus or E. coli).

2. Label three sterile serological tubes "6A," "6B," and "6C." Aseptically transfer 0.1 ml of culture from the highest dilution (1:_____) to each tube.

3. Repeat step 2 with the other dilutions using the same pipette. Label each tube appropriately. How many tubes do you have?

4. To each A tube, add 0.4 ml of unheated plant extract; to each B tube, add 0.4 ml of plant extract heated to 56°C for 30 minutes; to each C tube, add 0.4 ml of saline. What is the purpose of the B and C tubes?

5. Mix all tubes and incubate them at 35°C for 1 hour.

6. Label nine sterile Petri dishes in the same manner as the tubes: "1:10² A," "1:10² B," "1:10² C," "1:10⁴ A," 1:10⁴ B," and so on.

7. Remove 0.1 ml from the A tube of the highest dilution, and place it in the appropriate Petri dish. Using the same pipette, transfer 0.1 ml from the 1:10 A tube to the corresponding Petri dish. Repeat this procedure with the 1:10² A tube. Why are the samples transferred from the highest dilution first?

8. Using another pipette, repeat step 7 with the B tubes. With your remaining pipette, repeat step 7 with the C tubes.

9. Pour melted, cooled nutrient agar into each dish to a depth of approximately 5 mm. Gently swirl to mix the contents, and allow the agar to solidify.

10. Incubate the plates for 24 to 48 hours at 35°C. Record the number of colonies on each plate. Plates with fewer than 25 colonies should be reported as TFTC (too few to count), and those with more than 250 colonies as TNTC (too numerous to count). What should you do it none of your plates are countable?

11. Calculate the number of colony-forming units (cfu) per milliliter for the control. Choose one plate with between 25 and 250 colonies:

Calculate the number of colony-forming units per milliliter for the unheated and heated extract.

12. Calculate the percent of increase or decrease in bacterial numbers due to exposure to heated and unheated plant extract as compared to the control.