Procedure for Exercise D

Preparation of garlic extracts

  1. Peel 3 or 4 cloves of fresh garlic (F). Cut off the attachment scar on the bottom.
  2. Weigh out 10 g of garlic. Surface sterilize by dipping in 15 % bleach for 5 scconds and rinsing in distilled water.
  3. Using a sterile mortar and pestle, thoroughly grind the garlic in 10 ml of sterile distilled water. The results should be a pulpy slurry with no chunks. Transfer die slurry to an empty pctri dish. Label the petri dish A. This will be your full-strength sample.
  4. Weigh out 1 g of the full-strength slurry, and transfer it to a sterile test rube, and add 9 ml of sterile distilled water. Mix well Pour this into an empty pctri dish. This will be your 10% sample. Isabel the petri dish B.
  5. Transfer 1 ml of the 10% sample into a sterile falcon tube and add 9 ml of sterile distilled water. Mix well. Pour this into an empty pctri dish. This will be your 1% sample. Label the pctri dish C.
  6. Place 10 ml of sterile distilled water into an empty petri dish. This will be your control. Label the pctri dish D.
  7. Placc 6 sterile filter discs in each pctri dish. Allow the discs to incubate in the solution for at least 5 minutes before transferring the discs to the culture plates.
  8. Repeat steps 1-7 above with roasted garlic (R), but skip step (2)—that is, DO NOT dip in bleach or water.

Bacterial cultures

  1. Once you have prepared your garlic extract, begin preparing your culture plates.
  2. Obtain 12 petri dishes containing nutrient agar. Turn the plates over. With a permanent marker, mark the bottom into quadrants as shown in die following diagram. Label the quadrants A, B, C, and D.

/ A

b \

\ c

d j

  1. On die lid of the pctri dish, label the plates with the names of the bacteria as follows:
  2. Write near the edge of the dish, and also label each dish with vour initials. *
  3. On 6 plates, write E. coli (Escherichia coli). Then label 3 of these plates F and the other 3 R (for fresh and roasted).
  4. On the other 6 plates, write R. subrilis (Bacillus subrilis). Then label 3 of these plates F and the other 3 R-
  5. Using sterile technique, inoculate all agar plates with their respective test organism. Watch as your instruc tor demonstrates the method of inoculation. It is also described here:
  6. Dip a sterile cotton swab into a thoroughly mixed culture. Remove excess inoculum by lightly pressing the swab against the side of the tlask.
  7. Using this swab, streak the entire agar surface horizontally, vertically, and around the outer edge of the plate to provide a heavy growth over the entire agar surface.
  8. Allow the eulture plate to dry for about 5 minutes with the lid on.
  9. Repeat until all plates have been inoculated with the cultures.
  10. Be sure to use a different sterile swab for each organism.
  11. Using sterile forccps, apply the filter discs that arc soaking in your garlic extracts to the agar surface. Carefully and gently press each disc with the forccps to make sure the disc sticks to the agar surface. Be careful—do not press the discs into the agar.
  12. Apply discs soaking in the full-strength solution to all die quadrants labeled A.
  13. Apply discs soaking in 10% solutions to all the quadrants labeled B.
  14. Apply discs soaking in 1% solutions to all the quadrants labeled C.
  15. Apply discs soaking in control solutions to all the quadrants labeled D.
  16. Incubatc all bacterial plates at 37°C for 20 to 24 hours. Invert these cultures. The cultures should be checked tomorrow morning in case growth is extremely rapid.
  17. Following incubation, examine all cultures for the presence or absence of inhibition surrounding cach disc. Carefully measure cach zone of inhibition in millimeters. Record your results in the appropriate table on worksheet 14-3.
Zone Inhibition Petri Dish Diagram

8. Rccord the average inhibition zone for cach concentration (full strength, 10%, 1%, and control) for cach organism in the tables on worksheet 14-3. Which concentration was the best? Which were not effective in inhibiting the bacteria? How did the roasted garlic compare to the fresh?

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  • Emanuela
    How to measure the zone of inhibition?
    8 years ago

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