EXERCISE 8

MECHANISMS OF ANTIBIOTIC ACTION AND ANTIBIOTIC RESISTANCE

I. INTRODUCTION

Antibiotics selectively block a wide variety of metabolic activities:

• energy metabolism
• the function of bacterial membranes
• the synthesis of protein
• the metabolism of nucleic acid
• the synthesis of peptidoglycan

The following exercise demonstrates the difference between a bactericidal and a bacteriostatic antibiotic with respect to mode of action and sensitivity to penicillinase.

II. LAB WORK

• 1 tube of BACTERIAL CULTURE (dark blue cap)
• 1 tube of BROTH (large green cap)
• 1 tube of AMPICILLIN (pink cap)
• 1 tube of CHLORAMPHENICOL (black cap)
• 1 tube of PENICILLINASE (purple cap)
• 5 tubes, sterile (avocado cap)
• 4 sterile pipets, 1 ml
• 1 sterile pipet, 5 ml
• 1 Nutrient Agar plate (NA) - provided on Day 2

1. Label the 5 sterile tubes A - E as follows:
   • A. AMP
   • B. CHLOR
   • C. AMP + CHLOR
   • D. AMP + penicillinase
   • E. Control
2. Use the green PipetAid and a 5 ml pipet to transfer 1.0 ml of broth to each of the tubes A - E.
3. Use the blue PipetAid and one 1 ml pipet to transfer 0.1 ml of ampicillin to tubes A, C, and D.
4. Use a second 1 ml pipet to transfer 0.1 ml of chloramphenicol to tubes B and C.
5. Use a third 1 ml pipet to transfer 0.1 ml of penicillinase to tube D.
6. Use the last 1 ml pipet to transfer 0.2 ml of bacterial culture to all five tubes. Mix and incubate these 5 tubes at 37C until the next lab period.

7. Observe and record the turbidity in each tube. With a marking pen, mark off 5 sections on the bottom of the NA plate provided. Label the sections A - E. With a sterile loop, streak a sample from each tube onto the corresponding section of the agar surface. Sterilize the loop before each transfer. Incubate the plate at 37C until the next lab period.

8. Rate each section for amounts of growth by comparison to the control (E).

Experimental Results