EXERCISE 5

Microbial Control

Supplies Required: One sterile Petri plate; one T.S. deep; sterile filter paper disks; various antimicrobial agents; one Mueller Hinton agar plate; Kirby-Bauer antibiotic disks; cultures of Escherichia coli, Bacillus megaterium, Staphylococcus aureus, or Pseudomonas aeruginosa.

Protocol:

Period 1

A. Antiseptics and Disinfectants

1. Label your sterile empty plate in the usual fashion and with the name B. megaterium,

E. coli , S. aureus, or P. aeruginosa.

2. Remove your T.S. deep and cool to 50oC. Add 0.5 ml of your designated

organism to the deep and pour into the bottom of the sterile plate. Let the agar harden.

3. Using ethanol-flamed (sterile) forceps, aseptically pick up a sterile filter paper disc, dip it in the chemical to be tested, let it drain a bit and then place it on the surface of the plate (press the disc down slightly to adhere it to the agar). Immediately label underneath the applied disc with the letter "A", "B", "C", etc. and code the letter to the chemical in your notes.

4. Each person should do 5 different chemicals in their assay. To check the inhibitory effect of metal ions on microbial growth, ethanol sterilize a penny (a source of copper ions) and lay it on the surface of the agar in place of a filter paper disc. Incubate your plate at 32oC.

B. Antibiotics

1. Label your Mueller-Hinton plate in the usual fashion, with the name E. coli,

S. aureus, or P. aeruginosa.

2. Swab the entire surface of the plate with the test organism. Swab thoroughly--if spots are missed it will be difficult to interpret the results.

3. Dispense 4-6 different antibiotic-discs equally over the surface of the Mueller-Hinton plate using either the automatic dispenser or sterile forceps. As in part A, you will probably need to "seat" the discs on the agar by pressing them down gently with a sterile forceps (DO NOT break the surface of the agar). Incubate at 32o.

After 48 hours your plates will be refrigerated.

Period 2

1. Examine your plates. Using a ruler containing metric rulings measure the zone of microbial growth inhibition (in mm) caused by each of the chemicals or antibiotics used. Measure the zones from the bottom of the plate rather than opening up the plates and contaminating the rulers.

2. Exchange plates with your neighbors such that you can compare the action of each of the chemicals or antibiotics against each of the test organisms. Express your results as "zones of inhibition" measurements.