Fifth Lab - Experimental Protocol
1. Date and Time:
Fall 2015, 2 hours
2. Throat flora exercise
Conclude study of throat flora. Prepare a brief report listing the bacteria identified and the test results supporting your conclusions. (Lab Results, last page)
3. Isolation of Staphylococcus
3.1. Special Materials:
Examples of S. aureus and S. epidermidis plates with antibiotic discs, rulers.
3.2. Procedures:
Read antibiotic sensitivity plates. Use the rulers to measure the diameter of the zone of growth inhibition. Use the following table to determine if your isolates are susceptible to a particular antibiotic.
| Zone Diameter Interpretive Table |
|  | Diameter of Inhibition zone (mm) | ||||
| Antimicrobial Agent | Disc contents | Resistant | Intermediate or Indeterminant | Moderately Susceptible | Susceptible |
| Ampicillina (AM) | 10µg | â?¤13 | - | 14-16 | â?¥17 |
| Cefoxitin (FOX) | 30µg | â?¤14 | - | 15-17 | â?¥18 |
| Clindamycin (CC) | 2µg | â?¤14 | 15-20 | - | â?¥21 |
| Erythromycin (E) | 15µg | â?¤13 | 14-22 | - | â?¥23 |
| Gentamicin (GM) | 10µg | â?¤12 | 13-14 | - | â?¥15 |
| Penicillin Gb | 10 U | â?¤28 | - | - | â?¥29 |
| Penicillin Gc | 10 U | â?¤14 | - | â?¥15 | - |
| Vancomycin (VA) | 30µg | â?¤9 | 11-Oct | - | â?¥12 |
| aGram-negative enteric organisms | bStaphylococci | cEnterococci | |||
4. Fecal flora
4.1. Special Materials:
Nutrient agar plate, antibiotic discs, forceps, soft agar tubes with glucose, lactose and maltose, example of E. coli and Salmonella on MacConkey lactose plates
4.2. Procedure:
- Inspect MacConkey plates for growth and colony color. Also, note the relative number of colonies present.
- Score plates for tetracycline and ampicillin resistance.
- Gram stain colonies.
- Determine antibiotic sensitivity profile of a Lac+, Tetr or Lac+, Ampr clone. Use the same method you did for Staphylococcus aureus (fourth lab).
- The use of sugar fermentation tests facilitates identification of bacteria. Sets of tubes containing soft agar, a pH indicator, and various sugars are provided. The sugars are glucose, lactose, and maltose. Inoculate one tube of each sugar with a Lac+ colony. These tubes will be incubated for one or two days at 37°C.
BEFORE LEAVING, PUT AWAY EVERYTHING AND CLEAN BENCHES AND THEN YOUR HANDS !!!
Sample Web-based Course Designed by William R. Stephens, II