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Laboratory Exercise # 14: Antimicrobial Testing

The purpose of this exercise is to familiarize the student with the Kirby Bauer Disk Diffusion Method for testing antimicrobial sensitivities.
The testing of microbial sensitivity to antibiotics can be accomplished by several methods. In this exercise the Kirby Bauer Disk Diffusion method will be used. The
antibiotic to be tested is of only one specific strength and is impregnated into a filter
paper disk. A standardized inoculum is swabbed onto a Trypticase soy agar plate and the
antibiotic disks are placed on the agar. While the plate is incubating the antibiotic
diffuses away from the disk through the medium, and its concentration diminishes in the
agar the further it moves from the disk. After incubation, the area around the disk will
show a zone with no apparent growth if the microorganism is sensitive to the antibiotic,
or will grow completely up to the disk if it is resistant to the drug. The zone of inhibition
must be measured in millimeters and then compared with a zone standard, which allows
for the interpretation of whether a microorganism is resistant, intermediate, or sensitive to
a particular antimicrobial.

Materials: To be done in pairs

Trypticase Soy Broth cultures of: Staphylococcus epidermidis Procedure:
1. Label each plate with student name and the microorganism name.
2. Inoculate one of the Trypticase soy agar plates with Staphylococcus epidermidis and
the other with Escherichia coli by streaking for confluent growth using a swab that has been moistened with the appropriate broth culture. 3. Place the following antibiotic disks on each plate using sterile forceps. The disks should be spread out around the plate in order to prevent overlapping of the zones of inhibition. Gram positive organism:
4. Incubate the plates inverted at 37° C for 24 hours.
5. After incubation measure the zone of inhibition using a metric ruler. Record the results
in millimeters on the data chart.
6. Using the chart provided, interpret your results for each antibiotic and record whether
the organism is sensitive, intermediate or resistant for each disk.
S. epidermidis
Table 14.1: Evaluation of Zone of Inhibition
Antibiotic Content
Ampicillin (Staphylococci)
(Enterobacteriacae) Carbenicillin (Pseudomonas)
Resistant is the zone size indicated in the chart and any measurement less than that
Sensitive is the zone size indicated in the chart and any measurement greater than
that number.

1. Which antibiotic(s) is the most effective against Staphylococcus epidermidis?
2. Which antibiotic(s) is the most effective against Escherichia coli?
3. Which antibiotic would you not use for treating Staphylococcus epidermidis?
4. Which antibiotic would you not use for treating Escherichia coli?
5. Which of the antibiotics would be considered broad spectrum?


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