BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

This is a technique for assay of antibiotic levels in blood of patients undergoing antibiotic therapy. Blank paper disks are allowed to absorb sera containing known concentrations of an antibiotic, and then are placed on agar plates previously inoculated to give confluent growth.

The diameter of the zone of inhibition is plotted against antibiotic concentration to give a standard curve. The diameter of the zone of inhibition around the serum sample containing an unknown amount of antibiotic is measured and the antibiotic concentration in this sample is then calculated by reference to the standard curve.

Procedure:

1. The assay solutions consist of four vials with known concentrations of tetracycline (Tet) and a sample with an unknown concentration of tetracycline (Tet). Each sample will be tested in duplicate.
   
   
2. Using sterile forceps, place ten paper disks in an empty Petri dish in five rows with two disks per row. Using a micropipette, place 0.02 ml of a solution containing 24µg of Tet on the disks in the first row of two.

Continue as follows:

• 2nd row--0.02 ml with 12µg of Tet
• 3rd row--0.02 ml with 6µg of Tet
• 4th row--0.02 ml with 3µg of Tet
• 5th row--0.02 ml with an unknown concentration of Tet
  
  

3. Add 0.1 ml of a culture of indicator bacteria (Staphylococcus) to molten Mueller-Hinton medium. After thorough mixing, pour two agar plates and allow them to harden on the bench top.
   
   
4. Make a reference mark on the bottom of the seeded agar plates. Using the template provided, place the disks on the surface of the agar plates using sterile forceps. Gently press the disks onto the agar surface and record the position of each disk in relation to the reference mark. After all disks have been placed on the agar, incubate the two plates at 35^oC.
   
   
5. Following overnight incubation, measure the diameter of each zone of inhibition to the nearest whole mm.
   
   
6. Record the zone of inhibition average diameter for each of the disks and refer to standard table for interpretation.

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