MIC/E Test

Preparation of Doubling Dilution tubes for MIC

1. Add 1ml of the antibiotic stock solution to the tube containing 9.0 ml TSB and vortex. 2. Label your 8 TSB tubes (3.0 ml) from 1-8. 3. Add 3ml of the diluted antibiotic stock solution from Step #1 to tube #1 and vortex. 4. Using a new pipette, transfer 3.0 ml from tube #1 to tube #2 and vortex 5. Repeat the doubling dilutions through all 7 TSB tubes (refer to Fig. 1) 6. Discard 3.0 ml from tube 7 in the sink (this will leave 3 ml in all 7 tubes).

Discard the 3ml in the sink FROM WHICH TUBE

TUBE 7

Epsilometer Test (Etest)

The Etest utilizes Mueller-Hinton Agar. Bacteria are inoculated onto Mueller Hinton Agar to create a lawn, then the Etest antibiotic strip is placed in the center of the inoculated plate and gently pressed onto the surface of the agar so that the entire strip is in contact with the agar (fig. 3). The plates are then incubated to allow the bacteria to grow and to allow the antibiotics to diffuse into the agar.

MIC

The lowest concentra1on of inhibitor that completely inhibits growth

There are several different methods for testing antibiotic efficacy:

1. Kirby-Bauer Test (most commonly used test) 2. Determination of the Minimal Inhibitory Concentration (MIC) 3. Epsilometer test (Etest)

Inoculation of MIC tubes

1. Using aseptic technique, obtain an isolated colony from your streak plate and transfer it to the tube of 0.85% NaCl and vortex to create a suspension of cells in the diluent. Important Note: You DO NOT want the suspension to be turbid (cloudy). You should only be using one colony - two at most. 2. Using a 1.0 ml pipette, transfer 0.1 ml of the bacterial suspension to each of the MIC tubes (1-8). 3. Incubate at 37oC for 24 hours. 4. After 24 hours, vortex the MIC tubes and examine them for signs of bacterial growth (turbidity) and record the MIC in your notebook. In your write-up be sure to show how you determined your MIC and show your calculations.

Add no bacteria to which tube

tube 8

zone of inhibition on E test

The zone of inhibition will appear larger where the concentration of antibiotic is greatest and will taper toward the strip as the concentration of antibiotic and the inhibition of the bacteria's growth is decreased