Determination of MIC by broth dilution method

Minimum Inhibitory Concentration (MIC)

Definition: is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation.

WHY?
Clinically, MIC is not only used to determine the amount of antibiotic that the patient will receive but also the type of antibiotic used. This will lower the opportunity for microbial resistance to specific antimicrobial agents.

Common methods used for MIC determination

MIC by broth dilution method MIC by agar diffusion method

MIC by broth dilution method (Serial dilution method)

Principle
Add broth media in all tubes Serial dilution of selected AB Inoculation with the isolated pathogenic microorganism Incubation for 24 hour Observation of the turbidity as a parameter of the growth

Detailed procedure
Materials:
7 Wassermann tubes (Mark 1-6 Last: control) Test Tube with 10 ml of double strength nutrient broth Tube with 2 ml of AB solution Tube with Microorganism Culture One Sterile 5 ml pipette 1 Sterile 1 ml pipettes One sterile Pasteur pipette

Detailed procedure

If AB conc is 400ug/ml

1
1X 200

2
100

3
50

4
1/8 25

5
1/16 12.5

6
1/32 6.25

1- Aseptically distribute 1ml broth into each wasserman tube with 5 ml pipette
21Add 1ml AB in the first wasserman tube, mix and transfer 1 ml from tube 1 to 2, repeat till tube 6 using 1 ml pipette 3- Inoculate all tubes with one drop of the microorganism using

Precautions
Mark carefully all tubes. Adjust the volume in all tubes Dont open the broth tube more than 2 times !!DO NOT ADD AB TO THE CONTROL TUBE!!

Mixing should be at the bottom of the tube? To avoid air bubbles ?? Contamination !!!!!!!!

YOU MUST MUST MUST AVOID ANY CONTAMINATION

Expected results

MIC= AB concentration in 3rd tube+AB concentration in 4 th tube/2

Other Expected Results

All tubes are clear: MIC is less than least dilution of the antibiotic in tube No 6 All tubes are turbid: MIC is higher than the highest dilution of the antibiotic in tube No 1 N.B: turbidity in the control tube is usually more intense than in other tubes showing growth