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

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 .

repeat till tube 6 using 1 ml pipette 3.Detailed procedure If AB conc is 400ug/ml 1 1X 200 2 ½ 100 3 ¼ 50 4 1/8 25 5 1/16 12.Inoculate all tubes with one drop of the microorganism using .5 6 1/32 6.25 C 1. mix and transfer 1 ml from tube 1 to 2.Aseptically distribute 1ml broth into each wasserman tube with 5 ml pipette 21Add 1ml AB in the first wasserman tube.

Precautions  Mark carefully all tubes. Adjust the volume in all tubes  Don’t 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 .

B: turbidity in the control tube is usually more intense than in other tubes showing growth .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.