Page 82 - MNU PM502 Ph. Micreo practical

Pharm D- Clinical Pharmacy Program Third Level Pharmaceutical Microbiology& Antimicrobials (PM 502)

Determination of MIC by Microbroth dilution

The broth dilution method uses small volumes for routine testing. It utilizes

microliter plastic plates containing 96 wells. The advantage of the system is that

it utilizes small volumes of reagents and allows a large number of bacteria to be

tested relatively quickly.

Steps:

a) Prepare two-fold serial dilutions of antibiotics: Pipette 0.05 mL of Mueller-

Hinton broth into wells 2–11 (using multichannel pipette).

st
b) Pipette 0.05 mL of antibiotic solution into wells 1 (1 concentration) and 2.

c) Transfer 0.05 mL from well 2 to well 3 and continue through well 9. Be certain

to change tips between wells to prevent carryover of antibiotic.

d) Discard 0.05 mL from well 9. Well 10 and 11 serves as positive and negative

controls, receive no antibiotic.

Prepare culture for inoculation

e) Select 4–5 isolated colonies from an overnight culture and dilute in broth to a

turbidity comparable to that of a 0.5 McFarland turbidity standard (approximately

1.0

8
×10 CFU/mL). Within 15 min of the preparation of this inoculum add 0.05 mL of
bacterial broth suspension to each well except the eleventh (last) well, which is the

negative control tube.

f) The plate should be incubated overnight in an ambient air incubator at 35–37 ͦ C

for 24 h.

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