Agar Diffusion Assay Well Diffusion Assay Developing Resistance Soil Screening Isolating Active Compounds

Discover a New Antibiotic MIC Determination Christine L.Case

MIC Determination Serial dilutions

Prepare serial dilutions of the antimicrobic in small sterile tubes, a cell well plate, or a sterile microtiter plate. A 2-fold dilution series is shown in the figure. Be sure to change pipette tips after each transfter and mix the contents of each well. Add 2-5 ml of nutrient broth to each well and inoculate with the test bacterium. Cover the wells with parafilm and incubate for 24 hr.

Check for growth

. Check the bottom of the wells for a pellet of growth. Growth is shown by a white dot in the bottom of the well. Record the presence of growth

Subculture

Transfer an inoculum from wells showing no growth to nutrient broth. Incubate for 24 hr. and record the presence of growth.

Check for growth

If growth occurs in a subculture, the dilution was not bactericidal. The lowest dilution that inhibited growth is the MIC. The lowest dilution that killed the bacteria (i.e., no growth in the subculture) is the MBC.

What is the MIC? The MBC? Click here to check your answers. If you do not reach the MIC or MBC, change the dilutions and try again.

Developing Resistance

You can estimate whether resistance to the antimicrobial will develop quickly with the following procedure. Transfer an inoculum from the highest dilution showing no growth to new dilutions of the antimicrobic + nutrient broth.

Incubate

After incubation, observe for growth.

Results	This shows that the bacteria developed resistance.
	This shows that the bacteria have not developed resistance.

Repeat this experiment using a commerical antibiotic against the test bacteria to determine whether resistance develops more slowly to your new antimicrobic.