A Broth Microdilution Method for Antibiotic Susceptibility Testing of Gram-Positive Bacteria

Isolate Gram-positive bacterial colonies from a culture plate and suspend them in a medium.

Dilute the suspension to achieve the optimal bacterial concentration for the assay.

Inoculate the test wells of a microplate containing increasing concentrations of a lipoglycopeptide antibiotic and a growth control well containing only the medium.

Spread an aliquot from the control well onto culture plates to verify culture purity and for bacterial count.

Incubate to allow bacterial growth, during which the cell wall is synthesized.

The antibiotic binds to peptidoglycan, a cell wall component, and its precursors, inhibiting polymerization and weakening the wall.

The disruption allows osmotic pressure to cause cell lysis.

After incubation, confirm culture purity and optimal bacterial count.

Inspect the wells for bacterial growth.

The control well shows visible growth, while the the test well with the lowest antibiotic concentration that completely inhibits growth indicates the minimum inhibitory concentration (MIC).

In this demonstration, one freshly prepared plate will be inoculated. To prepare the inoculum, select several well isolated colonies from an 18 to 24 hour blood agar or other non-selective agar plate. Here, quality control S. aureus and E. faecalis strains are used.

Touch the top of each colony with a sterile swab and transfer some of it to a tube containing one to five milliliters of CAMHB or saline. Keep transferring until the turbidity matches a 0.5 McFarland standard within 15 to 30 minutes of preparation, dilute the inoculum one to 100 in CAMHB to arrive at a final concentration of five times 10 to the fifth CFU per milliliter. The acceptable range is two to eight times 10 to the fifth CFU per milliliter.

Immediately using a multi-channel pipette with sterile tips, transfer 50 microliters of the final inoculum to each appropriate well of the MIC panel plate. Here, four rows are inoculated with each of the two isolates for a total of four replicates per isolate. Next, to perform a purity check, use a pipette to transfer one to 10 microliters of the solution in the positive growth control to a non-selective agar, such as trypticase soy agar with 5% sheep blood.

Use a sterile loop to spread the solution over the surface of the plate. Turn the plate 90 degrees and streak again to assure that the inoculum is evenly distributed. Cover the plate and set it aside.

In preparation for a colony count, use a single channel pipette to transfer 10 microliters of the solution in the growth control well to 10 milliliters of saline mix, and then transfer 100 microliters to a suitable non-selective agar medium, such as trypticase soy agar with 5% sheep blood using a sterile loop, spread the solution over the entire agar surface. Repeat this process two times in different directions to assure even distribution of the inoculum. Next, stack up to four MIC panels in a plastic container ensuring that the top panel is tightly sealed with a lid.

Then place a damp paper towel inside the plastic container and close it securely with the lid within 30 minutes of inoculation. Incubate the MIC panels, the colony count plate and the purity check plate in an ambient air incubator at 35 plus or minus two degrees Celsius for 16 to 20 hours. The next day, read the minimum inhibitory concentration as the lowest concentration that completely inhibits bacterial growth in the wells as detected by the unaided eye.

Determine the number of colonies on the colony count plate. Multiply the number counted by the dilution factor. For example, 50 colonies times a dilution factor of 10, 000 equals five times 10 to the fifth CFU per milliliter.

An acceptable range is 20 to 80 colonies, which equates to two to eight times 10 to the fifth colony forming units per ml. The concentration of the inoculum is an important variable to control the susceptibility testing in order to obtain accurate results. Finally, check the purity plate.

If all colonies are similar to the colonies used as inoculum, then it can be considered pure. If there are any other colonies present, then there is potential for a contaminant to be present in the MIC panel and the test should be repeated.