Question

How to determine the concentration of a bacteria in a 1 ml culture, and how to determine if the antibiotics are bacteriostatic or bactericidal?

Answer 1

The concentration of bacteria in a given volume, say 1 ml, can be calculated by different methods like direct cell counting using a hemocytometer and viable cell counting by plating.

In direct cell counting, the sample is loaded on to a hemocytometer and the cells are counted in each of the grids. This method is manual and time-consuming. However, if you load the samples along with trypan blue stain, you would be able to differentiate between viable and dead cells. In a hemocytometer, the average cell count obtained from the edge quadrant grids multiplied by the Dilution Factor and a factor of 10000 would provide the number of cells/ml of sample.

Another method is serial dilution followed by plating to obtain the number of viable cells through counting the colony forming units or CFUs. Here, take 1 ml of sample and serially dilute it by a factor of 10, 100, 1000, 10000 etc. Now do a spread plate from each of these diluted samples and the original undiluted sample. The viable cells would grow and form colonies which can be counted under a colony counter.This number multiplied by the dilution factor would give the number of viable cells present in the volume used for plating, say 0.1 ml. Now this data can be used to obtain the concentration of cells per ml of the original sample.

Any antibiotic, when given at its MIC ( minimum inhibitory concentration , i.e the minimum concentration of the antibiotic required in a specific medium and at specified conditions of temperature, carbon dioxide levels and other environmentall parameters to inhibit or prevent visible cell growth in 24 hours) kills bacteria enough to prevent visible growth. A bacteriostatic antibiotic has an MBC to MIC ratio ≤ 4. Here, MBC or Minimum Bactericidal concentration is the concentration of the antibiotic that could achieve a 1000 fold reduction in bacterial density as compared to an untreated control at the same conditions. Any antibiotic with a higher MBC to MIC ratio is considered to be bacteriostatic.

These MBC and MIC values can be calculated by treating a bacterial culture plate with the antibiotics at different concentrations. Calculate the ratio of the obtained MBC and MIC values to decide whether the antibiotic is bacteriostatic or bactericidal.