GRAM STAINING

This staining procedure is the most important in Microbiology. Danish physician, Christian Gram in 1884, developed it. It separates most of the bacteria into two groups i.e. Gram positive which stains blue and Gram negative which stains red. This staining involves the following four steps.

Crystal violet dye stains both types of bacterial cells blue.

Than iodine solution is added to form a crystal violet-iodine complex, all cells

Continue to appear blue.

Than organic solvent such as acetone or ethanol extracts a blue dye complex from lipid rich thin walled Gram-negative bacteria to a greater degree then from a lipid poor thick-walled Gram-positive bacteria. After this step the Gram negative bacteria appear colorless whereas Gram-positive bacteria remains blue colored.

Than the red dye Safranin stains the decolorized Gram-negative cells red and Gram-positive bacteria remains blue colour.

IMPORTANCE:

This staining helps the bacteria dividing into two main categories i.e. Gram positive and Gram negative.

After identification of bacteria the choice of selecting specific antibiotic become easier.

There are two methods mostly used for in vitro sensitivity testing:

1. Disk Diffusion:

In disk diffusion method, paper disks impregnated with known amount of antimicrobials are placed on the surface of an agar plate that has been streaked with the standard inoculums of microorganisms. Than after an appropriate incubation period (18-24 hours) at available temperature, depending upon the type of microorganisms, the diameters of clear zones around the disk indicating no microbial growth, are measured.

The diameters are susceptible, intermediate or resistant for individual drug, by referring to standardized value. This technique provides qualitative or semi qualitative on inhibitory activity of an antimicrobial agent. Now a day’s “WELL TECHNIQUE” is being used by researchers for screening the compound for their antimicrobial activity.

2. Broth Dilution:

It is used to determine the quantitative susceptibility of an organism to an antimicrobial agent. In this method serial 2-fold dilution of the antibiotic in broth are inoculated with a standardized suspension of microorganism. For bacteria, after an hour over night incubation the results are expressed as under,

1. MIC:

Minimum inhibitory concentration may be defined as the lowest concentration of the drug that inhibit visible growth after an overnight incubation or after a suitable incubation period depending upon the species of the microorganisms.

2. MBC:

Minimum bacterial concentration can be determined by sub-culturing tubes that show no bacterial growth on to agar plates after an overnight incubation period. It is the lowest concentration of the drug that kills at least 99.9% of original bacterial inoculums.

The results of MIC & MBC should be correlated with the concentration of the drugs achieved in the plasma and other body fluids and tissues.