Most bacteria can be categorized under Gram-positive and Gram-negative bacteria. The categories can be differentiated based on the cell wall composition and the reaction with chemicals using the Gram Staining method. This method is introduced by Hans Christian Gram which is very popular among microbiologists as a primary test to the sample. The cell wall between these two groups of bacteria varies based on the thickness of peptidoglycan and the presence of the outer membrane. Generally, the presence of a thick layer of peptidoglycan in Gram-positive bacteria will stain violet or purple color on the cell, while thinner layers of peptidoglycan in Gram-negative bacteria stain red or pink color on the cell wall.
This procedure consists of three techniques. 1) Staining of the Crystal violet, 2) Applying the Decolorizer and 3) Counter Stain with Safranin. In mud crab, the samples can be collected from the blood, gill or hepatopancreas.
Samples are stained with crystal violet dye. Gram’s iodine then overlays to form a complex between crystal violet and iodine. This solution will form a larger molecule and be insoluble in water.
Decolorizer then added to the sample to dehydrate the peptidoglycan layer, shrink and tighten it. The peptidoglycan shrinking will not apply to the Gram-positive bacteria cell, as it is thicker. The bacteria will retain the crystal violet dye in their cell. On the other hand, the shrinking will occur to Gram-negative bacteria thus the crystal violet color will be faded.
Safranin then applied to the sample as a counterstain. Safranin is less dense compared to crystal violet, thus it will not disturb the cells that already stain the violet color. Yet, the red color will then stain on Gram-negative bacteria.
The shape and clear color of the sample will be observed under a compound microscope. Under 1000x total magnification, immersion oil is used to increase the resolving power of the microscope. Both Gram-positive and Gram-negative bacteria can be pathogenic to the host depending on the type of bacteria. In mud crab aquaculture, Vibrio Spp is a major Gram-negative bacteria that can cause death to the mud crab. It is a great concern because Gram-negative bacteria consist of lipopolysaccharide (LPS) molecules which have a strong resistance toward antibodies.
To conclude, the gram staining method is a basic laboratory technique that can be applied by the farmers. It is an easy technique to identify the presence of pathogens inside the culture organism.