Gram staining

 Aim

To perform Gram staining technique to differentiate gram positive and gram negative bacteria

Principle

Gram staining method, the most important procedure in Microbiology, was developed by Danish physician Hans Christian Gram in 1884.  This technique separates most bacteria into two groups on the basis of cell wall composition. Gram-positive bacteria stains purple and Gram-negative bacteria stains red/pink. This difference is due the differences in cell wall composition of Gram-positive and Gram-negative bacteria.

The staining technique uses four chemical agents that are applied sequentially to a heat fixed smear.  First reagent is called primary stain, crystal violet (CV). Mordant is Grams Iodine (I) which increases the cells affinity for crystal violet. Iodine interacts with crystal violet to form large crystal violet-iodine (CV-I) complexes within the cytoplasm and outer layers of the cell. Ethyl alcohol is used as decoloring agent and it serves dual function, as a protein dehydrating agent and a lipid solvent.  In gram negative cells alcohol increases the porosity by dissolving the lipids in the outer membrane and the CV-I complex can be easily removed from the thin peptidoglycan layer.  In gram positive cells, the pores in peptidoglycan are made smaller due to the dehydrating effect of alcohol.  So here the CV-I complex remains within the cell. saffranin is the counter stain.  While Gram negative cells take up the saffranin and appear in pink colour, Gram positive cells retain the purple colour of the primary stain. 

The most critical stage of the procedure is the decolorizing step.  Over decolourisation result in loss of primary stain causing Gram positive cell to appear Gram negative.  It is important that in between applications of reagents, slides be washed under slow running water.  Gram staining is to be done with fresh cultures, not older than 24 hours.  As culture old, Gram positive organisms tend to lose their ability to retain primary stain and may appear gram variable, that is some cells will appear purple while others will appear in red colour.

Materials required

Cultures - 24 hour nutrient agar slant culture of Escherichia coli, Bacillus spp

Reagents - crystal violet, Grams Iodine, ethyl alcohol and safranin

Equipments - bunsen burner, inoculating needle, staining tray, microscope

Procedure

1. Air-dried, heat-fixed smear was flooded for 1 minute with crystal violet
2. Slide was washed in a gentle and indirect stream of tap water for 2 seconds.
3. Slide was flooded with the mordant, Gram’s iodine for 1 minute.
4. Slide was washed in a gentle and indirect stream of tap water for 2 seconds.
5. Slide was washed with decolorizing agent by adding drop by drop until decolorizing agent running from the slide runs clear.
6. Slide was flooded with counterstain, safranin for 30 seconds.
7. Slide was washed in a gentle and indirect stream of tap water until no color appears in the effluent.
8. The smear was air dried and observed under oil immersion objective

Observation

Purple colored rods and pink colored small rods were observed.

Result

Gram-negative bacteria appeared as pink short rods and Gram-positive bacteria appeared as purple rods