Gram Staining Procedure

Gram stain was introduced in 1880 by the Danish bacteriologist Christain Gram.  Based on this staining reaction almost all bacteria can be separated into two large groups called Gram positive and Gram negative.

 

Aim

To differentiate between two principal groups of bacteria: gram positive and gram negative.

 

Principle  

It is a type of differential staining which uses four reagents and has got four steps. The heat fixed smear of the organism is first treated with the primary stain called Crystal violet.  It is a basic dye and its function is to impart its color to all cells.  At this stage all the organisms appear in violet color.  In the second step, after washing, the smear is treated with Gram’s Iodine.  This reagent acts as the killing agent as well as the mordant.  Mordant is a substance that increases the cell's affinity for a particular stain.  It binds with the primary stain and forms an insoluble crystal violet- iodine (CV-I) complex.  All cells appear purple black at this stage.  In the third step, the smear is treated with the decolorizing agent, absolute ethanol.  During this process, some bacteria loose the CV-I complex, whereas some retain the same.  In the final step, the smear is treated with the counter stain or secondary stain called Safranin.  The organisms which have lost the CV-I complex take up this red dye and appear red in color and are called Gram negative bacteria.  The organisms which did not lose CV-I complex will not take up the secondary stain and remain violet in color. They are called Gram positive bacteria.

 

The difference in the chemical and physical nature of the bacterial cell wall is responsible for this difference in response to gram stain.  The gram negative cell wall is thin, complex, multilayered and contains relatively high lipid contents, in addition to proteins and mucopeptides.  The dehydrating agent (absolute alcohol) readily dissolves the higher amount of lipids leading to the formation of large pores in the cell wall which do not close appreciably on the dehydration of the cell wall proteins.  Through these pores the CV-I complex leakage takes place and the cells take up the counter stain.  In contrast, the gram positive cell walls are thick and chemically simple, composed mainly of protein and cross-linked polypeptides.  When treated with alcohol, it causes dehydration and closure of cell wall pores, thereby preventing the loss of CV-I complex and cells remain violet colored.

 

Requirements

 

24 hr old cultures of Escherichia coli and Staphylococcus spp.

 

Gram staining reagents:

   Crystal violet

   Gram’s iodine solution

   95 % ethyl alcohol

   Safranin          

 

Wash bottle of distilled water, staining tray, droppers, inoculating loop, glass slides, blotting paper, Bunsen burner, microscope etc.

 

Procedure

 

·         On separate slides, make thin smears of the cultures provided, air dry and heat fix

·         Flood the smear with Crystal violet for 30 sec

·         Wash the slides with distilled water for few seconds

·         Cover each smear with Gram’s iodine solution for 30 sec

·         Decolorize the smears by adding ethyl alcohol drop by drop, until no more color flows from the smear

·         Wash the slides with distilled water and drain

·         Apply Safranin to smears for 30 sec

·         Wash with distilled water, blot dry with blotting paper and air dry

·         Observe under oil immersion objective (100X).

 

Observations

Escherichia coli- Rod shaped (bacilli) cells in chains and appeared red in color

Staphylococcus - Round shaped (cocci) cells in grape like clusters and appeared violet in color.

 

Result                                                                                                                                        In the given cultures, Staphylococcus spp. belongs to Gram positive bacteria and E. coli to Gram negative bacteria.