Gram staining

Gram staining is a differentiate staining technique which is used to differentiate bacteria into two groups such as gram positive and gram negative based on their ability to retain color during staining.

                              Gram negative bacteria have outer lipid membrane which surrounds the cell membrane. Only 10% of their cell wall is comprised of peptidoglycan.  Ex: Salmonella, Shigella, Escherichia coli (E. coli). Gram negative bacteria are stained in pink color.

                          Gram positive bacteria have no outer lipid membrane. 80-90% of their cell wall is comprised of peptidoglycan. Ex:  Bacillus, Clostridium, Lactobacillus, Listeria, Taphylococcus, Streptococcus, and Streptomyces. Gram positive bacteria are stained in purple color.

Procedure- A loopful from a broth or agar plate is taken into a glass slide. Then the smear is prepared by heat fixing at bunson flame. As the proteins of the cell wall are denatured by heat, it supports the bacterium cells to adhere to the slide. The smear is stained by flooding with crystal violet. It is allowed crystal violet to react with smear for 2 minutes. Gram’s iodine is added to from large Crystal violet iodine complex. It is allowed iodine to react for 1minutes. Excess Crystal violet iodine complex is drained from the slide.  The smear is decolorized by adding 95% ethyl alcohol without washing for 3o seconds. The stain from the cell wall is rinsed by water. Safarin is added as the counterstain. It is allowed safarin to react with smear for 30 seconds. It is rinsed by water and allows air dry. The stained smear is observed under dry high, mid or lower power objectives or oil immersion objectives.

Observations:

Crystal violet- Crystal violet is added as the primary stain. It is a basic stain. Its positively charged ions penetrate the cell walls of both gram positive and gram negative and bond to the negatively charged cell components. So both gram positive and gram negative cells are stained in purple color.

Gram’s Iodine- It is added as the mordant. It increases the affinity of ions of the primary stains to cell wall via reaction with crystal violet ions. It forms large Crystal violet iodine complex. Both gram positive and gram negative cells are still stained in purple color.

95% Ethyl alcohol- In the case of gram negative bacteria, ethyl alcohol is able to dissolve the outer lipid membrane. It left the cell membrane exposure. It increases the porosity of the cell wall. So after addition of 95% Ethyl alcohol, water is able to wash away large Crystal violet iodine complex from the thin peptidoglycan layer.  So after water resining, the smear appears in colorless.

                                    Ethyl alcohol acts as dehydrate agent in gram positive bacteria. It causes the pores in the cell wall to shrink. So water is unable to wash away large Crystal violet iodine complex from the thick peptidoglycan layer. So after water resining, the smear still remain in purple color.

Safranin- Safranin is added as counter stain. Gram negative bacteria appear in pink color. Gram negative bacteria are unable to stain in safranin and still appear in purple color.