Gram Stain Procedure

M/C & useful staining procedure to assist in the identification of bacteria

Separates bacteria into 2 classifications based upon their respective cell wall structure
Gram (+) Bacteria => turn PURPLE; stain gets trapped in thick, x-linked mesh-like structure (PG) surrounding the cell. Gram (-) Bacteria => the thin PG layer does not retain the stain; must use a counterstain, which stains cell wall RED

Gram Stain Procedure

Steps in Gram Stain Procedure
1. Crystal Violet Solution 1 min. application of this Primary Stain to a heat-fixed specimen on a slide
Both gram (+) and (-) cells stain purple b/c the basic stain gets into PG layer

2. 3.

After rinsing the slide w/ H2O, Grams Iodine (KI) Solution (Mordant Stain) is applied to the slide for 1 min.
All organisms appear purple

De-colorization via an alcohol wash or acetone wash (5 seconds) => gets crystal violet stain out of the cell; H2O wash follows.
Gram (+) bacteria retain the stain; crystals remain in cell wall (trapped), thus cell appears PURPLE Gram (-) bacteria give up the stain and appear CLEAR; cell wall is partially dissolved and outer membrane dissolved; thin PG: so dye is NOT retained; gram (-) bacteria are no longer visible.

Gram Stain Procedure

Steps in Gram Stain Procedure
4. Application of a Safranin Counterstain for 30 sec; this Secondary Stain is a red dye; H2O wash follows
No effect of Gram (+) bacteria; remains purple Gram (-) bacteria appear light RED/PINK; dye gets back into the cell wall => re-introduction of color into cell

Essentially, Gram Staining is a Dyeretention procedure!

Gram Stain Procedure Summary

Gram (+) Crystal Violet
(Primary Stain)

Gram (-) Purple Purple **CLEAR** Light Red or Pink

Purple Purple Purple Purple

Grams Iodine
(KI Solution) De-colorization
(Acetone/Alcohol wash)

Safranin
(Secondary Stain)