byDr. Khalid Yousuf Memon Lecturer Dept: of Pathology

Gram’s Staining

OBJECT: To study the identification & morphological features of micro-organisms by Grams’ staining

Gram’s Staining

REQUIREMENTS:Glass slide, specimen, wire loop, tripod stand, crystal violet dye, gram’s iodine, spirit, 1/10 % safranin, water, cedar wood oil & Microscope

THEORY:

The Gram stain procedure was originally developed by the Danish Physician Hans Christian Gram to differentiate Pneumococci from Klebsiella Pneumonia. It is also known as “ Differential staining”, as it divides micro-organisms into two main groups i.e. Gram’s +ve and Gram’s –ve micro-organisms.

Gram’s Staining

Crystal violet is a basic dye, it forms a complex with iodine & get fixed permanently in Gram’s +ve organisms. The dye iodine complex is not washed out from these organisms ever. They are not decolorized by spirit use.

Gram’s Staining

This is because these organisms contain thick and multilayered peptidoglycan, while Gram –ve organisms have single layered peptidoglycan, therefore the dye is not fixed permanently in them & is removed by spirit.

Gram’s Staining

Gram –ve organisms loose the dye and become colorless while the Gram + ve organisms retain the colour of crystal violet so when stained with counter stain i.e. 1/10 % Safranin , Gram –ve organisms appear pink in colour while Gram +ve organisms appear violet in colour by retaining the violet colour.

Gram’s Staining

On the basis of Gram’s staining micro-organisms can be divided into four groups; according to their color & shape Gram’s +ve cocci: Streptococci,

Staphylococci. Gram’s +ve bacilli: Corny bacterium

Diphtheria, Clostridia, Bacillus Anthrax. Gram’s –ve Cocci: Neisseria. Gram’s –ve Baccilli: E.Coli, Salmonella,

Shigella, Proteus, Pseudomonas

Procedure1. Take specimen on the wire loop &

spread it on the slide.2. Fix the smear by passing it over

the flame 2-3 times.3. Cover the smear with crystal violet

for 1-2 minutes.4. Wash it & than cover it with Gram’s

staining for 01 minute.5. Wash the smear with spirit till the

blue colour of the slide stops to appear.

Procedure

6. Than wash the slide with water & cover it with 1/10 % safranin red for about ½ minute. Than wash it with water.

7. Allow the slide to dry in air. Put a drop of cedar wood oil.

8. Than see the slide under oil immersion lens.

RESULT Gram +ve organisms appear

violet in colour, Gram –ve organisms appear pink in colour. The epithelial cells and pus cells are also pink in appearance.

Gram +ve Bacilli

Gram +ve & -ve organisms

Gram +ve in chains (S. Pneumoniae)

Gram +ve BacilliClostridium Species

Gram +ve cocciStaph: aureus

Gram +ve Diplococci( Pneumococci)

Gram +ve rods( Diphtheroids)

G+ve & G-ve Bacilli