Diagnostic Laboratory Section

DIFFERENTIAL STAINING

Gram staining

Introduction

• The Gram stain was introduced by Hans Christian Gram, a Danish Physician in1884. This is most frequently used differential stain that divides bacteria into two major groups as Gram positive and Gram  negative.

Method

1. Prepare a thin smear, allow it to dry and fix with heat.
2. Cover the slide with crystal violet solution and allow to act for 1 minute. Wash in water.
3. Flood the smear with Gram’s iodine solution for 1 minute. Wash in water.
4. Decolorize with absolute alcohol or acetone for few seconds, until the smear loses colour. Wash with water.
5. Counter stain with dilute carbol fuschin solution for 30 seconds.
6. Wash in water, blot, dry and examine under oil immersion.

Result

Gram positive organisms -Violet	Gram negative organisms - Pink

Uses

1. It helps to differentiate bacteria into two groups. i.e. Gram positive and Gram negative.
2. This differentiation is helpful in determining the use of subsequent culture media and biochemical tests.
3. It helps in identification of organism on the basis of arrangement and morphology.
4. Presence of spores and its position (unstained area) can be determined.
5. It helps in choosing antibiotic in chemotherapy.
6. It can identify non bacterial forms such as Trichomonads, Strongyloides larvae and Toxoplasma gondii trpohozoite 

Limitations

• The Gram positive organisms that have lost cell wall integrity because of antibiotic treatment, old cultures, action of autolytic enzymes or excessive heat fixation of smear may allow crystal violet to wash out with the decolorizing step and appear as Gram –variable.
  • Not all bacteria can be seen in the Gram stain.eg. Mycobacteria,  Treponema   pallidum, Mycoplasma, Leptospira, Rikettsiae, Chlamydia.
  • In over decolorization of smear, use of iodine solution which is too old (yellow instead of brown) the Gram positive organism appear as Gram negative organisms.

Factors affecting the Gram staining

• Age of culture
• Colour of Gram’s iodine
• Treatment of cells with ribonuclease, cell wall acting antibiotics
• Duration of action of decolouriser