Microbiology Test

Gram stain

Dr. Simi Paknikar
Medically Reviewed by Dr. Simi Paknikar, MD
Last Updated on Apr 05, 2014
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Test : Gram stain

Indications : The test is employed to identify the possible group of bacteria causing an infection.

Steps employed in the gram stain technique -

Take a small loopful of bacterial culture and spread over a small area of the micro slide. Allow it to air dry.

Heat- fix the cells by passing the slide over the Bunsen burner two or three times.

Then flood the slide with crystal violet stain. Wash briefly with tap water and drain.

Treat with a mordant - I2:KI mixture - to help fix the stain

Flood the slide briefly (10 seconds) with 95% alcohol to destain. Wash off with tap water. If smear is thick, it needs some more decolorization. This step is the trickiest of all the steps involved.

Flood the slide with a counter stain using a paler dye of different color (safranin). Wash off with tap water and don’t rub the slide.

Physiology : The Gram stain is a staining technique that is most commonly used in the bacteriology laboratory to distinguish between gram-positive and gram-negative bacteria.

The technique, which yields faster results than culture, is used as the first step in identifying bacterial types and is based on the fact that the gram positive and negative bacteria have different cell wall structure and function.

It is mostly done on biopsy samples or on body fluids, and could have a vital impact on a patient’s treatment and prognosis. Antibiotics effective in gram positive and gram negative infections are usually different; therefore the test can help to decide which treatment can be started.

Normal Range : Gram Positive/Gram negative

Interpretation : Gram-positive organisms - Retain the initial violet stain

Gram-negative organisms - Decolorized by the organic solvent and hence a pink color of the counter stain is shown.

The basic difference between gram-positive and gram-negative bacteria is in the ability of the former to retain the crystal violet color on its cell wall.

Sample : Bacterial culture

Test Method : Gram staining

Related Tests : Fungus smear, Acid fast stain

References : +



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