GRAM STAIN

Effective Date: 
Thu, 08/01/2013
Reviewed: 
Tue, 01/10/2017
Revised: 
Fri, 08/09/2013
Policy: 

The Gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and Gram reactions; in a clinical microbiology laboratory, it is additionally a critical test for the rapid presumptive diagnosis of infectious agents and serves to assess the quality of clinical specimens. The test was originally developed by Christian Gram in 1884, but was modified by Hucker in 1921.
Interpretation of Gram-stained smears involves consideration of staining characteristics and cell size, shape, and arrangement. These characteristics may be influences by a number of variables, including culture age, media, incubation atmosphere, staining methods, and the presence of inhibitory substances. Similar considerations apply to the interpretation of smears from clinical specimens, and additional factors include different host cell types and possible phagocytosis.

Gram stain permits the separation of all bacteria into two large groups, those which retain the primary dye (gram-positive) and those that take the color of the counterstain (gram-negative). The primary dye is crystal violet and the secondary dye is safranin O.  Gram positive bacteria appear violet due to the crystal violet-iodine complex. Gram negative bacteria appear pink due to the safranin counterstain.
The morphology of the bacteria can also be observed and organisms can be classified according to their shape, (i.e. rods or cocci). Rods are elongated, narrow forms whose ends can be rounded, squared or sharp. Cocci are generally round or oval forms that can be seen in chains (streptococci) or clusters (staphylococci).

 

Procedure: 

Procedure:

1. For swabs, dab the swab on a clean glass slide several times. (If the same swab is to be used for culture only dab the tip of the swab so the sides of the swab will maintain integrity.)
2. For other clinical specimens and culture colonies apply the specimen in a manner that will yield a thin, uniform smear.
3. Allow the smear to air dry.
4. Fix smear to slide by setting on the heat block for several minutes.
5. Cover slide with Crystal Violet Reagent for one minute.
6. Rinse slide with tap water.
7. Cover slide with Iodine Reagent for one minute.
8. Gently rinse the slide with tap water and allow to drain.
9. Rinse slide with Decolorizer until Decolorizer runs off the slide with no color ( less than 10 seconds).
10. Rinse slide gently with tap water.
11. Cover slide with Safranin Reagent for one minute.
12. Rinse slide gently with tap water. Allow slide to drain and dry gently with bibulous paper.
13. Examine slide under oil immersion lens.

Note: Use caution so that slides are not over decolorized, causing gram positive bacteria to appear gram negative.

Interpretation:

Gram positive bacteria appear violet due to the crystal violet-iodine complex. Gram negative bacteria appear pink due to the safranin counterstain.
The morphology of the bacteria can also be observed and organisms can be classified according to their shape, (i.e. rods or cocci). Rods are elongated, narrow forms whose ends can be rounded, squared or sharp. Cocci are generally round or oval forms that can be seen in chains (streptococci) or clusters (staphylococci).

 

Key Points: 

Specimen Requirement:

Clinical specimens submitted on a swab, body fluids, urine or sputum can be submitted for gram stain. The exact source or location the sample was obtained from must be submitted with the specimen.
Gram stains can also be performed on isolated colonies from blood agar plates as an aide in the identification of the organism.

Quality Control:

A quality control slide containing a gram positive cocci (Staph aureus ATCC 25923) and a gram negative rod (E coli ATCC 25922) should be stained and evaluated each day gram stains are performed on clinical specimens. The organisms must stain properly before any results on clinical specimens can be released.

Record result in Gram Stain Test QC log.

Reagents and Supplies:

Gram Stain Kit  (Hardy Diagnostics)
            Crystal Violet Reagent
            Stabilized Iodine Reagent
            50% Acetone/Alcohol Decolorizer
            0.4% Safranin
Microscope Slides
Microscope with oil immersion lens
Blotting paper
Heating block
 

REFERENCES

Hardy Diagnostics Gram Stain Kit Package Insert   Copyright 2002

Bailey and Scott, Diagnostic Microbiology. 11th Edition, 2002, C.V. Mosby Inc., pp 122-125