Gram staining is a general purpose bacteriological identification technique used in the bacteriology section of the microbiology laboratory to identify and differentiate bacteria into two groups i.e. Gram-positive and Gram-negative. It was discovered by Christian Gram (1853-1938), a Danish scientist in 1884. Christian Gram showed that the cells of some bacteria could be easily decolorized with organic solvents (e.g. ethanol) after staining with some basic dyes (e.g. crystal violet) while the cells of other group of bacteria (known as Gram-positive bacteria) resisted the decolorization after the staining process. Gram staining technique is generally used to identify bacterial pathogens from cultures and clinically important specimens based on their Gram reaction. Bacteria that are neither Gram-positive nor Gram-negative are termed Gram-variable organisms and such bacteria cannot be identified with the Gram staining technique. Gram variable bacteria are usually old bacterial cultures that have lost their cell wall structure over time. Gram indeterminate bacteria are those bacteria that cannot be detected by Gram staining technique. Typical examples of Gram indeterminate bacterium is Mycobacterium species that posses a waxy/lipid-containing cell wall (that contains mycolic acid).
In the Gram staining technique, Gram-positive bacteria stain purple or blue-violet (Figure 1) while Gram-negative bacteria stain red or pink (Figure 2). Gram staining is one of the most important differential staining techniques used by microbiologists worldwide to characterize bacteria based on their cell wall compositions. Crystal violet (primary stain), Lugol’s iodine (mordant), ethanol/acetone (decolorizer) and safranin (secondary or counter stain) are the stains or dyes used for carrying out the Gram staining technique in the microbiology laboratory. Gram-positive bacteria retain the primary stain after decolorization while Gram-negative bacteria do not. The mordant (iodine) increases the interaction of the bacterial cell wall with the primary stain while the decolorizer removes the primary stain from the cell wall of the test bacteria (i.e. if the organism is Gram-negative). Gram-negative bacteria retain the colour of the counter stain (safranin), and thus appear reddish or pinkish under the microscope.
Fig. 1. Illustration of Gram positive stain result. The stained organism is Gram-positive cocci – since it appeared purple (blue-violet) under the microscope.
Fig. 2. Illustration of Gram negative stain result. The stained organism is Gram-negative bacilli – since it appeared pink or red under the microscope.
STEP BY STEP PROTOCOL FOR GRAM STAINING TECHNIQUE
- Prepare a thin smear of the test bacteria on a clean glass slide. Smear preparation is described in detail in the top part of this section.
- Allow the prepared smear to dry while standing on a staining rack.
- Add crystal violet stain using a dropper. Allow for 60 seconds.
- Wash off the stain with clean water.
- Add or cover the smear with Lugol’s iodine using a dropper. Allow for 60 seconds.
- Wash off the stain with clean water.
- Add a drop of acetone or ethanol to decolorize the stained smear.
- Wash off with water immediately.
- Add safranin to the stained smear. Allow for 60 seconds or a maximum of 2 minutes.
- Wash off the stain with clean water. Allow to dry while standing on the staining rack. Ensure to carefully clean the back of the stained slide with a clean cloth or cotton wool.
- Add a drop of immersion oil on the stained smear.
- View the stained smear under the microscope using the oil immersion objective lens (100x).
- Gram-positive bacteria appear blue-violet under the microscope while Gram-negative bacteria appear red. Ensure to take notice of the bacteria shape (i.e. cocci, rod, coccobacilli or streptococci) when reporting the result of a Gram stained smear.
Heat-fixing is defined as the microbiological technique of attaching bacterial cells onto the surface of a glass slide by the use of a Bunsen burner flame or 70% of methanol. A gentle heating of a bacterial cell on a slide when passed through the blue flame of a Bunsen burner actually kills the bacteria but nevertheless, heat-fixing does not distort the cell structure of the organism. The cell structure of the bacterial cell becomes distorted if and only when the slide is over-heated. Heat-fixing of bacterial cells allows the internal and external structures of a microorganism to be intact during staining and microscopy.
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Basic laboratory procedures in clinical bacteriology. World Health Organization (WHO), 1991. Available from WHO publications, 1211 Geneva, 27-Switzerland.