Gram Staining


In 1884, Hans Christian Gram, a Danish bacteriologist, developed a method for distinguishing between two major classes of bacteria. This technique, the Gram Stain, continues to be a standard procedure in medical microbiology. Gram was a modest man, and in his initial publication he remarked "I have therefore published the method, although I am aware that as yet it is very defective and imperfect; but it is hoped that also in the hands of other investigators it will turn out to be useful."


Gram staining is an empirical method of differentiating bacterial species into two large groups (gram-positive and gram-negative) based on the chemical and physical properties of their cell walls.


Gram staining is probably the single most useful staining procedure in a bacteriological laboratory. The technique is widely used as a tool for the differentiation of gram-positive and gram-negative bacteria, as a first step to determine the identity of a particular bacterial sample.

Gram stains are performed on body fluid or biopsy when infection is suspected. It yields results much more quickly than culture, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.

Gram Staining Procedure:

1. Make a slide of tissue or body fluid that is to be stained. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide.

2. Add the primary stain crystal violet and incubate 1 minute, then wash with water for a maximum of 5 seconds to remove the unbound crystal violet.

3. Add Gram's iodine for 1 min. It is not a stain it is a mordant. It doesn't give color directly to the bacteria but it fixes the crystal violet to the bacterial cell wall.

4. Wash with Acid Alcohol. If the bacteria is gram positive it will retain the primary stain. If it is gram negative it will loose the primary stain.

5. Add the secondary stain, safranin, and incubate 1 min, then wash with water for a maximum of 5 seconds. If the bacteria is gram positive then it will retain the primary stain and will not take the secondary stain. It will look black-violet in a pink background. If it is gram negative then it will loose the primary stain and take secondary stain making it pink-red.

Gram Stain is 2 g of 90% crystal violet dissolved in 20 ml of 95% ethyl alcohol.

Gram's iodine is 1 g of iodine, 2 g of potassium iodide, dissolved in 300 ml of distilled water.

Acid alcohol is 70% ethyl alcohol containing either 0.5% or 1.0% HCl. Quite often acetone or an acetone-alcohol mixture is substituted.

In addition it now common to use basic fuchsin inistead of safranin.


The decolorizing mixture causes dehydration of the multilayered peptidoglycan in the gram-positive cell wall, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. But in gram-negative bacteria, the decolorizing mixture acts as a lipid solvent and dissolves the outer membrane of the gram-negative cell wall. The thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the gram-negative cell is decolorized. The decolorisation step is the crucial one, and requires some degree of skill, as being gram positive is not an all-or-none phenomenon.


Gram-positive bacteria (thick peptidoglycan layer): Black-violet
Gram-negative bacteria (thin peptidoglycan layer, and an outer membrane): Pink-Red

As a general rule of thumb (which has exceptions), gram-negative bacteria are more dangerous as disease organisms because:

- Outer membrane hidden by capsule or slime layer (which hides its antigens, or "foreign markers")

- Gram-negative bacteria have lipopolysaccharide in their outer membrane, an endotoxin which increases the severity of inflammation (so severe it may lead to septic shock).

Gram-positive bacteria infections are less severe because of human body's production of lysozyme, an enzyme that attacks the open peptidoglycan layer of gram-positive bacteria. Gram-positive bacteria are also much more susceptible to beta-lactam antibiotics, such as penicillin.


After the staining distinction, the bacteria are further distinguished via their shape - as rods or cocci.