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It is named after the inventor, the Danish scientist Hans Christian Gram (1853-1928), who developed the technique in 1884 to discriminate between pneumococci and Klebsiella pneumoniae bacteria.

1 The Gram stain procedure

1. First, an inoculum is taken from a culture using an inoculation loop and put on a slide. If the culture is solid, it is diluted by adding a drop of water on the slide and mixing with the loop. It is important here to take a very small inoculum so that the end result is a sparse single layer of bacteria. It is a common mistake for beginners to put way too much inoculum at this step.
2. The specimen is heat-fixed by passing the slide through a bunsen flame a few times, without allowing the slide to become hot to the touch.
3. A basic dye, crystal violet or gentian violet , is used to stain the slide. This dye is taken up by both Gram-positive and Gram-negative bacteria. Allow to stain for 1 minute. The slide should look purple to the unaided eye, and if examined microscopically at this point both Gram-positive and Gram-negative bacteria are purple.
4. Rinse off with water for a maximum of 5 seconds.
5. Add iodine solution (1 % iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye.
6. Rinse with water.
7. Apply 95% ethanol or a mixture of acetoneIn chemistry, acetone (also known as dimethyl ketone 2-propanone propan-2-one and beta-ketopropane is the simplest representative of the ketones. Acetone is a colourless mobile flammable liquid with a pleasant, somewhat fruity odor, melting at -95. 4 ° C and alcoholIn general usage, alcohol (from Arabic al-khwl , or al-ghawl ) refers almost always to ethanol, also known as grain alcohol and often to any beverage that contains ethanol (see alcoholic beverage . This sense underlies the term alcoholism ( addiction to a several times until no more colour appears to come from the sample.This leaves Gram-positiveGram-positive bacteria are those that are stained dark blue or violet by gram staining, in contrast to gram-negative bacteria, which are not effected by the stain. The stain is caused by a high amount of peptidoglycan in the cell wall, which typically lac organisms stained purple and Gram-negativeBacteria that are Gram-negative are not stained dark blue or violet by Gram staining, in contrast to Gram-positive bacteria. The difference lies in the cell wall of the two types; Gram-positive bacteria have a high amount of peptidoglycan in their cell wa organisms unstained (colourless).
8. Rinse with water.
9. Apply a suitable counterstain. Opinions vary as to the best choice but suitable stains include safranin or fuchsin . This stain is taken up by both Gram-positive and Gram-negative organisms, but does not alter the colour of Gram-positive organism much, as they are already purple. It does, however, make the Gram-negative organisms pinkish-red.
10. Blot gently and allow to dry. Do not smear.

1.1 Results:

Inspect the slide under a microscopeA microscope is an instrument for viewing objects that are too small to be seen by the naked or unaided eye. The science of investigating small objects using such an instrument is called microscopy, and the term microscopic means minute or very small, not

Gram-positiveGram-positive bacteria are those that are stained dark blue or violet by gram staining, in contrast to gram-negative bacteria, which are not effected by the stain. The stain is caused by a high amount of peptidoglycan in the cell wall, which typically lac organisms will appear blue-black or purple.

Gram-negativeBacteria that are Gram-negative are not stained dark blue or violet by Gram staining, in contrast to Gram-positive bacteria. The difference lies in the cell wall of the two types; Gram-positive bacteria have a high amount of peptidoglycan in their cell wa organisms will appear red.

organisms that cannot reliably be differentiated by this staining technique are said to be Gram-variable