The Gram Stain

A description of Gram staining

1. First, an inoculum is taken from a culture using an w:inoculation loop and put on a slide and then allowed to air dry. If the culture is solid, it is diluted by adding a drop of water or sterile saline 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 far too much inoculum at this step.
2. The specimen is heat-fixed by passing the slide, inoculum side up, through a bunsen flame 1-2 times, without allowing the slide to become hot to the touch. This prevents the bacteria from being washed away later and it also kills the bacteria
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. Lugol can also be used instead of crystal violet.
4. Rinse off with water for a maximum of 5 seconds.
5. Add iodine (Gram's 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 acetone and alcohol several times until no more colour appears to come from the sample. This washes away all the unbound basic dye, (usually crystal violet) and leaves Gram-positive organisms stained purple and Gram-negative organisms unstained (colourless).
8. Rinse with water immediately to prevent over-decolourisation.
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.

[edit]

Interpretation

Inspect the slide under a microscope

• Gram-positive organisms will appear blue-black or purple.
• Gram-negative organisms will appear red or pink.

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