Day 3 Handout: Gram Stain

History

In 1884 a Danish physician named Haas Christian Gram developed a procedure that allowed him to differentiate between various types of bacteria. Today this procedure known as Gram stain is one of the quickest and most widely utilized differential stains. In a matter of moments, an investigator is capable of distinguishing between various types of microbes even within a culture where several different bacteria exist.

Procedure

1. Prepare smears of bacteria and fix.
2. Stain with Primary stain (crystal violet) for 30 second.
3. Rinse with water
4. Cover slide with mordant (Gram's iodine) for about 30 seconds. The mordant increases the binding between the primary stain and the substances with which it will interact.
5. Rinse with water
6. Decolorize with 95% ethanol for 10-20 seconds.
7. Rinse with water
8. Counterstain with safranin. The counterstain will allow you to see those bacteria which do not stain with crystal violet.

Results and Explanations

You should illustrate what the various bacteria look like being sure to note what color they stain. Gram positive cells will stain purple while gram negatives will stain red. Why is this?

It is thought that the thick cell walls of gram positive cells trap the primary stain within the cells. The thin walls of gram negative bacteria when treated with the decolorizing agent no longer retain the primary stain.