The Gram Stain

In 1884, Hans Christian Gram, a Danish doctor working in Berlin, accidentally stumbled on a method which still forms the basis for the identification of bacteria. While examining lung tissue from patients who had died of pneumonia, he discovered that certain stains were preferentially taken up and retained by bacterial cells. Over the course of the next few years, Gram developed a staining procedure which divided almost all bacteria into two large groups - the Gram stain.

Individual bacterial cells are hard to see, partly because they are small, but also because they are almost transparent. In addition to magnification under a microscope, optical tricks must also be used to be able to see them:

• Phase contrast microscopy

• Staining

Either of these methods can make bacterial cells visible under the microscope. Other staining methods are described elsewhere in these documents, e.g. the Ziehl-Neelsen acid-fast staining procedure, but the Gram stain procedure is as follows:

1. Place a slide with a bacterial smear on a staining rack.
2. STAIN the slide with crystal violet for 1-2 min.
3. Pour off the stain.
   Note: fingers stain Gram-positive - use forceps!
4. Flood slide with Gram's iodine for 1-2 min.
5. Pour off the iodine.
6. Decolourize by washing the slide briefly with acetone (2-3 seconds).
7. Wash slide thoroughly with water to remove the acetone - do not delay with this step.
8. Flood slide with safranin counterstain for 2 min.
9. Wash with water.
10. Blot excess water and dry in hand over bunsen flame.