THE CAPSULE STAIN    

Source:  http://student.ccbcmd.edu/courses/bio141/labmanua/lab6/lab6.htmlDISCUSSION
This page is based on the above link but has been heavily modified to make it appropriate for our class and to avoid violating the copyright.

Under normal growth conditions, many bacteria produce a capsule or slime layer, generally a layer of slimy polysaccharide surrounding the cell. The capsule helps in non-specific attachment, protects cells from predators such as white blood cells and amoebas, helps in nutrient gathering and retards desiccation. The ability to produce a capsule is an inherited property of the organism, but the capsule is not an absolutely essential cellular component. Capsule production is often stimulated by the presence of sugars or by excess organic molecules that can be stored as extracellular carbon-rich material.  Capsules have very poor contrast and are not typically visible under the microscope.  Furthermore, they are often not stained by the types of stains we routinely use to see the rest of the bacterium. Negative stains will stain neither the capsule nor the cell, resulting in a "puffier-looking" cell, but one in which the cell and capsule can't be  distinguished from one another. In this procedure, we use a combination of a simple stain and a negative stain. The bacterium itself is stained and the background is stained, leaving a clear or slightly colored halo around the cell which is the capsule.

Organisms and Culture medium

We will be growing Enterobacter aerogenes as well as a couple of other potentially useful bacteria for you to look at. The medium is a 50:50 mixture of milk and Tryptic Soy Broth (TSB). This medium not only provides the nutrients for bacterial growth, but the dried milk on the slide is stained by the crystal violet used.  This is what makes this procedure partially a negative stain.

PROCEDURE (to be done in pairs)

1. Stir up the Skim Milk broth culture with your loop and place a loopful of your organism on the slide, spreading it out as usual.

2. Let it completely air dry. Do not heat fix. Capsules stick well to glass, and heat may destroy the capsule.

3. Stain with crystal violet for one minute.

4. Wash off the excess stain with copper sulfate solution (20%). Do not use water!

5. Blot dry and find the specimen under low power. Eventually observe using oil immersion microscopy (Gilmore's 2nd law of microscopy:  view stained bacteria under 100x). The organism and the milk dried on the slide will pick up the purple dye while the capsule will remain colorless or be tinged a light blue from the cooper sulfate.

6. To see how the result should look, look at this link to a picture of a  capsule .

The original web site from which this was derived uses the term "microbial flora". The words microbiotum, microbiota are replacing "microbial flora" since "flora" means plants.  The term "microbial flora"  is based on an ancient taxonomic scheme that classified bacteria as plants and is >50 years out of date.