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The Gram stain, performed properly, differentiates nearly all bacteria into two major groups. For example, one group, the gram-positive bacteria, include the causative agents of the diseases diphtheria, anthrax, tetanus, scarlet fever, and certain forms of pneumonia and tonsillitis. A second group, the gram-negative bacteria, includes organisms which cause typhoid fever, dysentery, gonorrhea and whooping cough. In Bacteria the reaction to Gram stain reagents is explained by different cell wall structures. Gram-positive microbes have a much thicker cell wall, while that found in Gram-negative microbes is thinner. Microbes from the Archaea domain contain different cell wall structures than that seen in microbes commonly found in the lab (Bacteria domain). However, they will still have a species specific Gram stain reaction, even though the underlying macromolecular structures are different.

The Gram stain is one of the most useful differential stains in bacteriology, including diagnostic medical bacteriology. The differential staining effect correlates to differences in the cell wall structure of microorganisms (at least Bacteria, but not Archaea as mentioned above). In order to obtain reliable results it is important to take the following precautions:

• The cultures to be stained should be young - incubated in broth or on a solid medium until growth is just visible (no more than 12 to 18 hours old if possible). Old cultures of some gram-positive bacteria will appear Gram negative. This is especially true for endospore-forming bacteria, such as species from the genus Bacillus. In this class, many of the cultures will have grown for more than 2 days. For most bacteria this is not a problem, but be aware that some cultures staining characteristics may change!

• When feasible, the cultures to be stained should be grown on a sugar-free medium. Many organisms produce substantial amounts of capsular or slime material in the presence of certain carbohydrates. This may interfere with decolorization, and certain Gram-negative organisms such as Klebsiella may appear as a mixture of pink and purple cells.

Gram stain procedure

Below is a procedure that works well in the teaching laboratories.

1. Cover the slide with crystal violet stain and wait one minute.

2. After one minute wash the stain off (gently!) with a minimum amount of tap water. Drain off most of the water and proceed to the next step. It may help to hold the slide vertically and touch a bottom corner to paper toweling or blotting paper.

3. Cover the slide with iodine solution for one minute. The iodine acts as a mordant (fixer) and will form a complex with the crystal violet, fixing it into the cell.

4. Rinse briefly with tap water.

5. Tilt the slide lengthwise over the sink and apply the alcohol-acetone decolorizing solution (dropwise) such that the solution washes over the entire slide from one end to the other. All smears on the slide are to be treated thoroughly and equally in this procedure. Process the sample in this manner for about 2-5 seconds and immediately rinse with tap water. This procedure will decolorize cells with a Gram negative type of cell wall but not those with a gram-positive type of cell wall, as a general rule. Drain off most of the water and proceed.

6. As the decolorized gram-negative cells need to be stained in order to be visible, cover the slide with the safranin counterstain for 30 seconds to one minute.

7. Rinse briefly and blot the slide dry. Record each culture as Gram positive (purple cells) or Gram negative (pink cells).

The above video demonstrates the Gram stain procedure, while Figure 3-11 shows the results of a Gram stain for gram-positive and gram-negative negative bacteria.

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