BISC209: The Gram Stain

The Gram Stain

Procedure:
1. Prepare a bacterial smear slide (Find protocol at BISC209: Preparing a bacterial smear slide).

To Gram stain the desired organism(s):
Use the staining trays and sink area. Be sure you evenly cover all the smears on the slide throughout this procedure.

2. Flood the smears with Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) and stain for 1 minute. (Crystal violet is the primary stain.)

3. Rinse the slide in a very gentle stream of water; drain off excess water by touching the edge of the slide to a paper towel.

4. Flood slide with Gram's Iodine (mordant), let stand for 1 minute, and rinse with a gentle stream of water.

5. Quickly, drip Decolorizing Reagent (80% isopropyl alcohol, 20% acetone) down the length of the slide to remove the excess dye. This step is tricky as it is easy to over- or under-decolorize. IMMEDIATELY rinse with a gentle stream of water.

6. Flood the slide with the Counterstain Solution (0.6% safranin in 20% ethanol) for 2 minutes; rinse with water.

7. Blot dry using the bibulous paper package in your benches. Do not tear out the pages, just insert your slide and pat it dry.

8. Clean up your area, disposing of the reagents properly and rinsing your staining tray.

9. Observe your stained microbes microscopically following the correct procedure for using the the oil immersion objective on your compound brightfield microscope BISC209: Microscopy found in the protocol page.

Background
The first of the dyes most useful to bacteriologists was a reddish violet dye, mauvein, synthesized in England by William. H. Perkin, and patented by him in 1856. This synthetic dye and others were immediately appreciated by histologists, but were not applied to bacterial cells until Carl Weigert (a cousin of Paul Ehrlich) used methyl violet to stain cocci in preparations of diseased tissue in 1875. Subsequently, the use of various synthetic dyes for bacteriological preparations developed rapidly when they were promoted through the publications of Robert Koch and Paul Ehrlich.

The synthetic dyes are classified as acid dyes, or basic dyes, depending on whether the molecule is a cation or an anion. The introduction of the terms acidic and basic was unfortunate because it would be more revealing to refer to them as cationic or anionic dyes. A look at the structural formula will quickly reveal the nature of the dye.

Each dye molecule has at least two functional chemical groupings. The auxochrome ionizes and gives the molecule the ability to react with the substrate, while the unsaturated chromophore absorbs specific wavelengths of light. The color of the solution obtained is that of the unabsorbed (transmitted) light. To be a dye, the molecule must have both auxochrome and chromophore groups. The auxochrome is usually an ionized carboxyl, hydroxyl, or pentavalent nitrogen group. The chromophore may have unsaturated nitrogen bonds such as azo (-N=N-) indamine (-N=), nitroso (-N=O) or nitro (O-N=O), groups; or unsaturated carbon to carbon, carbon to oxygen, or carbon to sulfur bonds, such as ethenyl (C=C), carbonyl (C=O), C=S, or the quinoid ring (= = =).

Resonance is also important to color. In crystal violet, an electron resonates between the three benzene rings. As the pH of the solution is lowered, the resonance becomes more and more restricted. When the resonance is restricted from three to only two benzene rings, the solution turns from violet to green, and then to red when resonance between the two rings ceases.

Cationic dyes will react with substrate groups that ionize to produce a negative charge, such as carboxyl, phenolic, or sulfhydryl groups. Anionic dyes will react with substrate groups which ionize to produce positive charges, such as the ammonium ion. Any substrate with such ionized groups should have an ability to combine with cationic or anionic dyes. Generally, the most important staining substrates in bacterial cells are proteins, especially the cytoplasmic proteins; however, other substances also have dye affinity. These include amino sugars, organic acids, nucleic acids, and certain polysaccharides.

Sudan III, or sudan black B, is a popular stain for fatty material. It does not have an auxochrome group, and is insoluble in water, but soluble in fatty material. When a solution of sudan black B in ethylene glycol is placed over bacterial cells, the fatty material will dissolve some of the dye and thus take on the color of the sudan black. The staining effect is purely a solubility phenomenon, and not a chemical reaction, or physical adsorption.

There are many stains that can reveal the morphology of the cell, and some, such as methylene blue, have special uses. The Gram stain is especially useful because it not only reveals bacterial morphology, but also is a differential stain, giving additional information about the cell wall composition. The Gram staining procedure as it is done today, involves: a) the primary staining of all cells with crystal violet, b) the precipitation of this dye on and within the cells by means of iodine as a mordant, c) the removing of the dye-iodine precipitate from some cells (the Gram-negative) with a decolorizer such as 95% ethanol, acetone, or n-propyl alcohol, and d) the counter-staining of the decolorized cells with safranin. Organisms that retain the primary dye are termed Gram-positive, while those which lose the primary stain and show the counter-stain are termed Gram-negative. The differentiation obtained is not an absolute one, and is based on the differences in the rate at which the primary dye is lost from the cells.

Not all organisms or substances that retain the primary dye of the Gram procedure can be termed Gram-positive. For example, the staining of Mycobacterium with crystal violet requires the use of heat (as in the-acid-fast stain). The staining which results will be resistant to decolorization even though acid alcohol is used as the decolorizer and regardless of whether or not iodine has been applied to the slide. This is, in effect, an acid-fast stain rather than a Gram stain.

Truly Gram-positive cells, such as B. subtilis or S. aureus, will not retain the primary dye if the iodine step is omitted. Criteria for a true Gram-positive state include the requirement of iodine following the crystal violet.

Analysis of Morphology from a Stained Smear
The morphological characteristics of bacteria, such as size, shape, and arrangement, can be demonstrated by staining a bacterial smear so that individual bacterial cells can be visualized. For most species, these characteristics are genetically determined and thus typical of the species. However, there are some species of bacteria that show considerable variation, or pleomorphism in these characteristics even within a single culture. For example, both Mycoplasma (a bacterium lacking the rigid cell wall of most bacteria) and Arthrobacter (a soil bacterium) show forms ranging from coccoid to rodlike to filamentous. Some pathogenic species such as Mycobacterium tuberculosis and Corynebacterium diptheriae are also pleomorphic.

Bacteria range in size from as small as 0.2 µ to 3.0 µ. They may show the following shapes: spherical (coccus), rod-shaped (bacillus), curved (spiral), or helical (spirochete). They may also assume a characteristic arrangement, based on the way cell division and subsequent separation of the cells occur in that particular species. This typical arrangement shows up better in broth cultures than on a solid medium because in growth on a solid medium the cells are packed together tightly, thus modifying the natural arrangement.

There are various types of arrangements: singly, as in most of the Gram-negative rods; in pairs, as found in the bacillus Klebsiella pneumoniae and the cocci Streptococcus pneumoniae and Neisseria: in chains, as in Bacillus and Streptococcus: in regular packets of four or eight as in some Micrococcus; in irregular clumps as in Staphylococcus; and in parallel lines and/or sharp angles as seen in Corynebacterium diptheriae. This last arrangement is called palisade or Chinese character formation. Because of their waxy cell walls, Mycobacterium species are difficult to emulsify and tend to stick together in clumps on the smear; the pathogen Mycobacterium tuberculosis may form long cords of cells in culture. Finally, individual cells may show deviations from the standard form. For example, cocci of Neisseria show flattened sides, making them bean-shaped when occurring in pairs; the rods of Corynebacterium and Mycobacterium often appear club-shaped with swollen ends or knobs. Both groups may show irregular staining. The diplococci of Streptococcus pneumoniae appear slightly elongated and lancet-shaped, with one flattened end and one tapered end.