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):
2. Flood the slide 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. Then 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
Introduction The dyes most useful to the bacteriologist are the aniline or synthetic dyes. The first of these was a reddish violet dye, mauvein, which was synthesized in England by William. H. Perkin, and patented by him in 1856. The synthetic dyes were immediately appreciated by the histologist, but were not applied to bacterial cells until Carl Weigert (a cousin of Paul Ehrlich) in 1875 used methyl violet to stain cocci in preparations of diseased tissue. Subsequently, the use of various synthetic dyes for bacteriological preparations developed rapidly, primarily motivated through the works 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 and it would be more revealing to refer to them as cationic (basic) or anionic (acid) 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 which ionizes and gives the molecule the ability to react with the substrate, and the chromophore which is a site of unsaturation and which absorbs specific wavelengths of light. The color of the solution obtained is that of the unabsorbed light which is transmitted through it. 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, as illustrated by crystal violet where an electron that resonates between the three benzene rings is present. 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, it is considered that the most important staining substrates in bacterial cells are proteins, especially the cytoplasmic proteins. However, other substances would also have dye affinity, such as amino sugars, organic acids, nucleic acids, and certain polysaccharides.

Sudan III, or sudan black B, are popular stains for fatty material. They do not have an auxochrome group, and are insoluble in water, but soluble in fatty material. When solutions of sudan black B in ethylene glycol are 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 this morphology but it is also a differential stain and thus gives additional information about the cell. The Gram staining procedure as it is done today, involves: a) the staining of all cells with crystal violet, b) the precipitation of this dye on and within the cells by means of iodine, c) the removal 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, those which take 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 cells 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.

If the Gram procedure is now completed, these cells will retain the primary dye. For these organisms a valid Gram staining result would be impossible. 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.

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.