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gram stain
History of the Gram Stain and How it Works

The Gram staining method, named after the Danish bacteriologist who originally devised it in 1882 (published 1884), Hans Christian Gram, is one of the most important staining techniques in microbiology. It is almost always the first test performed for the identification of bacteria. The primary stain of the Gram's method is crystal violet. Crystal violet is sometimes substituted with methylene blue, which is equally effective. The microorganisms that retain the crystal violet-iodine complex appear purple brown under microscopic examination. These microorganisms that are stained by the Gram's method are commonly classified as Gram-positive or Gram non-negative. Others that are not stained by crystal violet are referred to as Gram negative, and appear red.

Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. The cell walls for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram-negative bacteria. Bacteria cell walls are stained by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed easily. This step is commonly referred to as fixing the dye. However, subsequent treatment with a decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker Gram-positive cell walls, closing the pores as the cell wall shrinks during dehydration. As a result, the diffusion of the violet-iodine complex is blocked, and the bacteria remain stained. The length of the decolorization is critical in differentiating the gram-positive bacteria from the gram-negative bacteria. A prolonged exposure to the decolorizing agent will remove all the stain from both types of bacteria. Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable).

Finally, a counterstain of basic fuchsin is applied to the smear to give decolorized gram-negative bacteria a pink color. Some laboratories use safranin as a counterstain instead. Basic fuchsin stains many Gram-negative bacteria more intensely than does safranin, making them easier to see. Some bacteria which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic fuchsin, but not safranin. The polychromatic nature of the gram stain enables determination of the size and shape of both Gram-negative and Gram-positive bacteria. If desired, the slides can be permanently mounted and preserved for record keeping.

Besides Gram's stain, there are a wide range of other staining methods available. By using appropriate dyes, different parts of the bacteria structures such as capsules, flagella, granules, and spores can be stained. Staining techniques are widely used to visualize those components that are otherwise too difficult to see under a light microscope. In addition, special stains can be used to visualize other microorganisms not readily visualized by the Gram stain, such as mycobacteria, rickettsia, spirochetes, and others. In addition, there are modifications of the Gram stain that allow morphologic analysis of eukaryotic cells in clinical specimens.

Procedures 1. Prepare a Slide Smear: A. Transfer a drop of the suspended culture to be examined on a slide with an inoculation loop. If the culture is to be taken from a Petri dish or a slant culture tube, first add a drop or a few loopful of water on the slide and aseptically transfer a minute amount of a colony from the Petri dish. Note that only a very small amount of culture is needed; a visual detection of the culture on an inoculation loop already indicates that too much is taken.

If staining a clinical specimen, smear a very thin layer onto the slide, using a wooden stick. Do not use a cotton swab, if at all possible, as the cotton fibers may appear as artefacts. The smear should be thin enough to dry completely within a few seconds. Stain does not penetrate thickly applied specimens, making interpretation very difficult.

B. Spread the culture with an inoculation loop to an even thin film over a circle of 1.5 cm in diameter, approximately the size of a dime. Thus, a typical slide can simultaneously accommodate 3 to 4 small smears if more than one culture is to be examined.

C. Air-dry the culture and fix it or over a gentle flame, while moving the slide in a circular fashion to avoid localized overheating. The applied heat helps the cell adhesion on the glass slide to make possible the subsequent rinsing of the smear with water without a significant loss of the culture. Heat can also be applied to facilitate drying the the smear. However, ring patterns can form if heating is not uniform, e.g. taking the slide in and out of the flame.

2. Gram Staining: A. Add crystal violet stain over the fixed culture. Let stand for 10 to 60 seconds; for thinly prepared slides, it is usually acceptable to pour the stain on and off immediately. Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle. Note that the objective of this step is to wash off the stain, not the fixed culture.

B. Add the iodine solution on the smear, enough to cover the fixed culture. Let stand for 10 to 60 seconds. Pour off the iodine solution and rinse the slide with running water. Shake off the excess water from the surface.

C. Add a few drops of decolorizer so the solution trickles down the slide. Rinse it off with water after 5 seconds. The exact time to stop is when the solvent is no longer colored as it flows over the slide. Further delay will cause excess decolorization in the gram-positive cells, and the purpose of staining will be defeated.

D. Counterstain with basic fuchsin solution for 40 to 60 seconds. Wash off the solution with water. Blot with bibulous paper to remove the excess water. Alternatively, the slide may shaken to remove most of the water and air-dried.

3. Quality control: It is a simple matter to prepare a control slide by breadking a clean wooden applicator stick and picking a small amount of material from the interproximal space of one's teeth. This should be smeared into a drop of clean tap water on a clean glass slide. The slide may be stained as above. This material will consistently display a few neutrophils and a mixture of Gram (+) and (-) organisms. Neutrophil nuclei should be pink.

3. Examine the finished slide under a microscope. A caveat in the examination of the Gram smears is the distortion in morphology that can be caused by antimicrobial therapy. This is especially likely to occur in urine speciments. Filamentous and pleomorphic forms may be observed among the Gram (-) rod species. Gram reaction of the organism may also change after antimicrobial therapy, Gram (+) bacterial may become gram variable. Look at areas that are one cell thick only; observation of thick areas will give variable and often incorrect results. White blood cells and macrophages should stain Gram-negative, whereas sqamous epithelial cells are Gram-positive.

Gram-positive bacteria Gram-positive cocci Clusters: usually characteristic of Staphylococcus spp., such as S. aureus

Chain: usually characteristic of Streptococcus spp., such as S. pneumoniae, B group streptococci

Tetrad: usually characteristic of Micrococcus spp.

Gram positive bacilli

Thick : usually characteristic of Clostridium spp., such as C. perfringens, C. septicum,

C. tetanomorphum

Thin: usually characteristic of Listeria spp.

Branched: usually characteristic of Actinomycetes and Nocardia, such as A. israelii Note: Mycobacteria are not branched, and often stain poorly with Gram stain. Some mycobacteria do appear as Gram-positive rods, not unlike diptheroids, sometimes with Gram-positive beading.

Gram negative bacteria