Medical science depends on the staining of cells in tissues to make accurate diagnoses of a wide range of diseases from cholera to sexually transmitted diseases , to parasitic diseases and skin infections. Staining techniques performed routinely in microbiological laboratories include gram's stain, acid-fast stains, acridine orange, calcofluor white, toluidine blue, methylene blue, silver stains, and fluorescent stains. Stains are classified broadly as basic, acidic, or neutral stains. The chemical nature of the cells under examination determines which stain is selected for use.
Cell staining is important in the diagnosis of microorganisms because bacteria can be identified by the color differentiation of stains (dyes). Microscopic examination of stained cell samples allows examination of the size, shape, and arrangement of organelles, as well as external appendages such as the whip-like flagella , which are the cell's organs of motion . When sample cells are stained to show their chemical composition it is called differential staining.
Histochemistry is the specialty that studies the staining properties of cells. Histochemistry is used in other specialties such as histology (the study of tissues), biochemistry (the study of the chemical makeup of cells), cytology (the study of cells), and microbiology (the study of organisms that are too small to be seen without a microscope ).
In 1880, Hans Christian Gram of Denmark noted the differences in the way bacteria react to stains. Those bacteria that retained a deep purple stain, even after they were washed, were termed "stain positive." Those that lost the stain and responded again to another stain, were termed "stain negative." Today, bacteria are classified as "gram-positive" or "gram-negative" to distinguish the two major groups of bacteria. This staining test highlights differences in the structure of the cell wall of the two types of bacteria.
Penicillin G is used to treat gram-positive infections, but it is ineffective against gram-negative bacteria. Other antibiotics are only effective against gram-negative bacteria. Chloromycetin, which was discovered in 1947, was the first antibiotic to be effective against both gram-positive and gram-negative bacteria.
Bacteria are nearly colorless, so their features are difficult to distinguish when they are suspended in a fluid and viewed directly under a microscope. Stains are salts that color particular ions in the bacterial cell, and make more visible distinctions under the microscope. The chemical composition of the cell determines which stain is absorbed. Acidic parts of a cell absorb stains that are positively charged; alkaline parts of a cell combine with stains that are acidic or negatively charged.
Before tissues are stained, a thin layer of cells that have been sliced from the specimen (a smear) is prepared by fixing. Fixing a specimen that has been placed on a slide is done by either allowing it to dry at room temperature , or by passing the specimen quickly over a flame. Next the specimen is stained: either a simple stain, a differential stain, a negative or indirect stain, a stain for reserve materials, or for microbial structures is used. Most staining dyes are prepared from coal tar and those used in microbiology come from aniline, an oily liquid.
In simple (or direct) staining only one dye is used, which is washed away after 30–60 seconds, before drying and examination. Gentian violet, crystal violet, safranin, methylene blue, basic fuchsin, and others are the dyes used in this method. In differential staining, the gram stain and the acid-fast stain are used to distinguish different microorganisms.
There are four steps involved in the gram stain method, which is considered the most valuable cell-staining technique used in bacteriological cell analysis. In the first step, the specimen is stained with crystal violet or gentian, and one minute later, the second step is taken which involves washing the dye off and flooding the solution with iodine. The third step involves washing the iodine off 60 seconds after it is applied and then washing the slide with an ethyl alcohol solution of 95% or a 50:50 mixture of acetone and ethyl alcohol 15–30 seconds after this. The fourth and final step is to stain the slide for 30 seconds with a red or brown dye. The critical action in this process is the washing away of the stain, called decolorization stain, (sometimes called the Ziehl-Neelsen technique) is particularly useful in identifying the organism that causes tuberculosis . When these microorganisms are stained with a red dye (carbol fuchsin), the color remains even though the slide is washed with a strong solution of acid alcohol. Most organisms, other than the ones responding to acid-fast staining, would decolorize from this wash. Methylene blue is then used to differentiate any other organisms present in the smear.
Negative (or indirect) techniques stain the background of cell smears, rather than the organisms directly. In this technique, a drop of the stain is placed on a slide and organisms are added to the stain. After the specimen is smeared over the slide, it is allowed to air dry and is then examined under the microscope. Negative or indirect staining procedures are useful when examining the size and shape of microorganisms.
Staining for reserve materials in cells isolates specific structures in the cells of microorganisms (such as granules or other reserve substances in bacteria that cause diseases such as diphtheria ). In staining of microbial structures, the flagella, nuclear material of the cell, the cell wall, or capsule is stained for viewing under the microscope. These procedures use two or more stains.
Standardization of tests
Cell staining is one of a number of laboratory tests that are performed to aid in the analysis and diagnosis of disease . The work in these laboratories is performed for physicians as well as for government agencies involved in water purification and sewage treatment , and for industries such as the food industry involved in the manufacture of goods that need to adhere to strict health standards.
Standardization of these tests have been widely adopted throughout the microbiological laboratory community. The National Committee for Clinical Laboratory Standards (NCCLS), located in Villanova, Pennsylvania, continuously publishes standards for these laboratory tests. Among the factors that have been standardized in laboratory testing are temperature, pH (acidity or alkalinity), growth medium, antibiotics, quality control, and other factors.
Keynes, Milton. Handling Laboratory Microorganisms. Philadelphia: Open University Press, 1991.
Koneman, Elmer W. Color Atlas and Textbook of DiagnosticMicrobiology. 4th ed. Philadelphia: J. B. Lippincott, 1992.
Postgate, John R. The Outer Reaches of Life. Cambridge, England: Cambridge University Press, 1994.
Prescott, L., J. Harley, and D. Klein. Microbiology. 5th ed. New York: McGraw-Hill, 2002.
KEY TERMS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
- Acidic stains
—Stains that adhere to microorganisms having a high lipid (fatty) content.
—Washing away of the staining medium.
- Differential staining
—Staining technique that uses more than one stain to differentiate the structure of the microorganism.
—Preparing a cell specimen on a slide for examination under a microscope.
—Those cells that lose the color of the stain after they are washed with a 95% alcohol solution during the staining process.
—Those cells that retain the color of the stain after they are washed with a 95% alcohol solution during the staining process.