Cell Staining

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.