Staining procedures

Stain (ASTMD1328). This test measures the amount of stain on paper or other ceUulosic materials by asphalt. Variations of the cigarette paper stain procedure include the Barber stain, usually conducted at 54.4°C and 3.9 MN (400 g-force). Talc stain tests are also used. 

Under certain conditions the sample is clearly visible throughout the process. Other times it is necessary to stain the matrix to visualize the components. In cases where a final staining procedure is required, a small amount of dye is often added to the sample before the analysis. The dye typically migrates faster than any sample component. The position of the dye in the matrix indicates the speed of the resolution of the components of the sample. Typically, the electrophoretic medium is discarded after use. Good resolution can be obtained from 1 to 20 hours, using applied voltages of 10 to 2000 V and currents of 5 to 100 m A. 

Gram positive Bacterial cells which retain the crystal violet stain during a staining procedure. Most strains of bacilli are gram positive. 

A number of staining procedures have been described. Some stains, such as chlorazol black, require fresh specimens and are not widely used. A variety of stains for fecal smears preserved by Schaudinn or PVA fixative have been described, including various hematoxylin stains. The stain most widely used in the United States is the Wheatley trichrome stain, which is the only permanent stain described in this chapter. The trichrome staining procedure uses reagents with a relatively long shelf life and is easy to perform. Note that there are differences in staining times depending on whether the specimen is fixed in Schaudinn or PVA fixative, as penetration is slower in the latter. 

Table elsewhere outlines the steps in the trichrome staining procedure. 

Should the stain be unsatisfactory, the slide can be destained by placing it in xylene to remove the cover slip or immersion oil and then placing it in 50% alcohol for 10 min to hydrate the slide. Destain the slide in 10% acetic acid in water for several hours, and then wash it thoroughly first in water and then in 50 and 70% alcohols. Place the slide in stain for 8 min, and then complete the stain procedures. It is helpful to eliminate or shorten the destain step. 

Acid-fast staining for Cryptosporidium sp. has recently become important because this parasite is now recognized as a cause of severe diarrhea in immunodeficient patients such as those with AIDS, and it can cause transient diarrhea in immunocompetent individuals. The modified acid-fast stain recommended is similar to that used to stain Nocardia spp. in that it uses milder acid decolorization. A variety of acid-fast and fluorochrome staining procedures have been described for Cryptosporidium spp., and all the procedures appear to work. 

Thick and thin films are best stained with Giemsa stain, as it provides the most detailed and intense staining of parasites. Wright stain can be used for thin films but not for thick films, as it contains alcohol, which will fix the erythrocytes. Wright stain does not stain parasites as well as Giemsa stain. The staining procedure is outlined below. 

The cell wall has a low affinity for dyes, which means that it is probably not stained in some of the usual staining procedures. It is lightly stained by certain basic dyes such as basic fuchsin and the methyl violets. Where deep staining of the wall is desired, the use of a mordant, such as tannic acid, is necessary. The mordant not only increases the affinity of the cell for dye, but it may increase the thickness of the wall. 

Much of the confusion was caused by the inadequacy of the staining procedures. By means of the HCl-Giemsa staining technique of Piekarski, many observations have been reported demonstrating the presence of chromatinic structures in bacteria. 

The IHC stain procedure is a multistep staining protocol, the various steps intended to provide amplification of stain results. Therefore, a control system must include elements to control each step of the stain process. Such a control should also include a range of reactivities, and that range ideally would encompass the total expression range expected for the measured component. The control should also monitor each step of the multistep protocol. This author has devoted a number of years to this concept, resulting in a patented control for multistep staining processes.14 Such a control provides sufficient information to monitor every IHC stain run, and when the control is evaluated quantitatively, normalization of data from one stain run to another within the same laboratory, and even between laboratories. A process control is a measure of the stain protocol and does not take the place of a control for the primary antibody. While the primary antibody control should include range of expression level detection, a different primary control must be present for every primary antibody used in a stain run (Fig. 10.4). 

After heating process, wash slides with phosphate buffer solution followed by IHC staining procedure. 

The slides received in alcohol are stained in the Sakura Automated Stainer. Please see Non-Gynecologic staining procedure. The air-dried slides are stained manually in Diff-Quik stain. 

Once the staining procedure is completed, the slides are coverslipped with the Leica Automated coverslipper. 

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