Stains and fixatives

A number of stains and fixatives have been referred to throughout the book and these can be located by using the index. Here are listed some of the more common ones. 

To a a suspension of cells in BSS is added a drop of an aqueous 0.01% solution of acridine orange. On examination under the fluorescent microscope nuclei appear yellowish green and cytoplasm red. 

1 part glacial acetic acid 3 parts absolute ethanol 

The haematoxylin is dissolved in 95% ethanol and to 6 ml of a 17% solution is added 100 ml saturated ammonium alum solution and 0.5 g mercuric oxide. After boiling and cooling 25 ml of glycerol and 25 ml of methanol are added and the whole mixture filtered. 

The stain is 30 ml saturated ethanolic solution of methylene blue mixed with 100 ml 0.01% aqueous KOH. 

---

It is particularly hazardous to the eyes because of its ready reaction with organic matter, a property exploited in electron microscopy in order to stain and fix biological tissues. Although osmium is a d8 transition metal, its tetroxide is a coordinatively unsaturated 16-electron species and has a tetrahedral structure3. It is highly soluble in carbon tetrachloride (375 g/ 100 g), less so in benzene, and moderately soluble in water (7.24 g/100 g). 

The three principal microscopic examinations performed on stool specimens are direct wet mount, wet mount after concentration, and permanent stain. Although each examination can contribute to diagnosis, the yield of some methods is small with certain kinds of specimens. As a minimum, formed specimens should be examined by a concentration procedure. Soft specimens should be examined by concentration and permanent stain, and, if submitted fresh, by direct wet mount. Loose and watery specimens should be examined by wet mount and permanent stain. If specimens are received in fixative and the consistency is not known, concentration and permanent stain should be performed. Other examinations may be helpful. Special procedures which may assist in the diagnosis of specific parasites are noted below in discussions of the parasites. 

Measurements show some variation depending upon the staining solution used and the method of application. In dried and fixed smears, the cell wall and slime layer do not stain with weakly staining dyes such as methylene blue but do stain with the intensely staining pararosaniline, new fuchsin, crystal violet, and methyl violet. The great majority of bacteria have been measured in fixed and stained preparations. In some instances dried, negatively stained smears have been used. Therefore, the method employed should be specified when measurements of bacteria are reported otherwise the results will be of doubtful v alue. 

Liu H, Huang X, Zhang Y, et al. Archival fixed histologic and cytologic specimens including stained and unstained materials are amenable to RT-PCR. Diagn. Mol. Pathol. 2002 11 222-227. 

As for paraffin sections, it is advisable to mount cryosections also onto adhesive-coated slides in order to decrease the chances of sections dissociating from the slides in the course of immunohistochemical staining. Once mounted on slides, cryosections are air-dried and fixed, usually in acetone or methanol. Aldehyde... 

To obtain tissue preparations whose constituents were maintained as closely as possible to their state in vivo, the material had to be fixed, i.e. the enzymes inactivated so that cell structures were instantaneously preserved, an almost unattainable ideal. Formalin was the favored fixative, but others (e.g. picric acid), were also employed. Different methods of fixation caused sections to have different appearances. Further artifacts were introduced because of the need to dehydrate the preparations so that they could be stained by dyes, many of which were lipid-soluble organic molecules. Paraffin wax was used to impregnate the fixed, dehydrated material. The block of tissue was then sectioned, originally by hand with a cut-throat razor, and later by a mechanical microtome. The sections were stained and mounted in balsam for examination. Hematoxylin (basophilic) and eosin (acidophilic) (H and E staining) were the commonest stains, giving blue nuclei and pink cytoplasm. Eosinophils in the blood were recognized in this way. 

Samples may be fixed at step 2 of Subheading 3.2.1. by the addition of 100 pL of 37% formalin, and stored at 4°C until it is convenient to continue with the staining and analysis. After refrigerated storage, the samples are per-meabilized by the addition of 100 pL of 1 mg/mL lysophosphatidyl choline in PBS, and processed identically as described in steps 3-6 of Subheading 3.2.1. 

Mammalian cells in culture are exposed to the test substance. Established cell lines are treated both with and without metabolic activation. Cells are incubated for an appropriate length of time, then rinsed, fixed, and dried. Slides are developed, stained, and exposed silver grains are counted. The endpoint of UDS is measured by determining the uptake of labeled nucleosides in cells that are not undergoing scheduled (S-phase) DNA synthesis. The most widely used technique is the determination of the uptake of H-TdR by autoradiography. Primary cultures (e.g., rat hepatocytes), human lymphocytes, or established cell lines (e.g., human diploid fibroblasts) may be used in the assay. Multiple concentrations of the test substance over a range adequate to define the response, should be used. 

Approximately one half of the fetuses in each litter (i.e., every alternate fetus in the uterus) are eviscerated and fixed for skeletal examination. The skeletal examination is performed following maceration of the soft tissue with aqueous potassium hydroxide and staining of the skeleton. Single staining, of the ossified bones only, is performed for pharmaceuticals (see Chapter 16) and double staining, of the bone and cartilage is performed for chemicals (see Chapter 17). 

Fig. 21 Structures of hydrophilic 2PA fluorophores 49 used for cell staining, and confocal microscopy images of live NT2 cells incubated with a two-photon absorbing hydrophilic probe 49 and two-photon induced fluorescence image of fixed NT2 cells (60 x oil) stained with the hydrophilic probe 49...

A yellow to brown or black stain is produced,4 which is compared with a set of standard strips prepared under similar conditions. The chief difficulty encountered is to obtain a reliable and permanent set of standards especially is this the case with silver nitrate, the stains of which do not keep. The most satisfactory method5 of preparing such stains is to soak the filter paper in gum tragaeanth, dry it, soak it in silver nitrate solution, again dry it, expose to arsine under the requisite conditions and fix the stain by repeated soaking in very dilute ammonia and coating with paraffin. By the use of 66 per cent, silver nitrate solution, OT x 10-6 g, of As may be detected.6... 

Prior to the staining procedures, fix cells in 70% ethanol. Best results are obtained by resuspending the cell pellet in 1 mL of PBS and adding 9 mL of ice-cold 70% ethanol while vortexing to prevent agglutination. Solid pieces of tissue can be fixed as 5 mm3 pieces directly in ice-cold 70% ethanol. Cells and tissues are usually left at least 1 h prior to staining. Fixed cells or tissues should be stored at 4°C and, m the author s experience, will remain suitable for BrdU staining studies for at least 3 yr. 

---