Cytochemical Methods

The oldest and most extensively used method for determination of PolyPs in biological materials, although of course the least accurate, is based on the staining of cells and tissues by certain basic dyes such as toluidine blue, neutral red and methylene blue. The presence of condensed phosphates in the organisms is judged by the appearance in the cells of metachromatically stained granules, or volutin granules. 

It should be noted, however, that, despite the fact that in most cases the cytochemical detection of metachromatic granules is associated with the actual presence of PolyPs in the organism, such methods must nevertheless be carried out with great caution. This is primarily due to the fact that basic dyes are also capable of staining other polymeric compounds present in the cells. 

The more sensitive and convenient method of PolyP detection in situ is fluorescence microscopy using fluorochromes of the type 4/,6/-diamino-2-phenylindole.2HCl (DAPI), which is commonly used for DNA detection. At a high concentration (50 mg ml 1), it also stains PolyP granules and lipid inclusions (Allan and Miller, 1980 Tijssen et al, 1982 Streichan et al, 1990). DAPI-DNA fluorescence is blue-white, while DAPI-PolyP and DAPI-lipid fluorescence is yellow. The lipid fluorescence is weak and fades in a few seconds, while the PolyP granules appear bright yellow, thus allowing discrimination of the above types of cell inclusions (Streichan et al, 1990). 

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Hayat MA. Stains and Cytochemical Methods, Plenum Press, New York, 1993. 

Knight DR, Lewis PR. General cytochemical methods, in Cytochemical Staining Methods for Electron Microscopy. Practical Methods in Electron Microscopy (Glauert A, ed.), Elsevier, Amsterdam, 1992, pp. 79-145. 

Matsuzawa, T., Anderson, H. C. Phosphatases of epiphyseal cartilage studied by electron microscopic cytochemical methods. J. Histochem. Cytochem. 19, 801 (1971)... 

Other blood research techniques include the use of physical and chemicals agents, ultracentrifugadon. cytochemical methods, microincineration, and autoradiography,... 

Biochemically, apoptosis is characterized by the internucleosomal degradation of chromosomal DNA to form a series of double-stranded fragments that are multiples of 180 200 base pairs in length. These fragments give a characteristic DNA ladder pattern on gel electrophoresis [91, 92] and can be detected by several cytochemical methods, the most extensively used being the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) [93-95], The detection of ladder pattern and TUNEL positivity has been adopted as a marker of apoptosis. 

Interesting data on the structure and formation of PolyP granules in cyanobacteria were obtained by electron microscopic and cytochemical methods (Jensen, 1968, 1969 Jensen and Sicko, 1974 Sicko-Goad et al., 1975 Jensen et al., 1982). 

Seed, T. M., Aikawa, M., and Sterling, C. R. (1973). An electron microscope-cytochemical method for differentiating membranes of host red cells and malaria parasites. J. Protozool. 20,603-605. 

Takahashi, Y., and Sherman, I. W. (1978). Plasmodium lophurae Cationized ferritin staining, an electron microscope cytochemical method for differentiating malarial parasite and host cell membranes. Exp. Parasitol. 44,145-154. 

Comparison between the chromate inhibition test and a cytochemical method for the determination of glucose-6-phosphate dehydrogenase deficiency in erythroc5d es. 

Detection of glucose-6-phosphate dehydrogenase deficiency in erythrocytes a spectrophotometiic assay and a fluorescent spot test compared with a cytochemical method. 

Smetana, K. (1967). Basic histochemical and cytochemical methods. Methods Cancer Res. 2,361-443. 

Their cytochemical method was positive in 43% of the samples, but not in the same order of concentrations. 

Whether the occurrence of an enzyme in a particulate fraction really represents its in vivo location can be substantiated by the following properties (a) close binding to membrane lipids, cf. cyclopenase (D 8.4.2), which is a lipoprotein of the plasma membrane (b) cooperation with other enzymes, cf. the catalytic facilitation in the biosynthesis of cyanogenic glycosides, cinnamic and benzoic acids (A 3.1) and (c) in situ examination by cytochemical methods, cf. the localization of phenol oxidases (C 2.3.1), peroxidases (C 2.4) and thioglucosidases (D 9.4). 

The electron-cytochemical method for localization of DPP was optimized by Lojda (1981) and its modification for cytochemical reaction for DPP in glutaraldehyde prefixed yeast cells was described recently (Voffsek in preparation). In principle, the synthetic dipeptide substrate has its carboxyl terminus bound to 4-methoxy-2-naphthylamine. The primary reaction product was formed by coupling hexazonium pararosaniline (HPR) with the MNA liberated by DPP and was osmiophilic. Thus postfixation by osmium tetrox-ide revealed also the electron-dense final reaction product on the background of the general osmiophilia of the matrix phase and membranes. 

Morphological appearance Morphological examination including cytochemical methods... 

At the same time, this book is not merely a simple compilation, devoid of the author s personality. Professor Korochkin s works are well known. In his research he fruitfully combines good genetic analysis with modern biochemical and cytochemical methods of molecular biology. Professor Korochkin s works, with the aid of ultra microchemical methods, which allowed to formulate the original concept of multilevel gene regulation of individual development, are of great interest. 

The possibility that HbF and HbA can coexist in individual erythrocytes of adult and neonatal human blood has also been demonstrated by spectrophotometric and cytochemical methods (Matioli et al., 1962 Zipursky et al., 1962 Shepard et al., 1962). The switch from HbF and HbA thus consists evidently of a change in the direction of work of what is essentially the same single erythropoietic system, and not a complete replacement of one erythropoietic system by another new one, which might be expected from data showing changes in the localization of erythropoiesis in dif-ferentphases of embryonic development (Maximov and Bloom, 1957). 

Willingham MC (1999) Cytochemical methods for the detection of apoptosis. J Histochem Cytochem 47 1101-1109... 

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