The History of Staining Logwood Dyes

Except for the cochineal derivatives, logwood extract was the first of the important modern stains to be employed in histology. Certain other natural dyes, such as madder and indigo, had been used earlier, but they are of little significance in discussing the history of staining, because none of them nor even alizarin, the derivative of madder, are of any appreciable significance in these days of synthetic dyes. Hematoxylin, on the other hand, still continues a very important stain, and it has played an interesting part in the history of staining.     

The History of Staining Cochineal Dyes

Of the dyes used in early histological work, none was so highly prized as carmin. Even today biologists would be loath to part with it. It is still valuable to the histologist and embryologist as a bulk stain of great permanency, and for the preparation of in toto mounts. To the cytologist, also, it is invaluable as a medium for the rapid staining and examination of chromosomes in fresh material. Its permanence has always been a great advantage. To the early histologists, however, it was the dye, par excellence.     

The History of Staining Cochineal Dyes

Of the dyes used in early histological work, none was so highly prized as carmin. Even today biologists would be loath to part with it. It is still valuable to the histologist and embryologist as a bulk stain of great permanency, and for the preparation of in toto mounts. To the cytologist, also, it is invaluable as a medium for the rapid staining and examination of chromosomes in fresh material. Its permanence has always been a great advantage. To the early histologists, however, it was the dye, par excellence.     

The History of Staining the Development of Cytological Staining

Perhaps no other period has contributed more to our knowledge of the cell than the period 1875-1895. During these years most fundamental cytological phenomena were seen and described. Mitosis, maturation and fertilization, the great cornerstones of cytology, were firmly laid by the remarkable researches of Flemming, Strasburger, Van Beneden, Oscar and Richard Hertwig, Boveri and many others. Upon these researches experimental cytology developed and the significance of the morphological phenomena to inheritance and development was pointed out by such masters as W. Roux, Weismann, O. Hertwig, Boveri and E. B. Wilson.     

The History of Staining the Development of Bacteriological Staining Methods

The staining of bacteria is naturally of later origin than the use of dyes in histological work, as the systematic study of bacteria did not begin until after 1870. The use of dyes in this study followed very promptly after that date, however.     

Vital Staining with Two Dis-Azo Textile Dyes

Subcutaneous injections of 0.25% saline solutions of two dis-azo textile dyes, calcodur pink 2BL, C. I. 353, also known as benzo fast pink 2BL and amidine fast rose 2BL, and a blue dye, dianil blue G, C. I. 508, were made on alternate days on albino rats for one week. The blue dye is closely similar to Niagara blue 4B, C. I. 520, and dianil blue R, C. I. 465. Staining reactions were much like those of other vital blue disazo dyes. Although the pink dye exhibited a similar staining pattern, there were notable differences. The tissues of most glands were stained pink or red. Nuclei of the tubular epithelial cells of the kidney contained red granules as did the cytoplasm of the Kupfer cells. Most unusual was the bright red staining of the elastica interna of medium and large sized arteries.     

Thiazin Dyes in Supravital Staining of nerve Fibers

Supravital staining by thiazins of segments of small intestine and mesentery of young dogs was studied with reference to specificity for nervous tissue. Attempts to secure a purer form of methylene blue by alumina adsorption and alcohol elution of the commercial, medicinal dye yielded a product which appeared to be structurally different from the original dye. The treated dye had absorption maxima from 620 to 655 mμ in contrast with 665 for the untreated. Small nerve bundles were stained by the treated dye after 2 to 4 hours of immersion, but staining was always incomplete. Staining by untreated methylene blue was compared with that by the leucobase, thionol, methylene green, toluidine blue, new methylene blue and the azures. It was concluded that the specificity for nerve fibers resides mainly in the =N(CH₃)₂Cl radical, although some specificity appears to be effected by the methyl groups on the trivalent nitrogen, since azure A (dimethyl) and azure C (mono-methyl) stained weakly, but thionin did not. Methylene green showed some specificity but stained weakly. The leucobase was less active than the reoxidized dye obtained from it.     

History of Staining the Staining of Blood and Parasitic Protozoa

By the term “blood stain” one ordinarily means a compound dye formed from the chemical union of an acid and a basic dye, and usually a compound of the eosin-methylene-blue group. It is well known today that the sodium salt of a color acid (e. g. eosin) and the chloride of a dye base (e. g. methylene blue) may be converted by simple metathesis into sodium chloride plus the compound dye (e. g. methylene blue eosinate), the latter being insoluble in water unless an excess is present of either the acid or the basic dye. In modern blood stains a compound dye of this type is dissolved in methyl alcohol and mixed with water on the slide at the moment of staining.     

Chemical Structure as Affecting the Staining and Therapeutic Properties of Dyes

Toxicity resulting from the oxidation of methyl groups in certain therapeutic dyes seems to occur as the result of formaldehyde produced. It is suggested that ethylation be employed in lieu of the methylation to counteract this toxicity. In the triphenylmethane dyes it is found that apparent differences in staining and bactericidal value may possibly be accounted for by differences in structure, a rosaniline base in general being superior to the pararosaniline compounds.     

Chemical Structure as Affecting the Staining and Therapeutic Properties of Dyes

Toxicity resulting from the oxidation of methyl groups in certain therapeutic dyes seems to occur as the result of formaldehyde produced. It is suggested that ethylation be employed in lieu of the methylation to counteract this toxicity. In the triphenylmethane dyes it is found that apparent differences in staining and bactericidal value may possibly be accounted for by differences in structure, a rosaniline base in general being superior to the pararosaniline compounds.     

The Effect of Nucleases on Cytochemical Reactions for Amino Acids and on Staining with Acid Dyes

Crystallia ribonuclease has no marked proteolytic activity, since digestion of sections with this enzyme produces no appreciable decrease in the intensity of the cytochemical tests for arginine and tyrosine. Cytoplasmic basophilia is unaffected by treatment with cold trichloracetic acid or with boiling alcohol-ether mixture. Mononucleotides and fatty acids thus have nothing to do with basophilia. Digestion of sections with desoryribonuclease has no effect on the alkaline phosphatase or arginine tests, while it supresses the Feulgen reaction and the affinity of the chromatin for basic and for some acid dyes.     

A Histochemical Examination of the Staining of Kainate-Induced Neuronal Degeneration by Anionic Dyes

Anionic dyes, notably acid fuchsine, strongly stain the nuclei and cytoplasm of neurons severely damaged by injury or disease. We provide detailed instructions for staining nervous tissue with toluidine blue and acid fuchsine for optimal demonstration of injured neurons. Degeneration was induced in the hippocampus of the mouse by systemic administration of kainic acid, and the resulting acidophilia was investigated using paraffin sections of the Carnoy-or Bouin-fixed brains. The affected cells were bright red with the toluidine blue-acid fuchsine sequence. Their nuclei were stainable also with alkaline Biebrich scarlet and with the 1,2-naphthoquinone-4-sulfonic acid-Ba(OH)₂ method; all staining was blocked by benzil but was relatively refractory to deamination by HNO₂. These properties indicated an arginine-rich protein. The nuclei were strongly acidophilic in the presence of a high concentration of DNA (strong Feulgen reaction), and acidophilia could not be induced in normal neuronal nuclei by chemical extraction of nucleic acids. The cytoplasmic acidophilia of degenerating hippocampal neurons was due to a protein rich in lysine (extinguished by alkalinity, easily prevented by deamination, and unaffected by benzil). Stainable RNA was absent from the perikarya of the affected cells, but normal neuronal cytoplasm did not become acidophilic after extraction of nucleic acids. We suggest that kainate-induced cell death is preceded by increased production of basic proteins, which become concentrated in the nucleus and perikaryon. Groups of small, darkly staining neurons were seen in the cerebral cortex in control and kainite-treated mice. These shrunken cells were purple with the toluidine blue-acid fuchsine stain, and were attributed to local injury incurred during removal of the unfixed brain.     

Tissue Staining by Disazo Dyes, Teratogenicity and the Haemoglobin-Nitrite Sensitivity Reaction

Five disazo dyes related to trypan blue but differing in molecular structure by substitution, replacement or addition of radicals have been tested for their action in increasing the sensitivity of haemoglobin to oxidation by nitrites. None has been found to be as active as trypan blue itself. The activity of these dyes is not related to their ability to cause generalised tissue staining in vivo. Four dyes of known redox potential, neutral red, phenol-indophenol, phenol blue and janus green, have also been tested for their action on the haemoglobin-nitrite sensitivity reaction. Their activity is not related to their redox potential.     

Differential Dichrome Staining of Tissue Culture Monolayers: Alternate Dyes and Possible Mechanism

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Qualitative Analysis by Thin-Layer Chromatography of Some Common Dyes Used in Biological Staining

Thin-layer chromatography will resolve impurities in commercial dyes, and will do so much faster than paper chromatography. Solvent systems consisting of (a) n-propanol: n-butanol: NH₄OH (conc.): H₂O—4:4:1:1; (b) n-propanol: NH₄OH (conc.): H₂O—8:1:1 on silica gel G plates; and (c) n-propanol: NH₄OH (conc.): H₂O-7:2:1 on Adsorbosil plates were found to be the most effective. Dyes studied were azure A, azure B, azure C, methylene blue, toluidine blue O, thionin, pyronin B, pyronin Y, methyl green, crystal violet amido black 10B and buffalo black (NBR).