Polychrome Staining of Epoxy Semithin Sections Using Cacodylic Buffer as a Stain Solvent
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Abstract:
Many polychromatic stains are in use for epoxy-embedded tissues (Horobin 1983). We should like to report a quick and easy polychromatic staining procedure that we find useful for routine use. Formerly the stain we used was prepared in 20 ml water and 5 ml 96% alcohol, and gave polychromatic staining only at pH 7.4 obtained by the addition of 1 N NaOH. However, the stain gave satisfactory results only for two or three days. We found that stabilization of the staining solution through the use of an ethyl alcohol-cacodylic buffer solvent increased the life of the staining solution. This was convenient because the cacodylic buffer is used in our laboratory as a component of fixatives, and is not prepared specially for the staining.Keywords:
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Buffer (optical fiber)
The gallocyanin chromalum stain belongs to the classical DNA-RNA staining techniques in histochemistry. It has some important features for successful object orientated image analysis of whole sections of the human brain. To obtain reproducible staining results with these large sections, the method of Einarson was adapted to image analytical requirements. We discuss staining in a warm staining solution, pH adjustment, and optimal stain composition. The embedding procedure for whole human brains is considered as well.
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High contrast
Negative stain
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Objective: To compare the effect and their sensitivity of two different staining methods to DNA in the gels, and evaluate the possible application of SYBR GreenⅠin routine DNA staining as a substitute for ethidium bromide(EB). Methods: The new nucleic acid staining SYBR GreenⅠand EB were used to stain the DNA in the gel, perfectively stain before electrophoresis and after electrophoresis. Results: There was no distinct difference between the two methods. Both SYBR GreenⅠand EB stain could detect as little as 10 ng per band of DNA. Conclusion: The effect and sensitivity of the two staining methods are no distinct difference, and SYBR GreenⅠand EB may be used in these staining methods. SYBR GreenⅠ, which is a new nucleic acid stain may substitute for EB as a general dyestuff to stain the DNA in the gel.
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Ethidium bromide
SYBR Green I
Nucleic acid quantitation
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To the Editor:
—InThe Journal, April 14, p. 1092, Dr. Peter Mjedloff requests some further information concerning a bacillary stain in general use at the Kula Sanitarium. The stain to which he refers apparently is the stain designated as Much's granular stain. The stain which is in use at the sanatorium is identical with Much's stain, differing, however, in the fact that the Lugol's solution is used first on the prepared smear and this is then followed by the gentian-violet stain. After destaining with acid in the usual way, counterstaining in Much's technic is by means of a 1 per cent. methylene blue solution, although any other counterstain or dye, such as pyronin, may be used. In the usual staining technic this method of staining the tubercle bacilli is not considered superior to the carbolfuchsin or Ziehl-Neelsen method for detecting the bacilli if in their integrity. It is onlyStain
Tubercle
Ziehl–Neelsen stain
Methylene blue
Differential staining
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ABSTRACT The usefulness of the process of staining tissues with several dyes has been abundantly proved. The general effect aimed at is the staining of each separate part in a different colour, so that for the purposes of histological demonstration each shall be distinct and clear. In the use of certain of the most commonly employed and easily manipulated dyes, e. g. hematoxylin and picrocarmine, it is believed that a definite effect may be always calculated upon when they are used in combination. With anilin stains, however, the results arrived at appear to differ very materially if the methods of employment are made to vary in even a very slight degree, and this has been one of the causes of the restricted use of very beautiful staining colours. It has been shown by several experimenters that with combinations of anilin colours, there is a tendency at any rate for certain dyes to pick out and stain different parts of a tissue; but I think I am right in believing that no certain result has hitherto been expected, except in the case of a very few combinations.
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ALIZARIN RED
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AbstractThe importance of pH in staining tissue is emphasized. The effect of pH upon the selectivity and intensity of staining with iron hematoxylin, malachite green, and eosin Y is considered. Many difficulties may be avoided by staining in the higher alcohols and directions are given for the preparation of buffer solutions from pH 1.2-8 in alcohol. The concentration of stains, time of staining, and order of staining are discussed for progressive and regressive staining. At pH 8 in 95% alcohol very few tissues stain with malachite green at a concentration of 1/1000 saturated. At pH 6 most cytoplasmic elements stain with malachite green at a concentration of 1/1000 saturated or with eosin Y at 1/250 saturated. As the pH is lowered more tissue elements stain until the nucleus is completely stained. This behavior is in accord with the theory of chemical combination of dyes with proteins, which states that proteins combine with basic dyes on the basic side of their isoelectric points and with acid dyes on the acid side of their isoelectric points. With hematoxylin stain the pH range is much shorter. A satisfactory hematoxylin stain is composed of 0.1% hematoxylin, 0.1% FeCl3, and HCl to bring the pH to 1.2-1.6 in 80% alcohol. With this stain, which may be used immediately, the nuclei of most tissues begin to stain at pH 1.2 and much of the cytoplasm will be stained if the pH is raised to 1.4. The shortness of this effective pH range is thought to be due to the dissociation of the hematoxylin-iron-protein complex. The use of different dyes successively at different pH values, such as hematoxylin at 1.3, malachite green at 8, and eosin at 6, permits better differentiation of the tissue elements, and intelligent variations in the staining technic.
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Malachite green
Eosin
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Bacterial spore
Negative stain
Differential staining
Gram staining
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Many polychromatic stains are in use for epoxy-embedded tissues (Horobin 1983). We should like to report a quick and easy polychromatic staining procedure that we find useful for routine use. Formerly the stain we used was prepared in 20 ml water and 5 ml 96% alcohol, and gave polychromatic staining only at pH 7.4 obtained by the addition of 1 N NaOH. However, the stain gave satisfactory results only for two or three days. We found that stabilization of the staining solution through the use of an ethyl alcohol-cacodylic buffer solvent increased the life of the staining solution. This was convenient because the cacodylic buffer is used in our laboratory as a component of fixatives, and is not prepared specially for the staining.
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Buffer (optical fiber)
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Abstract Gram stain is a subjective and poorly controlled test, and the resultant errors often perplex laboratory scientists. To reduce errors and make Gram stain a precisely controllable and meritorious test, a standardized Gram stain procedure for bacteria and inflammatory cells was developed using an automated staining instrument in this study. Freshly expectorated sputum specimens, used as the optimized targets, were smeared on slides by laboratory technicians, defining each slide loaded with uniform matrix and monolayer cell. And then, the staining and decolorizing time, as well as the stain and decolorant volume, were optimized as 15, 105, 1, and 25 s and 1.1, 1.4, 0.3, and 0.7 ml, respectively. Culture‐positive blood specimens and original purulent fluids were used for confirming the developed standardized Gram stain procedure. Distinct tinctures of bacteria and inflammatory cells adhered to slide uniformly in a monolayer were observed, and the obtained staining results of these samples were highly consistent with their cultured results. Furthermore, according to the staining results under different staining conditions, an updated molecular mechanism of Gram stain for bacteria and the probable staining mechanism for inflammatory cells were also proposed in this study.
Gram staining
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Gram
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Negative staining is one of the many staining techniques that can be employed for viewing of bacterial cell morphology and size. The advantages of the negative stain include the use of only one stain and the absence of heat fixation of the sample. Negative staining employs the use of an acidic stain and, due to repulsion between the negative charges of the stain and the bacterial surface, the dye will not penetrate the cell. In negative staining, the results yield a clear cell with a dark background.
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Negative stain
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A simple and well reproducable modification of the Papanicolaou stain is reported. The most important modification is the replacement of the natural stain hematoxyline by the qualitively excellent synthetic stain thionin which is immediately added to the alcoholic fixation solution for a fixation stain. The high labor intense alternation between alcohol water and alcohol is thus eliminated. The quality of the nuclear staining is very good. The fixation staining with thionin without counter-staining of the cytoplasma may be used for a rapid thionin stain which gives sufficient cytoplasmatic staining by the thionin for a rapid cytological smear for cancer. The counter-stain of the cytoplasma was also simplified. The over-all staining of this simplification of the pap smear is comparable to that of the original papanicolaou stain. This modification of the papanicolaou stain fulfills requirements which a stain in competition with the original papanicolaou stain should have. 1. The modification is qualitively equal to the original method, 2. the staining method is simpler, 3. the staining method is well reproducable, 4. the staining method is more economical.
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