The Weigert-Pal Technic for Staining Myelin

The writer gives a schedule for carrying out the Weigert-Pal technic by which three difficulties of the original technic are overcome: the tendency of the sections to become brittle; the difficulty of observing the extent of the reaction occurring in the permanganate solution; and the slowness with which the reaction takes place. By the method proposed as many as 300 sections may be stained and differentiated in the time it formerly took to handle 50.     

A Dioxan Technic for Triple Staining

The first part of the paper summarizes the ways in which technicians have used dioxan in the various phases of microtechnic. The rest of the paper consists of a detailed account of the use of dioxan in triple staining. Specific directions are given for modifications of Castroviejo's method and Mallory's triple connective tissue stain. Special emphasis is placed upon the fact that dioxan will not wash the stains out of the sections in the dehydrating process to any great extent. This makes triple staining easier.     

A Technic for Staining Mouse Pituitary

Many technics for histological staining of the pituitary gland have been devised. Since most of the methods described have been for animals other than the mouse, and since the mouse hypophysis, for some unknown reason, is difficult to stain by the accepted technics, the present writer has worked out a modification of Mallory's triple staining method.     

An Improvement in Staining Technic for Protozoa

A modification of Donaldson's iodine-eosin stain for staining intestinal protozoa is presented. This modification consists of using high dilutions of colloidal iodine (Chandler)² instead of Lugol's solution as well as high dilutions of eosin. A better resolution of the external and internal structures is brought about by the new method.

The procedure is as follows: A portion of the fecal material to be examined is suspended in a 0.6% salt solution; the suspension should be of a consistency so that one drop will make a satisfactory microscope mount under a cover glass. To ten parts of this suspension, in a test tube, is added one part of the stain which is prepared as follows:—

10 parts of distilled water

6 parts of a suspension of colloidal iodine (Chandler) containing 4% iodine—20% iodine suspensoid, Merck

1 part of a 10% water solution of anilin red, Merck (eosin yellowish)

Technicians will find, because iodine in the form of colloidal iodine is readily released to the organisms, that the use of this material is far superior to Lugol's solution hi carrying out the technic for staining intestinal protozoa in the study of fresh mount preparations. Not only are organisms more deeply stained with iodine but by eosin as well, even when employed in high dilutions.     

A Feulgen-Carmine Technic for Staining Fungus Chromosomes

Apothecia of Pyronema confluens Tul. were stained by the Feulgen reaction and then squashed and mounted in propiono-carmine. Exceptionally clear differentiation of the chromosomes was obtained by this method. Snail stomach cytase was used as an aid in flattening the asci and a simple method for its extraction is described.     

A Flagella Staining Technic for Soil Bacteria

In an attempt to stain the flagella of soil bacteria, many of which have flagella so fine that they are hard to stain by most methods, a technic was developed which combines the best points of Hofer and Wilson's method with that of Bailey as developed by O'Toole. Satisfactory preparations have been obtained for organisms of the genera Pseudomonas, Phytomonas, Alcaligenes, Escherichia, Azotobacter, and Bacillus. This technic, therefore, is recommended as a rapid and constant method for routine flagella staining of all motile aerobic organisms; it combines Hofer and Wilson's method of cleaning the slides with O'Toole's technic of spreading the smears and with a modification of Bailey's mordant.     

A Rhodocyan Technic for Staining the Anterior Pituitary

A reproducible, one-step, differential staining technic which uses routine formalin-fixed tissue and gives brilliantly contrasting results is produced by incubating sections for 1 hr in a 60° C oven in the following dye mixture: 1% eosin B (CI#771), 8 ml; 1% anilin blue (CI#707), 2 ml; and buffer solution (0.1M citric acid, 1.1 ml; 0.2M Na₂HPO₄, 0.9 ml; distilled water, 28.0 ml) at pH 4.5. No differentiation is necessary. The method can be modified for duodenal enterochromaffin cells and alpha cells of pancreatic islets by adjusting the buffer to pH 3.6 and staining for only 3 min at 60° C.     

A Technic for Differential Staining of Nucleoli and Chromosomes

A procedure is described which enables a stain to be definitely located in the substance of the nucleolus. Material is fixed in either Navashin or Levitsky; the chromatin is stained by means of the improved Feulgen technic introduced by de Tomasi, and preparations brought thru the washing solutions down to distilled water. From distilled water the material is transferred to a mordant solution, 5% sodium carbonate in water, in which it is left for at least one hour. After mordanting wash well with water then stain for ten minutes in light green solution (90% alcohol, 100 cc, light green SFY, 0.5 g, aniline oil, 2 drops, well shaken); differentiate in alcoholic sodium carbonate solution, (70% alcohol saturated with carbonate); treat with 95% alcohol, absolute alcohol, equal parts xylene and absolute alcohol, clear in pure dry xylene and mount in neutral balsam. Cytoplasm and karyolymph should be quite clear, with magenta chromatin and well defined green nucleoli. The light green does not behave like a simple counterstain as in previous technics but as a definite stain for nucleolar material.     

Notes on Technic: Mahon's Myelin Stain for Celloidin-Embedded Nervous Tissue

Two methods commonly used to stain myelin sheaths are Kluver and Barrera's luxol fast blue (Kluver and Barrera 1953) and Weil's iron hematoxylin (Weil 1928). Both require differentiation of the stain; in addition, the Kluver-Barrera method specifies 16-24 hour staining. A third method for the selective staining of myelinated axons is that of Mahon (1938), which was introduced for use with paraffin-embedded autopsy tissue. The procedure possesses two distinct advantages since it requires: (1) no differentiation of the stain and (2) only 1 hour staining. Loyez's (1910) myelin stain for celloidin embedded tissue is similar to Mahon's but calls for long staining followed by differentiation. This report describes the application of Mahon's method to celloidin-embedded experimental tissue and emphasizes its utility for staining tissues to be used for reconstructing microelectrode penetrations (fig. 1) and for demonstrating the effect of experimental lesions (fig. 2).     

Staining Sections of Peripheral Nerves for Axis Cylinders and for Myelin Sheaths

Pieces of mammalian nerves 1 to 2 cm. long were placed under moderate tension and fixed 24–48 hours in: picric acid, saturated aqueous, 90 ml.; formalin, 10 ml.; and trichloracetic acid, 25% aqueous, 2 ml. They were washed in water, cut in two and one end stained with 0.04–0.06% osmic acid solution, while the other was dehydrated, embedded in paraffin, and mounted sections from it stained with protargol. The fixing solution used was selected from a number of combinations of acidified picro-formalin as the one most likely to give satisfactory results when followed by both silver and osmic acid. The use of osmic acid solutions of less than 0.1% concentration avoided the overstaining of myelin sheaths seen frequently when stronger solutions were used with material that had been fixed previously. Protargol, 0.5% solution with fast green FCF added to make 0.05% dye in the final concentration, was used to impregnate sections for axis cylinders. Reduction and toning were done as in Bodian's method.     

A Variation of the Pal Weigert Method for Staining Myelin Sheaths

A variation of the Pal-Weigert method for staining myelin sheaths is described, which is successful on some sections on which Pal's modification of the Weigert method fails; which does not require old ripened hematoxylin; and which can be completed in from three to five hours. It varies from the Pal-Weigert method by the insertion of a bath of ferric ammonium sulfate both before and after the staining with hematoxylin.     

Staining Sections of Peripheral Nerves for Axis Cylinders and for Myelin Sheaths

Pieces of mammalian nerves 1 to 2 cm. long were placed under moderate tension and fixed 24-48 hours in: picric acid, saturated aqueous, 90 ml.; formalin, 10 ml.; and trichloracetic acid, 25% aqueous, 2 ml. They were washed in water, cut in two and one end stained with 0.04-0.06% osmic acid solution, while the other was dehydrated, embedded in paraffin, and mounted sections from it stained with protargol. The fixing solution used was selected from a number of combinations of acidified picro-formalin as the one most likely to give satisfactory results when followed by both silver and osmic acid. The use of osmic acid solutions of less than 0.1% concentration avoided the overstaining of myelin sheaths seen frequently when stronger solutions were used with material that had been fixed previously. Protargol, 0.5% solution with fast green FCF added to make 0.05% dye in the final concentration, was used to impregnate sections for axis cylinders. Reduction and toning were done as in Bodian's method.     

Iodine131 Uptake in the Gram Staining Technic

The Hucker modification of the Gram staining technic, in which NaI¹³¹ was incorporated with the Gram's iodine solution, was performed as the basic procedure. The Gram positive test-bacteria were Staphylococcus aureus and Bacillus megaterium; the Gram negative were Escherichia coli and Pseudomonas aeruginosa. The uptake of I¹³¹ was measured after the addition of the Gram's iodine solution (NaI¹³¹) to the test-bacteria dried on a glass slide, after the decolorization process and after counterstaining. Radiation was measured by placing the slide under a GM-TGC-2 end-window counting tube after each procedure. The Gram positive test-bacteria retained approximately twice as much I¹³¹ after decolorization and counterstaining as did the Gram negative bacteria. In this, the basic technic, the uptake of I¹³¹ by the test bacteria appeared to be directly related to the crystal violet concentration in the primary staining solution. The uptake of I¹³¹ was not significantly altered by the time of application of the Hucker crystal violet staining solution (15-180 sec), or of the Gram's iodine (NaI¹³¹) solution (30-120 sec) or by the duration of the alcohol decolorization process (30-120 sec).

Variations (herein referred to as variations 2 and 3) of the basic procedure were carried out in which the primary staining solution contained crystal violet combined with NaI¹³¹ or Gram's iodine solution (NaI¹³¹). In variations 4 and 5 the effect of the order of application of the various staining reagents was investigated. In these variations (2-5) all test-bacteria were stained Gram negative. The initial uptake of I¹³¹ was decreased, though in variations 4 and 5 the percent retention of I¹³¹ was increased. In the staining of bacterial spores by different methods (variation 6), it was noted that the initial uptake and percent retention of I¹³¹ was greater than with the vegetative forms. When ovalbumin was stained by the Hucker technic and variations thereof, it was noted that the initial uptake of I¹³¹ was directly related to the protein (ovalbumin) concentration up to an ovalbumin concentration of 1%.     

A Modification of the Cresyl Violet Technic for Staining Nerve Cells

Kill root tips in 1 part glacial acetic acid to 3 parba RB Solute alcohol for 12 or more hours. Remove from king fluid a d place for 5 to 10 minutes in a solution consisting of 1 part 95% alcohol to 1 part concentrated HC1. Transfer to Carnoy's fluid for 5 minutes or longer. Cut a small piece (0.5 mm. or less) off the tip of the root Press directly on the piece of root with a small fiat scalpel; the cells will now separate and float free in the stain. Place cover slip over the drop of stain and apply gentle pressure. Heat carefully by paseing the slide 3 or 4 times thru the flame of an alcohol lamp. Seal with heated mixture of 1 part Parowax to 1 part gum mastie. Make permanent by the McClintock permanent method. and place on a clean slide in a small drop of iron-ace-sinin.     

A Modified Turchini Technic for the Differential Staining of Nucleic Acids

The preparation of the 9-methyl-2,3,7-trihydroxy-6-fluorone reagent for the selective staining of both desoxyribose and ribose nucleic acids is described. With slight variations this method follows the Duckert (1937) modification of the Liebermann and Lindenbaum (1904) reaction.

The present modification of the Turchini et al. (1944) staining procedure has been used on human autonomic ganglia fixed in Bouin's fluid, rat tissues, fixed in Bouin's, Zenker's, formol and for-mol-saline fixatives and mouse liver frozen and dried. The modified Turchini method has been examined primarily for its qualitative reliability by means of the following procedure. Ribonuclease treated sections were compared with adjacent sections immersed in distilled water. In succeeding steps half of these sections were stained by the modified Turchini process and the other half by Einarsson's gallocyanin chrome alum. Evidence gleaned from this and other tests indicates that 9-methyl-2,3,7-trihydroxy-6-fluorone may be used for the selective staining of desoxyribose and ribose nucleic acids.