The Dioxan Technic

Diozan is recommended in place of alcohols and clearing oils in paraffin embedding and in the staining of sections. It is unnecessary to dehydrate fresh dioxan before using and the insertion of other dehydrators and clearers into the series is illogical. Used dioxan (if employed for the sake of economy), should be dehydrated with CaO rather than with CaCl₂. A provisional dioxaniron-hematein method designed to avoid watery solutions is as follows: after removing paraffin in xylene, mordant 30 min. in 1% ferric chloride in 100% dioxan, rinse in 80% dioxan, stain in the following solution: dioxan, 40 cc.; water, 6 cc.; glacial acetic acid, 4 cc.; hematein, 5 g., saturated with potassium alum and filtered. Differentiate in 0.25% picric acid in 80% dioxan and alkalinize in 80% dioxan saturated with sodium bicarbonate. Rough determinations of the solubilities of various salts and dyes in dioxan are presented. A summary is given of the unpublished experiences of other workers with a variety of both plant and animal tissues. A brief historical account of the development of the dioxan technic is included. A summary of pharmacological studies indicates that dioxan is not dangerously toxic in concentrations likely to be encountered in the microtechnic laboratory.     

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.     

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 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.     

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 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.     

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 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.     

Notes on Technic: A Carbonate-Colloidal Iron Staining Reagent

The use of Congo red as an elective stain for eosinophilic granulocytes and their precursors in tissue sections and autoradiographs is demonstrated and discussed. The 0.5% alcoholic Congo red solution of Highman, normally used for the detection of amyloid, may also be used with only minor changes. This simple method may aid in the diagnosis of special hematological problems and facilitates the recognition of eosinophil granulocytes as well as proliferating and nonproliferating myelocytes in autoradiographs from paraffin sections.     

A Nile Blue Staining Technic for the Differentiation of Melanin and Lipofuscins

When paraffin sections are stained in 0.05-.01% Nile blue in 1 % sulfuric acid, washed thoroughly in water and mounted in aqueous media, lipofuscins color deep blue, melanins dark green, myelin and red cells lighter greens and background pale green. If, immediately after staining, the preparations are at once extracted with acetone with or without a 1% sulfuric acid rinse, melanins remain dark green, mast cells color purple, lipofuscins and background decolorize and nuclei may stain light green.     

A Rapid Histological Technic for Staining Latex in Roots of Taraxacum Kok-Saghyz

The freezing technic described in this paper provides permanent slides of sections of the root of Taraxacum kok-saghyz with latex preserved in place. The following schedule is used: (1) Prefreeze the piece to be sectioned before removal from the root. (2) Mount in ice and section with chilled microtome knife. (3) Plunge frozen sections into the combination coagulant and stain prepared from Calco oil blue N. A., acetic acid and ethyl alcohol. (4) Wash in water. Aspirate if necessary. (5) Mount on a slide using Karo.

This technic is rapid and simple. The sections are well adapted to making counts and measurements of latex tubes since there has been a minimum of latex loss. Latex is retained in place by keeping tissues frozen until introduced into the coagulant.     

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%.