A Modified Wright's Method for Staining Blood Smears

After the blood smear is treated for the proper length of time with Wright's stain, neutral distilled water is used for diluting the stain. After the slide has been treated with neutral distilled water until the smear becomes pinkish it is then treated with pure absolute methyl alcohol which destains the plasma. Neutral distilled water is again kept on the mount until the corpuscles are well stained. Then the slide is dehydrated with absolute ethyl alcohol, cleared with clove oil and completed in the usual way.

Blood smears of different groups of vertebrates were uniformly brilliantly stained with the above technic.

Several lots of Wright's dry stain have been tested with the modified technic and no difficulties have been encountered in its application.     

A Method for Staining Certain Bacteria and Antherozoids

A modification of Loeffler's method for staining the flagella of bacteria was employed in staining large forms of bacteria and antherozoids. The bacteria or the antherozoids are killed and fixed in a drop of water on a slide and set aside to dry, before the next step is undertaken. The slide is treated for a period of time, varying from about ten minutes to several hours, in a practically saturated solution of tannic acid. After the slide is thoroly rinsed in water, it is stained with either a single stain or a combination of stains. The slide is then dehydrated with absolute alcohol, cleared, with clove oil, and completed in the usual manner.

The body of the bacterium and that of the antherozoid are well differentiated and the cilia are distinctly brought out by means of the method herein described.

The technic is of especial value in staining the antherozoids of mosses, liverworts, and ferns.     

Staining Raw and Cooked Apple Tissues for Study of Cell Wall Structure

Permanent preparations were made of paraffin sections from raw and cooked apple tissues stained with microchemical color reagents for pectins and pentosans. Sections stained with ruthenium red to show pectins were dehydrated and covered in balsam, and sections stained with diphenylene diamine acetate (DDA) to show pentosans were washed with water and covered in Clearcol.

Cooking was accomplished by steaming cubed histological samples. Both raw and steamed specimens were fixed in FAA in a vacuum chamber, dehydrated and cleared in tertiary butyl alcohol, and embedded in paraffin. Paraffin sections first fixed to slides with Haupt's adhesive were further stabilized by immersing in a 1% celloidin solution after dissolving the paraffin.

Ruthenium oxychloride flakes were dissolved in a Coplin jar of water containing 2 drops of ammonium hydroxide. Rehydrated sections were stained in ruthenium red 30 minutes and rinsed in water. Three methods of further preparation follow: (1) Flood sections with 10% gum arabic; drain and air-dry thoroughly; immerse in xylene 5 minutes; cover in balsam. (2) Drain and air-dry sections; if desired, counterstain dry sections with Johansen's fast green solution; immerse in xylene; cover in balsam. (3) Dehydrate by dipping in 70%, 95%, and absolute ethyl alcohol; immerse in xylene; cover in balsam.

DDA was made by heating 15 g. of benzidine in 150 ml. of glacial acetic acid and 450 ml. of water until dissolved, then adding water to make 750 ml. of solution. Rehydrated sections were stained 4 hours in DDA, washed, stained 5 minutes in Congo red (Congo red, 5 g.; NaOH, 5 g.; water, 100 ml.), washed, and covered in Clearcol.

An Autotechnicon was used for dehydration, clearing, infiltration, deparaffinizing sections, and staining. Procedures that necessarily remained manual were fixation in a vacuum chamber, and all operations that followed staining.

Ruthenium red, though the best available indicator for pectins, may not be specific for these substances. DDA and ruthenium red stained identical structures in hypodermis and cortex. DDA also stained cuticle, hence was more useful than ruthenium red for delineating that portion. DDA sections were better for photomicrography, and for measuring thickness of cell walls. Neither stain prevented the study of cell walls in polarized light.     

Differential Staining Of Tissue In The Block With Picric Acid And The Feulgen Reaction

The authors have found a modification of the Feulgen reaction to be a satisfactory stain for tissue in the block.

Pieces of fresh mammalian tissue not thicker than 5 mm. are fixed for approximately 48 hours at 25° C. in a mixture of equal parts of 5% aqueous sulfosalicylic acid and saturated aqueous picric acid. They are washed for 30 minutes in three ten-minute changes of distilled water and placed in Feulgen's staining solution diluted to one-half strength with distilled water. The staining solution is allowed to act for 24 hours (2 to 3 mm. thick blocks) up to 48 hours for 5 mm. thickness. After staining, the specimens are transferred to a mixture of sodium bisulfite, 0.5 g. and N hydrochloric acid, 5 ml. in' 100 ml. of distilled water. Two changes of IS to 30 min. each in the acid sulfite are given and these are followed by dehydration through 50%, 70% and 95% alcohol. One to two hours are allowed for each change except the last 95%, in which the stained tissue is allowed to remain overnight. The dehydration is completed in two changes of absolute alcohol with subsequent clearing in xylene and embedding in paraffin. Sections may be cut 10 μ or other thickness desired, mounted on slides, paraffin removed, and covered in the usual manner. Nuclei stain reddish violet against a lemon yellow background when the stain is typical. Orange G, 200 mg. per 100 ml. may be added to the fixing fluid if a more polychromatic effect is desired.     

A New Methylene Blue Technic for Permanent Preparations

1. Tissues stained intra vitam with methylene blue are fixed in a 10% ammonium molybdate solution in physiological saline (or sea water if the tissue is from a marine animal). Fixation time is kept to a minimum. Washing also is reduced to a minimum.

2. Excess fluids are removed from tissues by blotting with a paper or cloth towel before they are put into the succeeding solution. Tissues are taken from the wash water, blotted and placed in a mixture of equal parts of absolute ethyl alcohol and n-butyl alcohol for 30 minutes. They are then blotted and transferred to n-butyl alcohol for 30 minutes. After blotting they are placed in a mixture of one part methyl salicylate and four parts xylene until cleared. Tissues may be mounted whole or prepared for sectioning by embedding in paraffin in the usual way.

3. Tissues fixed, washed, dehydrated and cleared as described retain nearly all of the stain; the time required is greatly reduced; there is no need to chill the dehydrating solutions; cell distortion is much reduced.     

Oil Blue Na as A Stain for Rubber in Sectioned or Ground Plant Tissues

Oil blue NA (Calco), a stain which colors rubber bright blue, has been used effectively in studying the distribution of rubber in several plant species. Fresh or fixed sections are cut, bleached with Javelle water or NaOCl solution, treated with 9% KOH in 95% ethanol, washed with several changes of water and finally with 95% ethanol, and stained with 0.05% oil blue NA in 70% ethanol. Sections are rinsed in 50%' ethanol, placed in 40% glycerin, and mounted in glycerin jelly.

For the detection of changes in the distribution and character of rubber in milled or ground tissues, much the same staining procedure is followed. The stained tissues usually are examined and dissected under a stereoscopic microscope, a procedure which permits rubber to be recognized by both its staining reaction and by a more specific property, elastic elongation.

A microscopic technic is presented whereby it is possible to determine approximately the relative proportion of dispersed and coagulated rubber latex in unstained tissues.     

Staining Methods For Studying Ingredient Distribution In Baked Cakes

A method is described for preparing cake crumb for sectioning and staining. Previous to embedding, the fat was stained and fixed by exposing small blocks of cake to the fumes from a 5%, freshly-prepared, aqueous solution of osmic acid (OsO₄). This was followed by dehydration in ethyl alcohol and tertiary butyl alcohol, removal of air under vacuum and infiltration with paraffin.

Sections were cut 20 and 9Op thick and mounted with water.

Wax was removed by immersion in xylene. The sections were rehydrated in a series of ethyl alcohol dilutions, from concentrated to dilute, then transferred to distilled water.

Protein was then stained pink by immersion of the slides in an acidified 0.04% water solution of eosin Y, or starch was stained blue with a dilute aqueous solution of iodine. Ten grams iodine and 10 g. KI were dissolved in 25 ml. distilled water. This stock solution was diluted for use one to two hundred times.

The relationship between protein and starch was demonstrated by staining the sections with eosin, differentiating in 50% alcohol and staining with iodine.

When slides of cake crumb were prepared in this way, the fat was stained black, the protein bright pink and the starch granules a dark blue.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

A Simplified Stain for Hemoglobin in Tissues Or Smears Using Patent Blue

The following technic, based on the patent blue V hemoglobin reaction, is useful for identifying hemoglobin in tissue fixed in neutral formaldehyde solution and embedded in paraffin:

Stain the deparaffinized, hydrated sections 3 to 5 minutes in the working reagent, prepared by adding 2 ml. of glacial acetic acid and 1 ml. of 3% hydrogen peroxide to 10 ml. of the filtered stock solution (1 g. patent blue, 10 g. zinc powder, and 2 ml. glacial acetic acid). Counterstain 30 to 60 seconds in 1:1000 safranin solution in 1% acetic acid, rinse, dehydrate with alcohols, clear in xylene and mount in clarite. Total time required, 37 minutes.

Blood and tissue and smears may be stained, following fixation in methyl alcohol, by applying the working reagent as above.     

A Nissl Method Using Buffered Solutions of Thionin

Experiments were performed in an attempt to obtain a rapid method for staining the chromophilic substance of formalin-fixed nerve cells differentially without resorting to over-staining and subsequent acid-decolorizing. A satisfactory procedure using thionin in dilute buffered solutions was developed: Prepare a stock solution of the dye using 1 g. in 100 ml. of distilled water. Prepare veronal-acetate buffers (Michaelis, 1931) in the range of pH 5 to pH 3.S. To each 10 ml. of the buffer add 0.5 ml. of the stock dye solution. After rinsing in CO₂-free distilled water place mounted or unmounted sections in this solution. (Freshly fixed material, 10μ to 20μ thick, is completely stained in 10 to 20 minutes but over-staining does not occur when longer times are allowed.) Return sections to distilled water (2 changes) and wash until diffusion of excess dye is no longer visible. Wash in 70% ethyl alcohol (2 changes) until diffusion of excess dye is no longer visible. Dehydrate in 95% ethyl alcohol and normal butyl alcohol, clear and mount.

Optimum staining of chromophilic material occurs at pH 3.65. Glial processes are well stained at pH 4.6. Nissl bodies and glial cytoplasm are purplish blue, nuclear chromatin is blue, background is clear at pH 3.65 but pale blue at pH 4.9.     

Staining Aspirated Human Bone Marrow with Domestic Wright Stain

The method employs the domestic Wright stain for the staining of aspirated human bone marrow. Freshly distilled water, pH 6.0 to 6.4, is used. Wright stain, 0.5 cc, is placed upon the air-dried preparation and permitted to act for two minutes. The stain is then diluted with 2 cc. distilled water and permitted to act for from 5 to 10 minutes. After washing off the stain with distilled water, the preparation is placed into a decolorizer (acetone 0.5 cc, pure methyl alcohol 5.0 cc, and 100 cc. distilled water, pH 6.0 to 6.4) for differentiation from 1 to 5 seconds, rinsed, washed under running water and permitted to air-dry. A well stained and differentiated preparation shows the “Romanovsky effect”, and the sharpness of minute structures obtained compares favorably with control preparations stained with German dyes.

The bone marrow should be prepared as described. The Wright stain marketed by the National Aniline and Chemical Co., N. Y. was found to be reliable as regards staining quality of registered batches. One photomicrograph, showing bone marrow cells from pernicious anemia, is included.     

A Buffered Giemsa-Bodian Stain for Neurological Material

A buffered Giemsa counterstain for the Bodian method is described. It is very useful for bringing out Nissl substance and nerve fibers in the same section. Bouin perfused or formalin fixed material from mammals, amphibia, reptiles and fish was used. After fixation, all tissues were decalcified for at least a week in 50% formic acid (1 part) and 20% sodium citrate (1 part). This was washed out thoroughly. The method described by Bodian (1936) was followed except for the following minor changes: Winthrop Protargol (Strong Protein Silver) Batch N346BJ was used exclusively; all glassware was cleaned with acid prior to setting up the stain; before developing, the sections were washed in warm tap water for 10 minutes; the gold chloride was chilled before use. The sections were then put into buffered Giemsa for 24 hours. Stock Giemsa: 0.75 grams powdered Giemsa (Coleman and Bell, Certification No. CGe-3) was dissolved in 50 ml. of glycerin overnight in the oven, then 50 ml. of methyl absolute alcohol were added. To 3 ml. of Giemsa stock solution, 87 ml. of distilled water buffered to pH 5.3 with 10 ml. of Sorensen's buffers were added and the solution filtered. Coleman buffer tablets gave best results at pH 5.0. Sections were then rinsed in 95% alcohol, two changes of absolute alcohol, two changes of xylene, and were then mounted in Clarite.     

The Use of Bismarck Brown Y in some new Stain Schedules

The staining quality of Bismarck brown Y may be improved and sterility maintained by adding 5% phenol to a 1% aqueous solution. Use the phenolic Bismarck brown in combination with iron alum hematoxylin except for stripped epidermis in the following procedures:

Stem and Root Schedule: Mordant sections from water in 4% iron alum for 10 minutes. Rinse in distilled water and stain in 0.5% aqueous hematoxylin for 1 minute or until darkly stained. Rinse in distilled water and destain in 2% iron alum until a gray color appears. Rinse thoroly in distilled water and intensify hematoxylin by transferring sections to 0.5% aqueous lithium carbonate until the desired black color appears. Rinse thoroly in distilled water and stain for 1-5 minutes in phenolic Bismarck brown. Rinse in distilled water, dehydrate successively in 30, 50, 70, 95 and 100% alcohol. Clear in methyl salicylate for 5 minutes, then to xylene for 3-5 minutes, and mount in balsam.

Middle Lamellae in Wood: Destain more thoroly in 2% iron alum than for the general stem and root schedule, and intensify in lithium carbonate for a longer period (about 1 hour).

White Potato Tuber Sections: Modify above schedule by reducing time of destaining in 2% iron alum to about 30-60 seconds and intensify hematoxylin until starch grains appear bluish in color. Stain in phenolic Bismarck brown for 1-2 minutes.

Wheat Grain Sections: Fix grain for sectioning when in “dough” stage. Use schedule the same as for potato tuber except for reducing time of staining in phenolic Bismarck brown to about 45 seconds.

Tradescantia zebrina Epidermis: Strip epidermis from leaf while submerged in water. Fix in 100% alcohol 10 minutes, pass thru 95, 70, 50, 30, and 10% alcohol to water. Stain in phenolic Bismarck brown for 10-20 minutes. Dehydrate, clear in methyl salicylate and mount in balsam.     

Orcein-Hematoxylin in Iodized Ferric Chloride as a Stain for Elastic Fibers, With Metanil Yellow Counterstaining

Autopsy and biopsy specimens of human skin were fixed overnight in alcoholic Bouin's solution, embedded in paraffin, cut at 7 μ, deparaffinized, hydrated to 70% alcohol, and treated as follows—stained 2 hours in a mixture consisting of: 0.2% orcein in 70% alcohol and 1% HC1 (conc.), 125 ml; 5% hematoxylin in absolute alcohol, 40 ml; 6% FeCl₃ in water, 25 ml; and aqueous I₂-KI (1:2:100), 25 ml—rinsed in distilled water until the excess stain was removed—differentiated in 1.2% FeCl₃, 5-15 sec—washed in running water, 5 min—differentiation completed in 0.01% HC1 acid-alcohol, 1 min—a dip in 95% alcohol—distilled water, 2 min—0.25% aqueous metanil yellow, 5-10 sec—a 95% alcohol dip—dehydrated in absolute alcohol, xylene, and mounted in a resinous medium. The technic combines the orcein of Pinkus' stain and the hematoxylin mixture of Verhoeff into a single staining solution and gives sharp and reliable results for both coarse and extremely delicate elastic fibers. These stain purple; nuclei, violet; and background, yellow. The stain allows the use of formalin, Bouin's fluid and Zenker-formol fixation. The results have been consistent in other primates as well as in man.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

Initial and Persisting Staining power of Solutions of Iron-Hematoxylin Lake

A study was made of factors affecting the initial staining power and the stability of iron-hematoxylin lake solutions. The findings were applied to the preparation of a superior hematoxylin staining solution. This is made up as follows: in 50 ml. water dissolve, in order, 1.0 g. ferric ammonium sulfate [FeNE₄ (SO₄)₂⋅ 12H₂O], 0.8 ml. sulfuric acid, 50 ml. 95% ethyl alcohol, 0.5 g. hematoxylin. Filter the solution to remove the insoluble, white crust of the ferric ammonium sulfate. The solution stains well ten minutes after it has been made. Peak performance is attained within 5 hours, and is maintained for 4 to 8 weeks. Staining time is 3 to 30 minutes. Excess stain can be rinsed off the slide and section by immersion in water, after which destaining, if necessary, can be accomplished with a solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.18 ml. sulfuric acid. The slides may or may not be placed next in a neutralizing solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.5 g. sodium bicarbonate. They may then be passed through 50 ml. water, 50 ml. 95% ethyl alcohol on the way to alcoholic counterstaining solutions, or through water leading to aqueous counterstains.

The nuclear stain produced is black, intense and very sharp and has proved to be consistently excellent on a variety of animal and human tissues following a number of different fixatives.     

Specific Staining of Glycogen with Haematoxylin and Certain Anthraquinone Dyes

Specific staining of glycogen in rat liver fixed in chilled 80% alcohol, chilled formol alcohol or 10% neutral formalin has been accomplished with acid alizarin blue SWR, alizarin brilliant blue BS, alizarin red S, gallein, haematein, and haematoxylin solutions. TO prepare a staining solution, 1 gm dye, 1 gm K₂CO₃ and 5 gm KCl were dissolved by heating in 60 ml of water. Concentrated NH₄OH (0.880 sp.gr.), 15 ml, followed by 15 ml of dry methanol were added to 20 ml of the cooled solution. Paraffi sections were stained for 5 min, rinsed in dry methanol, cleared in xylene, and mounted in D.P.X. The high specificity obviated the need for counterstaining: nuclei and cytoplasm were unstained. Precipitation of stain onto the slide was rare. As all the dyes carried, like carminic acid, numerous groups capable of forming hydrogen bonds, it is suggested that the staining mechanism involved hydrogen bonding.     

The Mechanism of the Gram Reaction. III. Solubilities of Dye-Iodine Precipitates and Further Studies of Primary Dye Substitutes

Solubilities of dye-iodine precipitates in alcohol and in aqueous safranin solution were determined by direct solubility methods and by photocolorimetric methods. It was found that, increasing precipitate solubility in alcohol or safranin solution gave decreasing differentiation between Gram-positive and Gram-negative bacteria. Dyes which did not stain the cells well as a primary stain did not give good Gram stains, regardless of the solubilities of their precipitates. Some dyes (typified by methylene blue) which gave relatively alcohol-insoluble iodine precipitates gave inferior Gram differentiation because these precipitates were readily soluble in the safranin counterstain.

Solubilities of precipitates of crystal violet and various iodine substitutes were determined photocolorimetrically. The ability of a substance to replace iodine in the Gram stain correlated with its ability to give a precipitate which was only slightly soluble in alcohol and relatively insoluble in aqueous safranin solution.

It was concluded that the usual Gram reagents are not truly specific for the differentiation. Any dye and mordant could be used if the dye was deeply colored, stained the cells well, and if the precipitate of dye and mordant was only slightly soluble in alcohol and relatively insoluble in the counterstain. These factors, combined with those influencing differences in cell membrane permeability, constitute the most important factors in the Gram stain differentiation.

Studies were made concerning the ability of dyes to substitute for crystal violet in the Gram procedure. Of 29 dye samples reported on here for the first time none proved to be good substitutes for crystal violet.     

Differential in Toto Staining of Bone, Cartilage and Soft Tissues

The following method for staining bone and cartilage allows study of the gross cleared specimen and does not injure the tissues for subsequent microscopic study: Fix in 10% neutral formalin; bleach thoroughly in 3% H₂O₂ in sunlight. Wash in distilled water. Stain bone 24 hours in 0.01 g. of Biebrich scarlet in 100 ml. of distilled water. Destain in 95% alcohol until soft tissues and cartilage are colorless. Stain cartilage 24 hours in a pH2 buffer solution of 2.1g. of citric acid per 100 ml. of water with 0.001 g. of methylene blue. Destain in pH2 buffer solution until soft tissues are pale. Dehydrate in two changes of 95% alcohol in preparation for clearing. (This completes the destaining and may remove too much stain from the cartilage if destaining in the pH2 solution has been carried too far.) Place in Groat's clearing fluid and cover loosely so that the alcohol may evaporate, or remove the alcohol in vacuo. Groat's Mixture No. 19 is usually satisfactory.

For a combined stain, first stain bone, as above, and then apply the cartilage stain.

Seal jars with an ordinary liquid wood glue such as LePage's.     

New Fuchsin-Hematoxylin-Eosin; A Stain for Acid-Fast Bacilli and Surrounding Tissue

This is a staining technique for histopathologic evaluation of tissue reaction in the environs of acid-fast tubercle bacilli (avian and bovine) in sections. Fresh tissue is fixed in 10% neutral formalin and processed in the usual manner for embedding in paraffin. Sections are cut approximately 6 μ. thick, dewaxed, hydrated, and stained with Harris' hematoxylin. They are rinsed in tap water, differentiated in add alcohol, washed in tap water, given a distilled water rinse and stained at 20-30° C in a 1% solution of new fuchsin in 5% phenol. Each slide is then handled individually by placing it directly into a saturated aqueous solution of Li₂CO₃ and agitated gently for a few seconds. This is followed by differentiation with 5% glacial acetic acid in absolute or 95% ethyl alcohol until the color stops running. Two rinses in absolute or 95% ethyl alcohol follow. The sections are then counterstained in the color add of eosin Y prepared according to the method of Schleicher (Stain Techn., 28, 119-23, 1953) and used as an 0.025% solution in absolute alcohol. Following passage through 2 changes of absolute alcohol, the sections are cleared in xylene, then mounted in Permount or similar synthetic resin. The add-fast barilli are emphasized by their bright retractile red color within a contrasting background of hematoxylin and eosin.