Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Modifications of Lebowich's Soap-Wax Technic

Lebowich's technic is outlined for simultaneous dehydration and infiltration of tissues by a medium composed of stearic acid, 56° C. paraffin, diethylene glycol, and monoethanolamine. The prices and places where these materials may be purchased are given.

Tissue for sectioning is placed in acetone, C.P., for 1 hour, then put directly into the soap-wax medium at 60° C. under reduced pressure, and finally embedded in new soap-wax.

Modifications include a simplification of the apparatus used by Lebowich. A preserving jar fitted with a rubber stopper serves as a vacuum chamber, and use of an aspirator accomplishes the reduction of pressure. With invertebrate embryos up to 1000 μ diameter no reduction of pressure is needed. Embryos are fixed, washed, placed in acetone, infiltrated in soap-wax, and embedded.

By this soap-wax method the alcohols, xylene, and overnight drying of affixed ribbons are eliminated. Tissue may be fixed, sectioned, stained, and permanently mounted within 6 to 8 hours.     

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.     

Carbowax for Embedding and Serial Sectioning of Botanical Material

Plant material infiltrated with gradually increasing concentrations of Carbowax 400, followed by Carbowax 1540 and finally a 19:1 embedding mixture of Carbowax 1540 and 4000 showed minimum shrinkage. Quantitative measurements of shrinkage in tissue of potato tubers gave the following amounts: fixation and washing, about 4%; transfer from water directly to 70% Carbowax 400, 5176; from water through a graded series (5, 10, 15, 20, 30, 40, 50 and 60% Carbowax) to 70%, only 2.5% shrinkage; with an additional 1.5% occurring in transition to the embedding mixture. Dry ribbons are placed on adhesive-coated (gelatin, 5 gm; water, 120 ml; glycerol, 40 ml; phenol, 2 gm) slides in a humidity chamber. In 10-15 min enough moisture is absorbed by the ribbon to float the sections out gently and bring them in contact with the adhesive. Slides are then dried 5-10 min at room temperature. To remove minor wrinkles, the sections are subsequently flooded with water, then redried 12-24 hr; after which, they are ready for staining.     

Carbowax for Embedding and Serial Sectioning of Botanical Material

Plant material infiltrated with gradually increasing concentrations of Carbowax 400, followed by Carbowax 1540 and finally a 19:1 embedding mixture of Carbowax 1540 and 4000 showed minimum shrinkage. Quantitative measurements of shrinkage in tissue of potato tubers gave the following amounts: fixation and washing, about 4%; transfer from water directly to 70% Carbowax 400, 5176; from water through a graded series (5, 10, 15, 20, 30, 40, 50 and 60% Carbowax) to 70%, only 2.5% shrinkage; with an additional 1.5% occurring in transition to the embedding mixture. Dry ribbons are placed on adhesive-coated (gelatin, 5 gm; water, 120 ml; glycerol, 40 ml; phenol, 2 gm) slides in a humidity chamber. In 10-15 min enough moisture is absorbed by the ribbon to float the sections out gently and bring them in contact with the adhesive. Slides are then dried 5-10 min at room temperature. To remove minor wrinkles, the sections are subsequently flooded with water, then redried 12-24 hr; after which, they are ready for staining.     

The Mounting of Carbowax Sections with Lighter-Fluid and Rubber Cement

Serial sections cut from plant tissues embedded in Carbowax have been affixed to slides with rubber cement. A rather thick layer of undiluted rubber cement was first spread on the slides. The Carbowax ribbons were added next. Lighter-fluid, essentially petroleum ether which can be substituted for it, was then run under the sections to dissolve the rubber cement and to float the ribbons. This notation medium did not dissolve the Carbowax and the ribbons could be manipulated in it for accurate location. The slides were dried on a 45° C warming table which also helped to flatten the sections. Adhesion was best when drying times were held to 4 hr or less. All excess rubber cement was washed away with xylene immediately prior to covering and the cover slips were carefully applied with a very thin resinous mounting medium to prevent dislodging the sections. Both aqueous and alcoholic stains have been used successfully and the slides have been left in them for as long as 3 days without loss of sections. The method was developed for fluorescence microscopy but serves equally well for visible light microscopy. Slides stained with a safranin-fast green combination have been used for both purposes, the safranin staining and fluorescing in a manner similar to rhodamine B.     

Red-blue staining of hydrolysed nucleic acids in paraffin sections

A previous treatment with 10% HC1 in tetrahydrofuran for 2-3 min at 37° C hydrolyses DNA while substantially preserving RNA in formol-fixed paraffin sections. If this treatment is followed by dyeing with basic fuchsin-thiazine or oxazine mixtures, the basic fuchsin stains DNA, the blue dye cytoplasmic RNA, though nucleolar RNA is not well preserved. A specimen sequence is to treat the hydrolysed section with a mixture of 1% aqueous trimethylthionin (Chroma), 15 ml; 0.1% basic fuchsin (G. T. Gurr), 4 ml; and glacial acetic acid, 1 ml. Stain for 15-30 min, dehydrate in acetone, then pass sections through xylene to polystyrene. The specificity of this stain for cytoplasmic RNA is sharper than that of methyl green-pyronin; hence the technic given can be a useful addition to the standard Unna-Pappenheim procedure.     

Normal Butyl Alcohol Technic for Animal Tissues with Special Reference to Insects

N-butyl alcohol is substituted in dehydration for the higher ethyl alcohols. No special clearing is necessary as n-butyl is miscible with paraffin.

The greatest advantage of this method is the elimination of both hardening agents (the higher percentage ethyl alcohols and xylol or benzol). Another advantage is the great time toleration of the processes of dehydration and infiltration. For example, tissues have been kept without deleterious effects in n-butyl alcohol for a year before infiltration. Also, aphids which have been kept in a hot (58°C.) paraffin bath for as long as four weeks, have sectioned well. For small insects and vertebrate tissues about five days proved necessary to insure satisfactory infiltration.

N-butyl alcohol was found to give better results than many other technics in serial sectioning of lightly chitinized insects, and in the preparation of embryological and other vertebrate tissues. This technic has been used as a routine method by beginning students in animal microtechnic with better success than attended the usual methods.     

Delafield's Hematoxylin and Safranin for Staining Meristematic Tissues

The following technic is suggested for staining permanent preparations of meristematic tissues: Prepare and mount the sections by the usual paraffin method. From water, stain them 2-10 minutes in a solution made by adding 2-4 cc. of Delafield's hematoxylin to a Coplin jar full of tap water. As staining is progressive, the sections should be examined from time to time with a microscope. When the cell walls have become a deep purple, transfer the preparations, thru the usual series, to a mixture of xylol-absolute-alcohol in equal parts, and from this to a counterstain made by adding 4-6 cc. of a saturated solution of safranin in absolute alcohol to a Coplin jar full of xylol (75%) with absolute alcohol (25%). This stains the nuclei. Leave the sections in the counterstain at least 2 hours and then rinse them in xylol-absolute-alcohol (1:1) to remove excess safranin. Transfer them to pure xylol and then mount them in neutral balsam.     

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.     

Staining Rickettsiae in Sections of Formalin-Fixed Tissue; a 12-Minute Wright-Buffer Sequence

Rhesus monkey tissues obtained at autopsy were fixed in neutral phosphate-buffered formalin, embedded in paraffin, and sectioned 3-5 μ thick. Sections were stained with Wright's stock stain for 6 min and differentiated in Wright's stock buffer for 6 min and mounted as usual. The rickettsial cytoplasm stained reddish pink; the nucleus, blue. This method was much simpler than that of Wolbach although the results obtained were nearly identical.     

Alcoholic Thionin Counterstaining Following Golgi Dichromate-Silver Impregnation

Brains of cats that had been fixed 2 months or longer in 10% formalin were cut into 3-6 mm. slices and impregnated by Golgi's dichromate-silver procedure (6% dichromate solution, 4-6 days; 1.5% silver nitrate solution 2 days). Sections 100 µ thick were cut after embedding in low melting point paraffin. Three changes of xylene and three of absolute alcohol were followed by staining 3-5 minutes in a saturated solution of thionin in absolute alcohol. The sections were dipped quickly in absolute alcohol and cleared in xylene, then differentiation was effected by an equal-parts mixture of absolute alcohol and xylene. A final clearing in three changes of xylene and mounting in Permount completed the process. Counter-staining was most successful when applied to freshly cut sections.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

A Staining Combination for Phloem and Contiguous Tissues

A technic is described for producing critically stained preparations of phloem tissue. The preparations promise to be relatively stable. Sections of fixed unembedded or of embedded (paraffin or celloidin) phloem, cambium, and xylem are (1) stained in Foster's tannic acid-ferric chloride combination; (2) treated with 1% NaHCOg in 25% or 50% ethyl alcohol for 30 minutes; (3) stained in a saturated solution of lacmoid (made alkaline by adding a few ml. of 1% NaHCO₃ in 25% alcohol) for 12 to 18 hours; (4) dehydrated and cleared in a series composed of 1% solution of NaHCO_s in 50% ethyl alcohol, 80%, 95%, and absolute alcohol, equal proportions of absolute alcohol, clove oil, and xylene, and finally pure xylene; and (5) mounted in a neutral resin. Callose and lignified secondary walls are blue or blue-green in color, cellulose walls and stainable protoplasmic contents are generally light brown. The technic has been successful with sections from 5 to 40μ in thickness, and the staining has been satisfactory for both color and black and white photomicrography.     

Infiltrating and Embedding Tissues with Mixtures of Polyethylene Glycols and Polyvinylacetate Resins

The use of water-soluble polyethylene glycol polymers (Carbowax, Hydrowax) as embedding media can be extended and facilitated by incorporating a water insoluble polyvinylacetate resin, AYAF (Union Carbide Co.). A combination of 7.5% resin added by heating to a 3:1 mixture of polyethylene glycols 1540 and 4000 gives blocks which may be cut at 2-3 μ. Sections can be floated and properly expanded on an ordinary water bath in a manner which may be impossible with Carbowax alone because of section fragility. This may require judicious adjustment of surface tension by the prior addition of minute quantities of the wax. On water, polyethylene glycol dissolves out of tissues, which remain supported by the resin. After attachment to albumen-coated slides, residual resin may, at option, be removed by a 1-2 min immersion in methyl alcohol without visible impairment of fat content. Abopon is used for mounting. The method appears suitable for the study of intracellular lipids, particularly in tissues which cannot be conveniently handled after Carbowax alone.     

Method of combined detection of nerve fibers and blood vessels in nerve trunks]

A new method for investigation of neuro-vasal relationships in nerve conductors is proposed based on a combination of the fine injecting of their blood vessels and staining the nerve fibres. The vessels are injected with the chloroform emulsion of finely ground Paris blue (5-10 g per 100 g of solvent). Pieces of nerves 0,5 cm thick are fixed in 12% neutral formalin for 3 days, kept in a dark vessel in Weigert's mortant for 5 days, dehydrated and imbedded in paraffin. Thin slices 4-5 micronkm thick are stained in hematoxylin after N. N. Kulchitski, differentiated for 2-12 h in the mixture containing 1% solution of potassium ferricyanide and saturated solution of lithium carbonate (1:10) and after passing through alcohols of increasing concentration and xylene imbedded in balsam. In the preparations the fibres of different caliber are stained grey and the vessels are stained blue.     

Staining Procedures for Ultrathin Sections of Tissues Embedded in Polyester Resin

Tissues were fixed for 30 min In cold (0-2° C) 1% OsO₄ (Palade) buffered at pH 7.7, to which 0.1% MgCl₂ was added. Dehydration was in a graded ethanol series (containing 0.5% MgCl₂) at 0-2° C, and terminated with 2 changes of absolute ethanol. Tissues were then transferred by a graded series to anhydrous acetone. Infiltration of the tissue with Vestopal-W (a polyester resin), is gradual with the aid of graded solutions of Vestopal-W in acetone. The infiltrated tissue is encapsulated and initial polymerization is done under ultraviolet light at room temperature for 8-16 hr. This is followed by final hardening at 60° C for 36-48 hr. Sections (0.2-1 μ) were cut, dried on slides, placed in acetone for 1 min and then treated by either of the following staining procedures: (1) Thionin-azure-fuchsin staining: Flood the preparation with 0.2% aqueous thionin and heat to 60-80° C for 3 min; if the preparation begins to dry, add stain. Rinse in distilled water. Flood the slide with 0.2% azure B in phosphate buffer at pH 9. Heat to 60-80° C for 3 min; do not permit the preparation to dry. Rinse in distilled water. Dip the slide in MacCallum's variant of Goodpasture's carbol-fuchsin stain for 1-2 sec. Rinse in distilled water. Check the preparation microscopically for intensity of the fuchsin stain. Repeat dips as may be needed to obtain the desired intensity. Rinse in distilled water. Dehydrate quickly in 95% and absolute alcohol; clear in 2 changes of xylene and cover in Permount or similar synthetic resin. (2) Thionin-azure counterstain for the periodic acid-Schiff reaction: Oxidize the tissue in 0.5% periodic acid for 15 min and transfer to Schiff's leucofuchsin solution for 30 min. Counterstain with 0.5% aqueous thionin for 3 min; wash in distilled water; stain in 0.2% azure B in phosphate buffer at pH 5.5; wash in distilled water; dehydrate; clear and cover as in the first method. For temporary preparations let dry after absolute alcohol and apply a drop of immersion oil directly on the section.     

The Use of Picric Acid with the Gram Stain in Plant Cytology

The technic described involves the use of a saturated solution of picric acid in absolute alcohol in the process of dehydration following the gentian-violet-iodine stain as applied to plant cytological material. The method is suitable for both paraffin sections and smears of pollen mother cells fixed in Navashin's or Flemming's solutions. Differentiation in clove oil is very easy since cytoplasm destains immediately, while chromatic material destains very slowly following picric acid. Chromosomes are stained more distinctly than with the usual Gram stain and do not fade.