Comparative Study of Dehydration

The paraffin method has frequently been criticised because of its hardening and shrinking effect on tissue. The author believes this distortion is due to the dehydration and not to the immersion in melted paraffin. An experimentally controlled series of various tissues was dehydrated in different dehydrating reagents, dioxan, isobutyl alcohol, and ethyl alcohol with chloroform. Except for the dehydration, the tissues were treated identically. In every case, dioxan proved to be a better dehydrating reagent with less shrinkage and brittleness than any of the others. Ethyl alcohol with chloroform produced the greatest degree of distortion.     

Effect on Tissue Volume of Various Methods of Fixation, Dehydration, and Embedding

A new indirect method is described for following volume changes of homogeneous pieces of tissue during fixation, dehydration and embedding, and area changes during sectioning, staining and mounting. Pieces of rabbit kidney cortex were compared after fixation in Destin's, Orth's, Petrunkevitch's cupric-paranitrophenol, Bouin's, SUSA, Zenker-formol, 10% formalin in distilled water, formalin in saline, Burke's pyridine formalin, CaCOy neutralized formalin, MgCO₃-neutralized formalin, Bensley's vacuum distilled neutral formalin in distilled water, and Bensley's neutral formalin in saline; during dehydration in ethyl alcohol, dioxan, and tertiary butyl alcohol and clearing in xylol and chloroform; and after infiltration and embedding with parowax, with paraffin-nitrocellulose double embedding technic, with hot nitrocellulose, and with cold nitrocellulose. The H-ion concentration of these fixatives was followed during tissue fixation.

Altho all fixatives showed specific merences, SUSA and Bouin's gave the best general results and neutral formalin mixtures, especially pyridine-formalin, the poorest. Isotonic saline was found superior to distilled water as a formalin diluent, reducing tissue swelling during formalin fixation. Reagents producing marked decreases in tissue volume render such tissues less susceptible to shrinkage during subsequent procedures. Shrinkage of tissue during dehydration and infiltration with hot parffin may exceed that produced by fixation alone. Excessive heat causes tissue distortion and shrinkage. Inijltration with hot paran causes considerable shrinkage, with hot nitrocellulose Iess, and with cold nitrocellulose the least sbrinkage.     

A Rapid Combined Fixing and Staining Method for Plant Chromosome Counts

A new method for rapidly preparing slides suitable for chromosome counts by the use of a combined fixing and staining solution involves the substitution of anthraquinone for picric acid in Bouin's formula and the addition of alizarin red S with a metallic salt as a mordant. The fixed smears, after being dehydrated to 95% alcohol, are differentiated in 0.5% sulfuric acid in 95% alcohol saturated with picric acid, washed, cleared and mounted in xylol-balsam. Cymene may be used to intensify the stain. Root tips fixed in the above solution may be dehydrated in dioxan, a paraffin solvent; infiltrated, embedded, sectioned and mounted in the usual way. The sections are subsequently differentiated in picro-sulfuric acid alcohol and cymene. An alternative method of differentiation for this stain is also described.     

An Ammoniated Silver Carbonate Technic for Nervous Structures in Mounted Paraffin Sections

Specimens of both vertebrate and invertebrate nerve-containing tissues were fixed 2-3 days in Bouin's fluid, soaked 2 days in alcohol containing 2% strong ammonia water, dehydrated and embedded in paraffin. The sections were mounted with gelatin adhesive according to Masson's procedure, dewaxed, passed through graded alcohols to water, then back to 2% ammoniated 80% alcohol for 12-24 hours. The slides were rinsed 3-5 seconds in distilled water, impregnated about one and a half hours in 40% AgNO₃ at increasing temperature up to 45°C. The slides were flooded with 62.5% formalin and this solution allowed to remain 3-5 minutes; they were then blotted with filter paper. A second impregnation in ammoniated silver carbonate, controlled under the microscope, was followed by a 10-minute treatment with 10% aqueous acetic acid, toning with gold chloride, then thiosulfate and finally washing. Counterstaining with ponceau red or acid fuchsin, eventually followed by aniline blue or fast green, dehydration and covering, completed the process.     

Experimental Cyanine Red: A New Stain for Nucleic Acids and Acid Mucopolysaccharides

A new cationic dye, experimental cyanine red (du Pont), with an absorption maximum of 536 mμ and a pH of 2.9 in 0.5% aqueous solution, is shown to be suitable for staining nucleic acids and tissue materials presumed to contain acid mucopolysaccharides. Mammalian tissues fixed in 10% neutral buffered formalin or Bouin's fluid are dehydrated, embedded in paraffin, sectioned, mounted, deparaffinized, passed through ethanols to water, and stained for 3-30 min in 0.5% experimental cyanine red in water. Differentiation and dehydration in 3 changes (about 1 min each) of n-butanol is followed by clearing in xylene and mounting in resin.     

Isopropyl-Alcohol-Paraffin Methods for Plant Tissues

Two methods using isopropyl alcohol for dehydration prior to paraffin infiltration of plant tissues are reported: (1) a modified Rawlins-Takahashi (1947) schedule in which the preliminary dehydration is effected by concentrating glycerol and (2) isopropyl alcohol as the sole agent for dehydration. With the latter method the fixed tissues are dehydrated successfully in 60%, 85%, and 99% isopropyl alcohol. Paraffin infiltration is accomplished by placing the tissues in isopropyl alcohol over solid paraffin in a vial and heating to 56°-58° C. The tissues settle into the melted paraffin as infiltration progresses. Several changes of pure paraffin are then made, with the last change under reduced pressure. The embedded tissues are trimmed and soaked 2-4 hours at 40° C. in either water or a glycerol, acetic acid, 70% alcohol mixture (10:15:75) to reduce static and insure uniform ribboning during microtomy.     

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.     

Possible uses of Dioxan in Botanical Microtechnic

Dioxan has been well established as an advantageous dehydrating agent for plant tissues. It dehydrates equally well after fixatives containing formalin, acetic acid, chromic acid, chromates, mercuric chloride, osmic acid, and alcohol. Better infiltration of paraffin after dehydration may be obtained by passing the material thru (1) a cold bath composed of 30 cc. of dioxan, 5 cc. of xylol and 20 cc. of melted soft paraffin and, (2) a warm bath of 50 cc. of dioxan, 50 cc. of paraffin, and 10 cc. of xylol. Transfer from (2) to soft paraffin. A dioxan fixative consisting of dioxan 50 cc., formalin 6 cc., acetic acid 5 cc., water 50 cc. was devised for delicate subjects. The fixed material is transferred directly into dioxan and mounted in dioxan-diaphane or dioxan-balsam. Very delicate objects require dioxan dilution of the balsam and slow concentration of the mounting medium by evaporation.

Entire plant parts or epidermal peelings are fixed in any desired fixative, washed if necessary, transferred to dioxan and mounted in diluted dioxan-balsam or diaphane. Dioxan may be used to mount hyalin objects whose refractive indexes approach those of balsam in media of higher index than balsam. It may be used in place of alcohol in finishing parafin sections, and since it exhibits different stain solubilities than alcohol it offers an important new tool in obtaining and maintaining stain balances.     

An Arginine Histochemical Method Using Sakaguchi's New Reagent

A mounted paraffin section of material fixed in Bouin's, Carnoy's or 10% formalin is allowed to stand 15 minutes at room temperature in a 0.3% solution of 8-hydroxyquinoline in 30% ethanol. The slide, with adhering solution, is placed in 0.15 N hypochlorite (with enough KOH added to make the solution 0.015 N KOH) for 60 seconds, then (without draining) into a solution containing: 10 ml. of 0.15 N KOH; 15 g. of urea; 70 ml. of tertiary butyl alcohol, and water to make 100 ml. Here it is gently agitated for 10 sec. and then kept in a second change of the same solution for 2 min. Two changes of pure tertiary butyl alcohol, 10 sec. and 4 min.; one in aniline, 3 min.; and one of 10 sec. in xylene, complete the procedure. Permount containing 0.02% aniline is used as a mounting medium.     

The Use of N-Butyl Alcohol in the Paraffin Method

Several modifications in the use of n-butyl alcohol are suggested. These modifications include a revised series of dehydration solutions for exacting work, an abbreviated schedule of limited usefulness, and a simple method for more rapid paraffin infiltration. The use of a triangular coordinate graph may be valuable in designing dehydration procedures for special purposes. Changes in the primary fixation image are significantly less severe by dehydration with butyl alcohol than with many other reagents. Such deleterious effects may be further minimized by reducing the time and temperature factors to the practical limit and by substituting acetone for ethyl alcohol in a dehydration series such as that of Zirkle.     

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.     

The Use of N-Butyl Alcohol in the Paraffin Method

Several modifications in the use of n-butyl alcohol are suggested. These modifications include a revised series of dehydration solutions for exacting work, an abbreviated schedule of limited usefulness, and a simple method for more rapid paraffin infiltration. The use of a triangular coordinate graph may be valuable in designing dehydration procedures for special purposes. Changes in the primary fixation image are significantly less severe by dehydration with butyl alcohol than with many other reagents. Such deleterious effects may be further minimized by reducing the time and temperature factors to the practical limit and by substituting acetone for ethyl alcohol in a dehydration series such as that of Zirkle.     

Elimination OF Distortion and Poor Staining in Paraffin Sections OF Plant Tissues

Three fixing solutions causing least distortion and bright staining of plant tissues are named. Glycerin dehydration causes less distortion than a series of alcohol concentrations; 95% alcohol removes some of the glycerin, sets the protoplasm and improves the staining. Absolute alcohol causes distortion and should be avoided. Pure chloroform, as a paraffin solvent, is followed by brighter staining but more distortion than are the butyl alcohols. A schedule resulting in minimum distortion is given. The results are shown in photomicrographs. Brightest staining follows the use of C. P. iron alum and hematoxylin. The use of a paper cup for very gradual change from one liquid to another and as a labor saver is described.     

The New Domestic Cresyl Echt Violet

Specimens of both vertebrate and invertebrate nerve-containing tissues were fixed 2-3 days in Bouin's fluid, soaked 2 days in alcohol containing 2% strong ammonia water, dehydrated and embedded in paraffin. The sections were mounted with gelatin adhesive according to Masson's procedure, dewaxed, passed through graded alcohols to water, then back to 2% ammoniated 80% alcohol for 12-24 hours. The slides were rinsed 3-5 seconds in distilled water, impregnated about one and a half hours in 40% AgNO₃ at increasing temperature up to 45°C. The slides were flooded with 62.5% formalin and this solution allowed to remain 3-5 minutes; they were then blotted with filter paper. A second impregnation in ammoniated silver carbonate, controlled under the microscope, was followed by a 10-minute treatment with 10% aqueous acetic acid, toning with gold chloride, then thiosulfate and finally washing. Counterstaining with ponceau red or acid fuchsin, eventually followed by aniline blue or fast green, dehydration and covering, completed the process.     

A Rapid Method for Preparing Paraffin Serial Sections of Teeth and Their Supporting Structures in the Dog

A method of preparing dogs' teeth for serial sectioning involving the following steps is described. The tissues are fixed in 10% formalin followed by partial dehydration in 80% iso-propyl alcohol; decalcified in 5% nitric acid in 10% formalin; washed for 5-8 hours in running water and neutralized in 5% aqueous sodium sulfate for 24 hours, followed by further washing in running water for another 24 hours. They are then dehydrated over a period of 4 days in increasing grades of iso-propyl alcohol and embedded in tissue-mat in sub-atmospheric pressure of 10 lb. Sections so obtained were better suited for cytological study than celloidin preparations, and serial sections could be obtained in a much shorter time.     

Pasternack's Paraffin Method Modified for Plant Tissue

This method for preparing paraffin sections of plant material is a modification of Pasternack's one-hour method for animal tissues. Fixation in Randolph's CRAF fixative is hastened by heat and increased vapor pressure obtained by the use of screw top vials. Dehydration with Zirkle's butyl alcohol series likewise is hastened in the same manner. The rapid penetration of paraffin by the use of 1/2 paraffin and 1/2 butyl alcohol in heated screw top vials shortens the embedding process. Sections are held on the slide thru staining by albumen fixative and a coating of 0.2% celloidin in absolute alcohol and ether. Good penetration with freedom from shrinkage or distortion is obtained and root tip chromosome counts can be made in approximately 3 hours.     

A Paraffin Method for Refractory Plant Materials

A modification of Zirkle's n-butyl alcohol method for dehydrating refractory plant material and embedding it in paraffin is described. The material is cut into small pieces and killed and fixed in CRAF III. It is dehydrated in Zirkle's n-butyl alcohol series, slightly modified. Infiltration is accomplished by adding chips of paraffin of low melting point (52°C.) to the bottle containing butyl alcohol and the tissues at a gradually increasing series of temperatures. The butyl-alcohol-paraffin mixture is gradually replaced by pure paraffin (melting point 56-58°C). The material is embedded in Fisher Tissuemat (56-58°C). Before microtoming, the block of embedded tissue is trimmed so that part of the specimen is exposed, and it is soaked in water until it cuts easily.     

Pollen size in Carex: The effect of different chemical treatments and mounting media

Pollen size and pollen aperture size for ten species of the genus Carex L., native to Estonia, have been measured using light microscopy. The species selected represent different sections of the genus, a range of habitats and different chromosome numbers. The effects of two basic chemical treatments, two mounting media and the effect of chemically induced dehydration with tertiary butyl alcohol (TBA) on the size of pollen grains were then recorded.

In general pollen size and pollen aperture size of the species examined is highly variable at both intraspecific and interspecific levels. Carex hirta has notably larger pollen grains than any of the other species investigated and, although correlations between size and chromosome number in the species examined are limited, it also has the highest chromosome number. Statistically significant size differences resulted from variations in chemical treatment, mounting media and tertiary butyl alcohol (TBA) induced dehydration. Acetolysed pollen grains are larger than potassium hydroxide (KOH) treated pollen grains. Pollen grains dehydrated after chemical treatment with TBA are larger than pollen grains not dehydrated. Pollen grains mounted in silicon oil are smaller than grains mounted in glycerine. But considering the great size variation of Carex pollen grains, the size changes caused by preparation procedures fall within the size variation range of the species examined.

All the samples contained a high number of deformed pollen grains and pollen grains with hardly distinguishable or no lateral apertures.     

Laboratory hints from the Literature: Department Devoted to Abstracts of books and papers from other Journals Dealing with stains and Microscopic Technic in General

A modification of Zirkle's n-butyl alcohol method for dehydrating refractory plant material and embedding it in paraffin is described. The material is cut into small pieces and killed and fixed in CRAF III. It is dehydrated in Zirkle's n-butyl alcohol series, slightly modified. Infiltration is accomplished by adding chips of paraffin of low melting point (52°C.) to the bottle containing butyl alcohol and the tissues at a gradually increasing series of temperatures. The butyl-alcohol-paraffin mixture is gradually replaced by pure paraffin (melting point 56-58°C). The material is embedded in Fisher Tissuemat (56-58°C). Before microtoming, the block of embedded tissue is trimmed so that part of the specimen is exposed, and it is soaked in water until it cuts easily.     

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.