A Tissue Basket for Paraffin Infiltration

The processes of washing, dehydration and paraffin infiltration when large numbers of tissues must be individually identified are always laborious. Washing and dehydration can be carried out in individual glass vials with relative ease and fairly rapidly if gravity flow reagent bottles are used to fill the vials. The use of similar vials for paraffin infiltration is usually complicated by hardening of the paraffin if too many vials are removed from the oven at once, or by cooling of the oven itself if it is too frequently opened. The same criticism applies to the process of embedding tissues when large numbers of vials must be handled simultaneously.     

植物材料快速石蜡制片方法

真空干燥箱已越来越广泛地应用于现代生物学研究领域。该文利用真空干燥箱温度和负压的可控制性能,将固定、脱水、透明和石蜡渗透等过程在真空干燥箱中进行,建立起一套可行的植物组织快速石蜡制片方法。结果显示,真空干燥箱的应用加速了多种试剂的渗透速率,提高了切片质量,达到了优化实验步骤、节省实验时间和减少室内有毒化学气体污染的目的。     

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.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

N-Butyl methacrylate and paraffin as an embedding medium for light microscopy.

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18--24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Isopropyl Alcohol in the Paraffin Infiltration Technic

A method is described for using isopropyl alcohol for dehydration of animal tissues preceding melted paraffin infiltration. Advantages of the technic are: simplicity, low cost, low toxicity, and diminished distortion and hardening of the tissues.     

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.     

An Ice-Solvent Method of Drying Frozen Tissue for Plant Cytology

A freeze-dry method where cold absolute ethanol is used as a dehydrating agent in place of vacuum dehydration has been applied to various plant materials with good cytological results. The method involves: (a) freezing rapidly small pieces of tissue 1 cubic mm or less in partly frozen isopentane cooled with liquid nitrogen, (b) transferring quickly to vials of cold absolute ethanol at -41° to -45°C, and (c) holding within this temperature range for 3 days to dissolve the ice. A simply constructed cryostat is used to maintain the vials of absolute alcohol and tissue at the cold temperature. This consists of a semi-frozen constant temperature bath of either 65% ethanol or pure diethyl oxalate in a tightly covered beaker which fits within a large dewar flask half filled with dry ice. The bath is arranged so that it will be on top of and in contact with the dry ice but properly insulated to prevent freezing completely.

The resulting dried tissue is very unstable in either water or hot absolute ethanol; therefore, to prevent loss of cytological detail during further processing, the tissue must be treated to render the proteins insoluble. Either (a) replace the cold absolute ethanol in the tissue vials with cold (approx. -40°C) 75% ethanol, warm slowly to 60°C, and hold for 1 hour, or (b) replace with cold acidulated 95% ethanol (100 ml. of 95% ethanol + 0.30 ml. of glacial acetic acid), warm to room temperature, and hold for 30 minutes. Following either treatment the tissues are dehydrated to absolute alcohol and embedded in paraffin by the usual technics. Sections are attached to slides by flattening over warm water and drying.

When epidermis from onion bulbs was used as a basis of comparison of fixed and living material with the phase-contrast microscope, the mitochondria, plastids, and other fine structures in fixed preparations appear to be nearly identical with the living. Fat droplets disappear. With larger tissues such as onion root tips, thin freehand sections must be prepared before freezing to obtain good cytological results. The application of the method to cytochemical studies is discussed and in many ways it seems to be as useful as the freeze vacuum-dry method.     

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 Comparative Study of some Dehydration and Clearing Agents

An experiment to determine the advantages of diozan, iso-butyl alcohol, tertiary butyl alcohol, and ethyl alcohol as dehydrants and chloroform, toluol, xylene, benzol, methyl benzoate, methyl salicylate, and acetone as clearers is described. Materials fixed in Bouin's fluid, Zenker formol, and 10% neutral formalin were dehydrated, embedded, sectioned, and stained. Bouin's fluid produces less hardening, shrinkage and distortion than the other fixatives employed. Slow dioxan is the best method of dehydration. All the picric acid need not be removed from tissues to be embedded in paraffin. Tissue blocks not more than 4 mm. thick may be dehydrated and impregnated with paraffin by slow dioxan in 13 hours, fast dioxan in 10 hours, iso-butyl alcohol and tertiary butyl alcohol in 14 hours, and ethyl alcohol-chloroform in 17 hours without incurring any distortion due to rapidity of dehydration and infiltration.     

Birefringence and Paraffinophilia of Cell Nuclei

Birefringence of cell nuclei was present in most tissues but seen exclusively in paraffin sections. Only after staining with an acridinc derivative (rivanol) was it found in smears and frozen sections. Although retention of paraffin in the nucleus contributes, for the most part to its anisotropy, present evidence supports the hypothesis that the chemical nature and the physical state of nuclear material, especially of the DNA, plays the important role as a substrate, which selectively binds of paraffin molecules. This evidence is based mainly on the blocking effect on birefringence which occurs when small pieces of tissues are treated in toto, before paraffin embedding, with DNA-extracting procedures and nuclear stainings. Moreover, the variability in degree and extent of birefringence noted in different tissues corroborates this view. Factors in the preparatory procedure, i.e, deparaffinization, hydration and dehydration, were found to affect markedly the binding of paraffin to nuclear substance. Nevertheless, if paraffin affinity for nuclei is not considered, it may introduce inaccuracies into methods designed to determine the nuclear mass quantitatively, after staining.     

Differentiation of Myofibrillae, Reticular and Collagenous Fibrils in Vertebrates

A procedure for the differentiation of the mesenchymal derivatives, myofibrillae, reticular and collagenous fibers is presented. Formol-Zenker fixation (5-12 hours) is followed by the washing, iodinization, dehydration and paraffin embedding steps routine for that fixative with the following modifications. Zirkle's butyl alcohol series is used for dehydration and infiltration with paraffin as well as in the alcohol slide series. Embedding paraffin used is Parawax plus 8-10% bayberry wax. Tissue-exposed surface of paraffin block is soaked in water overnight before cutting serial sections at 3-5μ. Sections are mounted using the dilute albumen method, and the slides, thoroughly dried at 37^oC. overnight, are left at 60^o for 10 minutes to melt the paraffin of the sections. Before staining, the sections are given a preliminary treatment with potassium permanganate and oxalic acid. For reticular staining a 10% silver nitrate bath is succeeded by an ammoniacal silver carbonate solution followed by reduction in 1% neutral formalin, toning in gold chloride and fixing in sodium thiosulphate. Myofibrillae, the sacroplasmic limiting membrane and other sarcous elements are stained by Heidenhain's azocarmine solution, adult tissues at room temperature and fetal tissues at 50 ^oC. Differentiation in phosphotungstic acid is followed by the staining of collagenous fibers. For adult tissue, light green SF (C.C.) is used and for fetal tissue, fast green FCF (C.C). A discussion of the preparation of ammoniacal silver solutions is included. Both stock and used solutions of ammoniacal silver have been in use by the author for over a period of two years.     

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.     

Alcohol-Xylene Versus Dioxane in the Shrinkage of Tissue

A direct measure of tissue volume changes using fluid displacement was employed to compare the effects of the ethyl alcohol-xylene with the dioxane method of tissue processing. Pieces of mouse kidney, spleen, testis and liver were compared after fixation in 10% neutral formalin and during clearing, dehydration and paraffin infiltration. It was found that 24 hr after fixation there was a 25% increase in volume. There was a progressive shrinkage during dehydration and clearing with dioxane to the volume of the tissue before fixation and a subsequent shrinkage of about 20% during paraffin infiltration. With the ethyl alcoholxylene method, tissue volumes returned to initial levels during alcohol dehydration, and progressively shrank with xylene treatment and paraffin infiltration. The final degree of shrinkage was about the same with both methods. This was confirmed from microscopic analysis of tissue components. It is concluded that one cannot use gross tissue shrinkage as the only criterion for selecting one method of tissue processing over another.     

桦树松萝的石蜡切片方法改良及形态学研究

以传统石蜡切片方法为基础,在固定、软化、包埋、染色等具体方法上进行了适合松萝属植物特点的改良。结果表明:采用改良制片方法获得了染色清晰、组织完整的桦树松萝切片。桦树松萝地衣体横切面结构从外到内依次划分为皮层、藻层、髓层和中轴;皮层是由横向分裂的菌丝交织成类似于高等植物的假厚壁组织,藻层由大量共生藻的藻细胞和菌丝所组成;髓层由疏松的菌丝体组成;中轴由致密的菌丝体组成。     

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.     

Studies in lipid histochemistry

Summary A considerable portion of polar lipids survives the routine dehydration procedure for paraffin embedding with ethanol, acetone and xylene and can be detected in dehydrated blocks of tissue. Sphingomyelin, cerebrosides, sulphatides and gangliosides can be demonstrated with appropriate histochemical methods and chromatographically even in ordinary paraffin sections especially when the amount of these lipids in tissues is sufficiently high, e.g. in lipidoses and in normal myelin. In blocks of tissue dehydrated with acetone and cleared with benzen a considerably higher amount of polar lipids is present. Factors governing the preservation of polar lipids in paraffin sections are discussed.     

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.     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Rapid fixation and embedding for electron microscopy

A method has been developed for rapid processing of animal tissues for electron microscopy. The whole process of fixation staining dehydration, infiltration and embedding including polymerization is completed in less than 4 hr. A variety of human and animal tissues such as liver, spleen, muscle, kidney and embryonic chick heart were processed by this method and the results were excellent. The rapid fixation and embedding method is strongly recommended when relatively soft tissues are to be studied. This method is especially useful for examining pathological tissues for rapid diagnostic purposes.