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 A summary is given of the unpublished experiences of other workers with a variety of both plant and animal tissues.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.     

Simple Aids to Microscope Illumination

The following rapid but reliable method of making permanent preparations from temporary mounts has proved to be very useful.

Pollen mother-cell smears: Smeared anthers are treated hi the usual way with Belting's acetocarmine, except that the cover slip is left off. When correct differentiation is attained the stain is thoroly washed off with 50% acetic acid and the slide flooded with dioxan. This is followed by 2 changes of dioxan for 2 minutes each. A drop of Canada balsam dissolved in dioxan is added and a cover slip applied. In cases where a cover slip has been used at the acetocarmine stage it can be floated off in a staining jar of 50% acetic acid and dehydration with dioxan carried out as above.

Insect salivary gland chromosome smears: The glands are crushed under a cover slip in acetocarmine on a slide coated with dried egg albumen. After 20 minutes the area around the cover slip is flooded with 50% acetic acid and the cover slip floats loose so that it can be removed. The above described dioxan dehydrating procedure is then employed.

Squash preparations: Root tips are fixed in some suitable fixative and the Feulgen technic applied. The stained root tips can either be dehydrated by passing thru 3 changes of dioxan and mounting in dioxan-balsam where they are divided into small longitudinal sections by sharp needles, or they can be put immediately into a mixture of 1 part of 50% acetic acid to 1 part of corn syrup where shredding with needles is carried out. A cover slip is put on and separation of the cells completed by tamping or by applying pressure to the cover. This squash method is useful with anthers which are difficult to smear when in the early prophase stages of meiosis.     

Permanent Preparations from Rapid Cytological Technics

The following rapid but reliable method of making permanent preparations from temporary mounts has proved to be very useful.

Pollen mother-cell smears: Smeared anthers are treated hi the usual way with Belting's acetocarmine, except that the cover slip is left off. When correct differentiation is attained the stain is thoroly washed off with 50% acetic acid and the slide flooded with dioxan. This is followed by 2 changes of dioxan for 2 minutes each. A drop of Canada balsam dissolved in dioxan is added and a cover slip applied. In cases where a cover slip has been used at the acetocarmine stage it can be floated off in a staining jar of 50% acetic acid and dehydration with dioxan carried out as above.

Insect salivary gland chromosome smears: The glands are crushed under a cover slip in acetocarmine on a slide coated with dried egg albumen. After 20 minutes the area around the cover slip is flooded with 50% acetic acid and the cover slip floats loose so that it can be removed. The above described dioxan dehydrating procedure is then employed.

Squash preparations: Root tips are fixed in some suitable fixative and the Feulgen technic applied. The stained root tips can either be dehydrated by passing thru 3 changes of dioxan and mounting in dioxan-balsam where they are divided into small longitudinal sections by sharp needles, or they can be put immediately into a mixture of 1 part of 50% acetic acid to 1 part of corn syrup where shredding with needles is carried out. A cover slip is put on and separation of the cells completed by tamping or by applying pressure to the cover. This squash method is useful with anthers which are difficult to smear when in the early prophase stages of meiosis.     

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.     

不同染色条件对淡黄绒泡菌Physarum melleum菌核形成过程的差异显示

为了了解非细胞黏菌菌核形成过程中原质团组分及形态的变化,探索在番红-固绿二重染色、番红-焦油紫-橙黄G三重染色条件下,淡黄绒泡菌Physarum melleum菌核形成过程中原质团形态及显微结构变化的显示差异。结果表明：三重染色法可以显示原质团中纤维结构及细胞核,并且菌核随着休眠胞成熟而对焦油紫具有更强的亲和力。     

A novel staining method for quantification and 3D visualisation of capillaries and muscle fibres

The aim of this study was to introduce a combined fluorescent staining that clearly demonstrates capillaries and distinguishes them from the basal lamina of muscle fibres in skeletal muscle tissue. The triple staining with CD31, Griffonia (Bandeira) simplicifolia lectin (GSL I) and laminin efficiently distinguishes vascular endothelium from the basal lamina of skeletal muscle fibres in physiological and pathological conditions. The presented triple staining method has several advantages, which facilitate quantitative analysis of the capillary network, and its relation to individual muscle fibres.     

Adaptation of the electro-olfactogram in the frog

The recovery of the electro-olfactogram in the frog after adaptationwas investigated with different odorous substances (meta-xylene,toluene and dioxan) and with different concentrations of thesame substance (dioxan). Adapting and test stimuli both hadthe same intensity. The results can be summarized as follows: 1. There was a negative relationship between stimulus intensityand recovery rate. 2. The olfactory receptors were very resistant to adaptation;despite the strong stimulus concentrations we found for meta-xyleneand toluene that the recovery was almost complete after 25 sec.The recovery after adaptation to dioxan proceeded somewhat slower. 3. There was a negative relationship between the recovery rateand the duration of rising phase and decay of the EOG. ^*Present address: Department of Psychology, Physiological PsychologySection, Tilburg University, Tilburg, The Netherlands     

The beta-glucosidase from Botryodiplodia theobromae Pat. Kinetics of enzyme-catalysed hydrolysis of o-nitrophenyl beta-D-glucopyranoside in dioxan/water.

1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Botryodiplodia theobromae Pat. has been studied in the presence of added dioxan. 2. At donor saturation, the maximum rate of hydrolysis in the presence of up to 50%(v/v) dioxan was pH4.3-4.5 (pH of the buffer system in water) in McIlvaine's buffer. 3. Increasing dioxan concentrations progressively decreased the maximum rate of hydrolysis. 4. The rate of enzyme-catalysed reaction was enhanced at high donor concentrations, but inhibited at low donor concentrations in the presence of glycerol, methanol, fructose of sucrose. 5. The hydrolytic reaction was found to proceed with retention of configuration at the anomeric carbon atom. 6. The kinetics of the enzyme-catalysed process in the presence of added acceptors indicated that water was necessary for the maintenance of the active enzyme conformation apart from its acceptor function.     

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.     

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.     

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.     

Discriminative Staining Methods for the Nervous System: Luxol Fast Blue-Periodic Acid-Schiff- Hematoxylin Triple Stain and Subsidiary Staining Methods

This paper describes a new series of staining methods which can discrimina-tively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.     

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.     

Staining Paraffin-Embedded Plant Tissues with Acridine Orange

Stem, leaf, and bud tissue of sweet potato, tomato, and pepper were embedded in paraffin, sectioned, mounted, and stained with 0.01, 0.1 and 1% aqueous and 0.1% alcoholic solutions of acridine orange. Temporary and durable mounts were prepared and irradiated under short and long wave ultraviolet light. Intensity and specificity of the fluorescence imparted to tissues were chiefly affected by type of fixative. Best results were obtained with fixatives containing formalin but not acetic acid. Tests on the effect of pH obtained with McIlvaine's buffer between 4.5 and 8.3, and made only with the aqueous stain, showed 6-8 to be optimal. Aqueous staining 1 hr in 0.1% solution, pH 6-8 is recommended for temporary mounts. Durable mounts in a nonfluorescent resin can be made after differentiation in buffer and dehydration in dioxan solutions.     

Surface areas of 1-palmitoyl phosphatidylcholines and their interactions with cholesterol.

The activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. was affected by added NaCl in such a way that an initial phase of stimulation was followed by a phase of rapid non-linear decrease in velocity and finally by a phase of slow linear decrease in velocity as the concentration of NaCl was increased. In the presence of 0.014 M-sodium acetate/acetic acid buffer (pH 5.0) there was a slight increase in enzymic activity in the presence of low concentrations of dioxan (up to about 10% dioxan) and a rapid decrease in enzymic activity at higher dioxan concentrations, but both effects were mitigated in the presence of 0.1 M buffer. The order of efficiency of added glucosyl acceptors in beta-glucosidase-catalysed reactions was found to be fructose greater than sucrose greater than glycerol greater than methanol. The enzyme was inactivated by the active-site-directed compound conduritol-B-epoxide; but this inactivation was concentration-dependent, was prevented by 10 mM-glucose, and involved an acidic group with pKa 4.3. A rate equation has been derived on the assumption of a mechanism of action involving a solvent-separated and an intimate glucosyl cation-carboxylate ion-pair intermediate and an alpha-glucosyl enzyme intermediate [Umezurike, G. M. (1981) Biochem. J. 199, 203-209]. Calculations based on the application of the derived rate equation and the calculated kinetic parameters show that the rate equation explains the peculiar properties of beta-glucosidase in the presence of added glucosyl acceptors or of NaCl.     

Curtis' Substitute for Van Gieson Stain

A triple staining method is described in which nuclear staining is by Weigert's hematoxylin. The cytoplasmic and collagen staining is effected by the Curtis substitute for Van Gieson, in which ponceau S is substituted for acid fuchsin. Nuclear staining is sharper than with Delafield's hematoxylin. The red of the collagen fibers is probably not subject to fading. Unlike Van Gieson, this method gives staining of reticular as well as collagen fibers. The advantages of the method are its simplicity and reliability. The use of this method is made possible by a new source of reliable samples of the ponceau S called for in this method.     

Use of triple stain technique for simultaneous assessment of vitality and acrosomal status in boar spermatozoa

The object of this study was to adapt the triple stain technique to diluted and incubated boar spermatozoa. Freshly ejaculated semen was resuspended in MR-A diluent to contain 3x10(7) cells/ml (diluted spermatozoa) and was subsequently capacitated (incubated spermatozoa). Experiments were conducted to show the conditions required for optimal staining quality and validation of triple stain technique. The most satisfactory staining solutions for diluted spermatozoa were 2% Trypan blue at 37 degrees C for 15 minutes, 0.8% Bismarck brown in 30% ethyl alcohol (pH 2.8) at 40 degrees C for 10 minutes and 0.8% rose Bengal in 0.1 M of Tris (pH 4.3) at 21 degrees C for 20 minutes. Satisfactory results were obtained for incubated spermatozoa with rose Bengal when the staining time was 10 minutes. Triple stain technique seemed to be a useful method for the simultaneous assessment of sperm vitality and acrosomal status; consequently, it should be valuable tool, for use in porcine in vitro fertilization systems.     

A new triple crossover triangle (TXT) motif for DNA self-assembly

A new triple crossover triangle (TXT) motif was conceived on the basis of the DNA triple crossover (TX) motif. The new motif is rigid and triangular prism-shaped, out of which 1-D, 2-D, and 3-D DNA structures can be assembled. The 1-D TXT array was self-assembled and was observed by the TEM with negative staining by uranyl acetate. The architectural schemes for 2-D and 3-D structures are presented.     

Triple staining of brain tissue for neuronal classification.

The described triple staining (PAP, aldehydefuchsin, and cresyl violet) enables the distinction of different neuronal types with regard to their content of neurotransmitters or neuropeptides and their lipofuscin pigmentation respectively. The use of filters improves the contrast of immunohistochemical and lipofuscin staining, and facilitates the differentiation of neurons under the microscope. An additional important point is the advantage that perikaria can be measured in the usual morphometric manner.     

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.