A Method for Making Aceto-Carmin Smears Permanent

Anthers are collected and placed in a solution of 1 part acetic acid to 3 parts of absolute alcohol. The contents of the anther are squeezed out on a slide in a drop of Belling's iron-aceto-carmin solution and a cover glass placed over the drop. Care should be taken to remove all anther walls and flower parts. Heat the slide over an alcohol flame for a second, repeating 4 or 5 times. Place the slide in a petri dish filled with a 10% solution of acetic acid. When the cover glass has risen away from the slide gently remove the cover glass and place in a Coplin jar containing equal parts of alcohol and acetic acid. Likewise, place the slide in this solution. Run both cover and slide thru the following solutions: 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic acid to 9 parts absolute alcohol, absolute alcohol and finally equal parts of absolute alcohol and xylol. Recombine the cover and slide in xylol-balsam directly from this solution.     

A Method for Making Aceto-Carmin Smears Permanent

Anthers are collected and placed in a solution of 1 part acetic acid to 3 parts of absolute alcohol. The contents of the anther are squeezed out on a slide in a drop of Belling's iron-aceto-carmin solution and a cover glass placed over the drop. Care should be taken to remove all anther walls and flower parts. Heat the slide over an alcohol flame for a second, repeating 4 or 5 times. Place the slide in a petri dish filled with a 10% solution of acetic acid. When the cover glass has risen away from the slide gently remove the cover glass and place in a Coplin jar containing equal parts of alcohol and acetic acid. Likewise, place the slide in this solution. Run both cover and slide thru the following solutions: 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic acid to 9 parts absolute alcohol, absolute alcohol and finally equal parts of absolute alcohol and xylol. Recombine the cover and slide in xylol-balsam directly from this solution.     

An Improved Technic for the Examination of Chromosomes in Root Tip Smears

Young, vigorous root tips are fixed in aceto-alcohol (1 part glacial acetic acid, 2 parts absolute alcohol) and are left in the fixative from 24 to 48 hours. If it is desired to store the material, the root tips can be transferred to 80% alcohol and be kept indefinitely. In preparing the smears, the root tips are placed on a slide and are sliced as thinly as possible with a sharp razor blade. Then the slices are smeared on the slide and immediately flooded with aceto-carmin, followed by a cover glass. Using absorbent paper and exerting considerable pressure, the excess aceto-carmin can be removed and the material flattened at the same time. Finally the slide is warmed gently to a point slightly below boiling. By sealing with gum mastic and paraffin, such preparations can be kept from 5 to 10 days.     

A Plastic-Seal Method for Mounting Sections of Ground Bone

If sections of ground bone are first coated with a plastic solution before applying the usual mounting medium and a cover glass, excellent optical differentiation results. The solution consists of 28 g. of Parlodion dissolved in 250 ml. of either butyl or amyl acetate. Bone sections are prepared by grinding to the desired thinness, dehydrated with alcohol, air dried, dipped in the solution, freed of surface bubbles by agitation, and placed on a slide. After allowing the preparation to dry thoroughly, the customary mounting medium and cover glass are applied. The plastic seal prevents the escape of air from the lacuni and canaliculi, preserves the natural differentiation of bone tissue, and also permits it to be viewed with a polarizing microscope.     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

Contributions to semithin sectioning on a conventional rotary microtome.

Procedures for obtaining sections 1 micrometer thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen holder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections 1 micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

Contributions to Semithin Sextioning on A Conventional Rotary Microtome

Procedures for obtaining sections 1 μ thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen bolder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections I micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

A New and Rapid Method for Making Permanent Aceto-Carmin Smears

Smear the pollen mother cells of a single anther from each flower bud on a clean dry slide, using a small scalpel. Flood the slide with Belling's acetocarmin and heat for a second over an alcohol flame. Examine under the microscope to determine the stage of microsporogenesis. If the stage is satisfactory, smear the remaining anthers in the same manner, but fix and stain them by immediate immersion, face downward, in a petri dish full of hot (steaming) acetocarmin for from 1 to 10 minutes. Then rapidly transfer thru the following mixtures: two parts 99% (glacial) acetic acid plus one part absolute ethyl alcohol; one part acetic acid plus two parts absolute alcohol; and finally one part acetic acid plus nine parts absolute alcohol. The slides are then to be dehydrated completely by 1 to 2 minutes immersion in pure absolute alcohol, and cleared 2 to 3 minutes in a mixture of xylene and absolute alcohol in equal parts. The preparations are then made permanent by mounting each with balsam and a cover glass. The whole process takes from 5 to 15 minutes and is particularly recommended for chromosome counts.     

An Improved Pollen Grain Smear Method for Wheat

Anthers containing actively dividing pollen grains were treated 1 hour at 18-20° C. with 0.2% solution of colchicine, washed 1 hour in water, soaked in 0.002 M aqueous solution of 8-oxyquinoline at 10-14° C. for 1 hour, washed in water for 1 hour and then fixed in Carnoy's solution (alcohol, chloroform, acetic acid, 6:3:1) for 6 hours to overnight. They were washed successively in acetic-alcohol (1:1) 10-15 minutes, 70% alcohol 10-15 minutes and in water 30 minutes before hydrolysing them in bulk in 1 N HCl at 60° C. for 10-15 minutes. “Finally, they were stained in leuco-basic fuchsin for 15-30 minutes. Pollen grains were squeezed out of a stained anther in a small drop of egg albumen on a slide and the albumen smeared uniformly on the slide. The slide was dipped successively for a few seconds in glacial acetic acid and 45% acetic acid respectively. The smear was covered by a cover glass in a drop of aceto-carmine and pressed gently between folded filter papers. The cover glass was sealed with paraffin and stored overnight. To make the preparation permanent the paraffin was removed and the cover glass separated in a 1:1 mixture of acetic acid and n-butyl alcohol. The slide and the cover glass were then passed through n-butyl alcohol, 2 changes, and finally remounted in balsam.     

Sandwich Embedding of Monolayers of Cells in Epoxy Resin for Ultrathin Sectioning

In this procedure for embedding monolayers of cells, the usual glass slides are replaced by plates of resin 1-1.5 mm thick. Unlike the open-face embedding technique, the present procedure uses only a few drops of unpolymerized resin, which are applied to the fixed and dehydrated cells. During polymerization this small amount of liquid resin spreads across a relative large area, leaves the cells covered by a very thin layer, and permits phase contrast observations through it. Ultrathin sections of a particular cell encircled by a rotary scriber can be obtained by sectioning the resin slide, which has been trimmed and mounted directly in the specimen holder of the ultramicrotome.     

Semipermanent microscope slides of microfungi using a sticky tape technique

A technique is described for making semipermanent microscope slides of fungi using sticky tape. After being touched to a fungal colony, a modified segment of sticky tape is touched to ethyl alcohol and then immersed in a 50% glycerine solution containing cotton blue stain. Finally, it is transferred (sticky side up) to a microscope slide, covered with a cover with a cover glass, and sealed.     

Agar-Screen Specimen Carrier for Bulk Processing of Biopsy Materials for Electron Microscopy

A specimen carrier for processing large numbers of biopsy materials for epoxy embedding and electron microscopy is described. Commercially available 18-mesh stainless steel or 16-mesh aluminum wire screening is used. The screening is cut into 1 × 3-inch strips. One corner is snipped off for orientation purposes. Four drops of warm 4% agar is placed on a prewarmed standard microscopic glass slide. A thin agar support film is formed on the bottom side of the horizontally held wire screen by lightly running it against the agar. Tissue blocks trimmed to 1 mm³ are blotted on filter paper and placed in a prearranged order on the top surface of the support film. A thin top coating of agar is applied on the specimen by touching it with the tip of a pasteur pipette containing warm 4% agar. The agar-screen unit with the mounted specimens is stabilized in 4% buffered formalin and rinsed with Sorenson's phosphate buffer, pH 7.4, with 6.8% sucrose. It is then processed as a unit through routine osmium tetroxide postfixation, alcohol dehydration, and Epon 812 infiltration. The tissue blocks are plucked off the agar support film with fine-tipped tweezers and embedded in individual capsules. No difficulty in thin sectioning was encountered and examination of the sections under the electron microscope showed good infiltration by the epoxy resin.     

Hematein-its Advantages for General Laboratory Usage

The advantages of hematein over hematoxylin are that it is easy to prepare, easy to use, and saves time; while it gives equally good results. The writer has been employing for some time a pre-war imported hematein and until within the last few months has been unable to locate a satisfactory product of recent manufacture, either domestic or foreign. At the request of the Biological Stain Commission, however, an American manufacturer has at last put on the market a C. P. hematein which gives splendid results. The technic is as follows:

Paraffin or celloidin sections of Bouin or Zenker-formol material are run down to water and stained about 5 minutes in Mayer's hemalum (0.5 g. hematein ground up in a glass mortar with 10 cc. 95% alcohol and added to 500 cc. of 5% aqu. sol. potassium alum.) Rinse 1 to 3 seconds in tap water. Dip 1 to 3 seconds in eosin B (1 part 0.5% sol. in 20% alc. added to 2 pats dist. water; filtered from time to time). Wash several minutes in running water or in several changes of tap water. Dehydrate and mount; with unattached celloidin sections this may be done by running up to 95% alcohol, spreading on slide, blotting, wetting with absolute alcohol, draining and mounting in euparal.     

Permanent Mounts Of Chromosomes After 8-Oxyquinoline and Squashing

Fresh young root tips or free-hand cross sections thereof were placed in 0.002 M 8-oxyquinoline (aq.) at 10-14^oC. for 3 hours. After rinsing in water 1-2 minutes, they were soaked in N HC1 at 55^oC. for 25 minutes, rinsed again and squashed under a cover glass on a dry slide. Slide and cover glass were separated by placing in 70% alcohol and allowed to remain therein at least 0.5 hour after separation. Both slide and cover glass were passed through 50% and 30% alcohol to water and stained by the Feulgen procedure (without further hydrolysis) or with crystal violet after mordanting in 1% chromic acid overnight and washing in running water 3-4 hours. Dehydration and mounting in balsam completed the process. The smear on the slide was covered with a clean cover glass and the cover glass, bearing stained material, mounted separately.     

Surface Printing of Plant Leaves for Phylogenetic Studies

A solution of plastic consisting of toluene, 720 ml; methanol, 180 ml; ethyl cellulose (Ethocel, standard 7 CPS), 250 gm; and Dow resin 276 V-2, 75 gm is applied to a leaf surface which has been dampened with toluene. The plastic is spread to a thin film with the edge of a card and allowed to dry. After drying, the plastic may be peeled from the leaf surface and either stored dry in a small envelope or mounted permanently on a microscope slide. Permanent mounts are prepared by placing a small section of the peel from the upper and lower surfaces of the leaf on a microscope slide and covering with a No. 1 cover glass. A small spot of balsam on each corner of the cover glass secures the glass in position. This air mount has proved to be superior to water, glycerol or balsam mounts. Fresh leaves are washed with a mild detergent before application of the plastic. Herbarium specimens are soaked in water overnight to restore the leaf to a semiturgid condition. Five species of different plant families have been illustrated to show the diagnostic features of the surface of the cuticle. An isolated layer of epidermal cells obtained by chemical maceration permitted cell and imprint comparison. The remarkable amount of detail shown by the prints is an aid for phylogenetic studies and may make the recognition of fossil cuticles possible.     

Grinding Thin Sections of Plastic-Embedded Bone

The method describes an adaptation of a metallurgical procedure whereby dry, calcined bone may be simultaneously infiltrated and embedded in a transparent plastic, Transoptic, and then ground to the desired thinness for microscopic observation with transmitted light. A 2 mm.-thick specimen of bone, ground smooth on one side, is placed ground side up in a 1' mold assembly of a metallurgical specimen mount press. About 5 ml. of the plastic medium is added, the temperature raised to 130° C, and the pressure raised to 100 pounds. When 130° C. is reached, the heater is disconnected, the pressure immediately raised to 3500 pounds and maintained at that level until the mold cools to 68° C. The pressure is then released and the 5 mm.-thick plastic disc, with the embedded specimen therein, expressed from the mold. Grinding, as well as polishing, is dry; abrasives in fluid media are not used. The disc and specimen are coarse ground on #340 grit dry silicon carbide paper until histological details begin to appear. Final fine grinding is done on #600 grit dry silicon carbide paper. The disc is then polished and may be mounted on a 1 × 3 Plexiglas slide.     

An Application of the Frozen Sectioning Technic for Cutting Serial Sections Thru the Brain

The availability of CO₂ ice makes it practical to cut large blocks of cerebral tissue by the freezing method. If the tissue is first treated with 20-30% ethyl alcohol for sufficient time to secure uniform penetration of the alcohol (about 24 hours), formation of hard ice crystals can be controlled and serial sections 25-100 μ thick can be cut with negligible loss. The alcohol can be added to the fixative used for perfusion, or it can be added at any time later in the firing process, or after fixation is completed. The sections are cemented to the slide and groups of slides are manipulated thru staining processes in glass trays. Ordinary cell and fiber stains give satisfactory results. The method is particularly useful for certain neurophysiological purposes such as defining the location of electrode tracks and lesions and certain types of retrogrades. The Prussian blue test for electrolytically deposited iron can be conveniently applied in conjunction with other stains, to determine the point at which a given action potential response was observed, if steel electrodes are used.     

Open-Face, Epoxy Embedding of Single Cells for Ultrathin Sections

Intact stamens of Tradescantia were fixed, dehydrated, and infiltrated with an epoxy resin. Each stamen was then put into a drop of resin on a microscope slide, which was transferred to the stage of a dissecting microscope so that individual hairs could be detached from the filament with fine tungsten needles. The detached hairs were transferred to drops of resin ca. 2 mm in diameter (6 or 7 in each of two rows) lying on a slide heavily coated with evaporated carbon. Polymerization was carried out in an oven until the resin attained a degree of viscosity that permitted orientation of the isolated hairs (by using a compound microscope) without their subsequent dislocation. When the small drops of resin had hardened after further polymerization, the positions of the hairs were marked by circumscribing the cells with India ink. The block was pried from the slide after rapid cooling with solid CO₂, and was then trimmed and sectioned. Cells suspended in culture medium were embedded in much the same way; they were centrifuged to obtain a pellet, which was fixed, dehydrated, and infiltrated. A small fragment of the pellet with a little resin was placed on a microscope slide, where the cells were dissociated under a dissecting microscope at ca. 100 × magnification. Individual cells were then picked up with tungsten needles and transferred to droplets of resin on a carbon-coated slide. The subsequent steps were similar to those described for the staminate hairs. Pieces of tissue in the 50-500 μ range were also handled by the foregoing technique. However, after infiltration they were put into large drops of resin on a slide coated with silicone mold-release rather than on a surface coated with carbon.     

Orseillin Bb for Staining Fungal Elements in Sartory's Fluid

A method is described for the squashing and permanent mounting of preparations, similar to the nucleal squash technic, to be used after hematoxylin, coal tar dyes and other stains. The colored pieces are immersed in glycerin, squashed or smeared, the glycerin is slowly removed with water by capillarity and the water is afterwards similarly substituted by alcohol. The cover glass is then removed and after a rapid rinsing in alcohol the materials are permanently mounted in alcohol-soluble resin.