Permanent-Type Mounting of Aceto-Iron-Haematoxylin Squashes in Corn Syrup

Preparations obtained by the aceto-iron-haematoxylin technique reported previously (Stain Tech., 37, 27-30, 1962) can be made relatively permanent either by ringing the cover slip with Karo corn syrup, or by mounting the squash in this syrup after separating slide and cover slip by the solid CO₂ freezing technique. In the latter procedure, both slide and cover slip may be placed briefly in 45% acetic acid for further differentiation of the stain, then recombined with a drop of syrup.     

A Quick-Freeze Method for Making Smear Slides Permanent

Many types of smear slides can be made permanent rapidly and effectively by substituting for the usual dehydration series a single-step process of freezing the slide on a block of dry ice, placing it immediately in 95% or absolute alcohol, and then mounting it. Advantages of the technic are its speed, the ease of separation of cover slip from slide with a minimum loss of cells, and the superiority of the resulting permanent slides.     

Collodion Membranes in Pollen Tube Technique

Membranes are formed by allowing a drop of collodion-acetone solution to come into contact with the surface of warm sugar solution in a petri dish. Pollen is germinated upon the smooth areas of the membrane when all traces of acetone have evaporated. Semipermanent preparations are made by isolating the pollinated area of the membrane, floating it onto a slide, and, after the removal of excess sugar solution, adding a drop of acetic-stain fixative, followed by an albumenized cover slip. The preparation can be made permanent by inverting a slide in a mixture of 1 part glacial acetic acid and 3 parts absolute alcohol, when the collodion membrane will dissolve and allow the cover slip and adhering grains to fall free. The cover slip is then passed through absolute alcohol (2 changes), xylene, and mounted in neutral mountant on a clean slide. By substituting a drop of the alcohol-acetic acid mixture in place of acetic-stain fixative, the grains adhering to the cover slip may be stained by the Feulgen method.     

A simple method of preparing permanent squash preparations using cellophane]

A technique to prepare permanent squashed preparations of cell nuclei and chromosomes is proposed. Fix a piece of material on the slide with acetic alcohol (1:3), macerate with a 45% acetic acid, cover with hydrophilic cellophane previously soaked in a 45% acetic acid and then with a cover slip and filter paper to squash finally as it is routinely performed. After that soak off the cover slip with alcohol, post-fix the squashed preparation together with cellophane in alcohol for 5-10 min, unstick the cellophane, pass the preparation through alcohol once again and dry it. The subsequent treatment of the squashed preparation depends on the purpose of investigation. The slide may be tinctured overlaid with photoemulsion for autoradiography, or processed by different ways.     

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.     

Elimination of Material that Obscures Stained Chromosomes in Squashes of Vicia faba Root Tips

A procedure for elimination of cytoplasmic debris from Vicia faba root tip cells is: (1) a root tip previously fixed in 3:1 absolute alcohol-acetic acid and stained by the Feulgen method is placed on a slide and squashed in a small drop of water, (2) a cover slip is applied and the cells are flattened with a hand-operated lever device supplying 35 pounds pressure onto a 22 × 22 mm cover glass, (3) the slide is quick-frozen, the cover slip is removed, and the slide is dropped immediately into water, (4) the slide is cleared through an alcohol-xylene dehydration series and permanently mounted. The significant result of this procedure is the consistent presence of clear, flat cells showing excellent definition of chromosomes.     

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.     

Unstained chromosomes. A simple method for observation

A technique is described by which unstained metaphases may be observed using phase contrast microscopy. The cells are fixed on a cover slip which then is turned over and onto a slide, leaving a thin air layer sandwiched between. Observation in phase contrast shows metaphases comparable to those observed after Giemsa staining.     

Serial Sections of Smears for Electron Microscopy

Ultrathin sections for electron microscopy may be prepared from smears or squashes embedded in methacrylate. The cover slip or glass slide with the attached fixed cellular material is passed through alcohols to methacrylate monomer and finally to monomer containing a catalyst. The portion of the smear to be sectioned is covered with a gelatin capsule containing partially polymerized methacrylate. When polymerization is completed at 47°C, the hardened block is separated from the cover slip and trimmed under the compound microscope so as to encompass the desired area. Photographs are made of the intact smear to afford a basis for identification of cellular materials in electron micrographs of the individual ultrathin sections.     

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.     

Measurement of live bacteria by Nomarski interference microscopy and stereologic methods as tested with macroscopic rod-shaped models

A new method is proposed to measure bacterial cells under growth conditions. Bacterial cells, suspended in their growth medium, were attached to a cover slip with poly-L-lysine. The cover slip was inverted and placed on a glass microscope slide. To prevent dehydration of the medium, the edges of the cover slip were sealed to the microscope slide with clear fingernail polish. The bacteria on the slide were then quickly photographed with a Leitz light microscope, using Nomarski optics. The photographic negatives were then projected at a standard distance through a lens system, and the projected images of the whole cells were outlined by hand onto graph paper. The profile images so transcribed onto the graph paper were in effect transverse sections of each of the cells. Using stereologic grid and point counting techniques, the area of the cell transverse section as well as the perimeter or circumference of the transverse section were estimated. Formulae were developed so that both the volume and surface area of the whole cell could be ascertained from these area and circumference measurements. Since the efficacy of any measurements of surface area and volume of microscopic rod-shaped bacterial cells could be questioned, macroscopic rod-shaped models were used to test the theory and formulae and to compare this method with other commonly used cell-sizing techniques. This technique could be used in any study of bacterial cell size or changes in cell size (e.g., osmotic shifts).     

Measurement of live bacteria by Nomarski interference microscopy and stereologic methods as tested with macroscopic rod-shaped models.

A new method is proposed to measure bacterial cells under growth conditions. Bacterial cells, suspended in their growth medium, were attached to a cover slip with poly-L-lysine. The cover slip was inverted and placed on a glass microscope slide. To prevent dehydration of the medium, the edges of the cover slip were sealed to the microscope slide with clear fingernail polish. The bacteria on the slide were then quickly photographed with a Leitz light microscope, using Nomarski optics. The photographic negatives were then projected at a standard distance through a lens system, and the projected images of the whole cells were outlined by hand onto graph paper. The profile images so transcribed onto the graph paper were in effect transverse sections of each of the cells. Using stereologic grid and point counting techniques, the area of the cell transverse section as well as the perimeter or circumference of the transverse section were estimated. Formulae were developed so that both the volume and surface area of the whole cell could be ascertained from these area and circumference measurements. Since the efficacy of any measurements of surface area and volume of microscopic rod-shaped bacterial cells could be questioned, macroscopic rod-shaped models were used to test the theory and formulae and to compare this method with other commonly used cell-sizing techniques. This technique could be used in any study of bacterial cell size or changes in cell size (e.g., osmotic shifts).     

Different populations of Scomber australasicus in New Zealand and south-eastern Australia, demonstrated by a simple method using monogenean sclerites

A simple method for distinguishing fish populations using monogenean sclerites is described. Monogeneans are left in a drop of 45% acetic acid on a microscope slide for a few minutes, and squashed under a cover slip to spread the hamuli and genital hooks in one plane. Hamuli and hooks are then measured with the aid of measuring eyepieces. Measurements of sclerites of Kuhnia and Pseudokuhnia fixed directly in 10% formalin or fixed in 10% formalin after freezing for several days gave the same results. Prerequisites for the use of the method are that monogeneans occur in sufficient numbers and show distinct geographical variation. The method is easiest to use if the size of sclerites does not change with body growth. Kuhnia scombri (Monogenea: Polyopisthocotylea) from the mackerel, Scomber australasicus , in New Zealand and New South Wales differ significantly in the length of the hamuli; it is concluded that there may be different populations of mackerel at the two localities. There also are different populations of Scomber japonicus in Japan and Ecuador, and sympatric populations of S. japonicus and S. australasicus in Japan harbour identical populations of the monogeneans Pseudokuhnia minor and Kuhnia sprostonae.     

Sudan Black B And Acetocarmine as a Combination Stain

Epidermis stripped from either fresh or fixed plant organs, or sections of paraffin-embedded or fresh material are placed on a slide and covered with a drop or two of iron-acetocarmine. The stain is intensified by warming the slide over a flame. After a few minutes a drop or two of a saturated solution of Sudan black B in 45% acetic acid is added and a cover slip applied. The preparations cannot be made permanent, but last a few weeks if sealed with a compound such as gum mastic-paraffin, or if the combined stain is drained off and a drop of Karo syrup is added before the cover slip is applied. The acetocarmine produces its usual staining effects, i.e., nuclei dark red and some components of the cytoplasm of certain cells a less intense red. The Sudan black B colors lipid structures an intense blue.     

A Rapid HCl/Toluidine Blue Squash Technic for Plant Chromosomes

Fresh or pretreated root tips are simultaneously fixed and hydrolysed in 5 N HC1 for 15 min at room temperature. After washing they are macerated on a slide in a drop of 0.05% toluidine blue made up in McIlvaine citric acid-Na₂HPO, buffer at pH 4.0. Pressure on the cover slip completes the squash preparation. It is made permanent by removing the cover slip on dry ice, air drying and mounting in Euparal.     

The Negative Staining of Bacteria

The author does not propose new methods but calls attention to the value of Burri's India ink technic and Fischer's use of nigrosin for the negative staining of bacteria. The latter is particularly useful. The technic is as follows: Boil 10 g. nigrosin in 100 cc. water about 30 minutes. Filter several times thru the same filter paper, adding 10 drops of formalin before the last filtration as a preservative. Place a small loopful of this solution on a clean slide and add the bacteria with a needle or loop. After mixing spread the mixture a little irregularly on the slide and dry either at room temperature or slowly over a low flame. The preparation may be examined in oil immersion without the use of a cover slip.     

Squashing Under Scotch Tape No. 665 for Autoradio-Graphic and Permanent Histologic Preparations

Removal of the cover slip from squash preparations, for coating with auto-radiographic emulsion, or other purposes, is made easy if squashing is performed with a piece of Scotch double-coated adhesive tape No. 665, used as a cover slip. The material to be squashed is placed on a slide lightly coated with an adhesive consisting of 1% gelatin with 0.1% chrome alum added. The piece of tape is applied with the surface originally on the outside of the roll next to the specimen. Specimens should be soaked before squashing in aqueous 45% acetic acid, with or without added dye, such as carmine or orcein. After squashing, the tape is easily removed without damage to the cells. This allows autoradiographic emulsion to be applied, or, unstained material can be stained after squashing by technics suitable for microtome sections.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Staining Plant and Animal Chromosomes by the Feulgen-Acetocarmine Sequence

A technic, some fundamentals of which were first worked out on brome grass, has been considerably extended and adapted to the somatic chromosomes of salmon. Fresh salmon eggs were quickly pierced in 45% acetic acid and fixed therein for 4 minutes. The eggs were then placed in N HCl at 60°C. for 8 minutes and thereafter transferred to Feulgen stain for 30 to 45 minutes. Subsequently, each stained embryo was dissected out and divided in two, each half being placed on a slide in a drop of acetocarmine stain. The pieces were well macerated and, after covering with a cover slip, maceration was completed by tapping. Heavy pressure was gradually applied to the cover slip in order to flatten the chromosome complements. A square screw-type laboratory hose clamp was then used to maintain this pressure while a liquid gelatin seal was applied around the edges. The slide, with the clamp on, was placed in the refrigerator overnight. Before the slide was scanned, the clamp was removed permanently. After each scanning period the slide was returned to the refrigerator. Photomicrographs of well-spread chromosomes in one optical plane were enlarged and tracings made from them. These tracings together with the photomicrographs were used for chromosome analysis.