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.     

An Improved Technique for Sequential R-, Q- and C-Banding of Bone Marrow Chromosomes

A procedure for the sequential staining of metaphase cells to provide R-banding, Q-banding and C-banding of chromosomes is described. The combined techniques allow more precise identification of marker chromosomes and more accurate definition of breakpoints in the consistent chromosome translocations that are associated with certain human malignancies.     

Carbol Fuchsin as A Stain for Human Chromosomes

Cells derived from cultures of bone marrow or leucocytes were treated with hypotonic citrate solution, squashed in 45% acetic acid frozen with CO₂ to allow removal of the cover glass without disturbing the smear, and stained by the following schedule: absolute alcohol, 5 min; coat with 0.2% parlodion and air dry; 70% alcohol, 5 min; distilled water, 5 min; stain 2-5 min in a mixture of 45 ml of a 0.3% solution of basic fuchsin in 5% phenol, 6 ml of glacial acetic acid, and 6 ml of 37% formaldehyde. Differentiate and dehydrate in absolute alcohol, clear in xylene and cover. The stain is durable for several weeks if slides are stored in darkness when not in use. Results resemble those obtained by Feulgen or aceto-orcein methods.     

Chlorazol Black E as A Stain for Root-Tip Chromosomes

A simple method of shining root-tip chromosomes which gives excellent results in photomicrography is described. The schedule is as follows: run paraffin sections of root tips killed in Bouin's fixative down to water; stain for approximately two hours in 1% aqueous chlorazol black E; wash in water; run up to xylene.     

A Technique for C-banding Chromosomes with Pinacyanol Chloride

A technique is presented for rapid C-banding of eukaryotic chromosomes with pinacyanol chloride. This technique has given excellent definition and clarity to chromosome bands in plant and animal chromosomes. In permanent mounts the chromosomes did not fade after storage for up to two years.     

A Modified Giemsa C-Banding Technique For Hordeum Species

A Giemsa C-banding technique with a hot 1 N HCI hydrolysis step has been developed for barley chromosomes. This step makes it easy to obtain well separated C-banded chromosomes. To compare this technique with other C-banding techniques, chromosomes of H. vulgare cv. York were stained by both this technique and a modification of the technique of Kimber et al (1976). With respect to centromeric and intercalary bands, both techniques produce a similar banding pattern, but telomeric bands observed by the modified technique of Kimber et al (1976) were not detected by our procedure. This indicates that telomeric heterochromatin may be different chemically and/or structurally from the centromeric and intercalary heterochromatin and its appearance dependent upon the C-banding technique. The procedure described provides a relatively rapid technique for C-banding of barley chromosomes.     

Staining Aspirated Human Bone Marrow with Domestic Wright Stain

The method employs the domestic Wright stain for the staining of aspirated human bone marrow. Freshly distilled water, pH 6.0 to 6.4, is used. Wright stain, 0.5 cc, is placed upon the air-dried preparation and permitted to act for two minutes. The stain is then diluted with 2 cc. distilled water and permitted to act for from 5 to 10 minutes. After washing off the stain with distilled water, the preparation is placed into a decolorizer (acetone 0.5 cc, pure methyl alcohol 5.0 cc, and 100 cc. distilled water, pH 6.0 to 6.4) for differentiation from 1 to 5 seconds, rinsed, washed under running water and permitted to air-dry. A well stained and differentiated preparation shows the “Romanovsky effect”, and the sharpness of minute structures obtained compares favorably with control preparations stained with German dyes.

The bone marrow should be prepared as described. The Wright stain marketed by the National Aniline and Chemical Co., N. Y. was found to be reliable as regards staining quality of registered batches. One photomicrograph, showing bone marrow cells from pernicious anemia, is included.     

A Quadruple Stain Combination for Plant Tissues

A new procedure for the quadruple staining of paraffin sections of plant tissues is offered, involving the use of safranin, methyl violet, fast green and orange G. Differentiation is practically automatic and is controlled by the use of special washing solutions. Striking color-contrast effects permit the identification of every type of cell structure and cytoplasmic inclusions.     

Rapid Fluorescent-Antibody Stain Technique with Group A Streptococci

A rapid fluorescent-antibody stain technique used with young broth cultures of trypsinized group A streptococci is described. Results from 1,214 trypsinized cultures of group A streptococci employing the rapid stain method were equivalent, both in specificity and sensitivity, when compared to results obtained from identical non-trypsinized cultures stained with the longer standard technique of Cherry, Goldman, and Carski. Moreover, the rapid method requires less than 2 min to complete.     

Note from the Biological Stain Commission: Certification of Wright Stain Solution

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

A One-Solution Tannic-Aced-Iron Stain for Plant Tissue Sections

After completing the bulletin on “Actinomycetes in various parts of the potato and other plants” (Lutman, 1945) the author found the beautiful plates in the atlas to Olivier's monograph (1881) on root structure in which the same intercellular inclusions were shown. Olivier stated that he had stained his sections in “tannate of iron”. Attempts were made by the author to prepare and use such a combination but they were unsuccessful owing to the precipitate that was formed.

The formula used by the U. S. government for ink for official use was tried. This combination is composed of tannic and gallic acids with ferrous sulphate and is acidified with hydrochloric acid. When used double strength, as suggested for special blackness and permanence, the stain was very successful on sections of potato roots and tubers. It stained the Actinomyces hyphae very differentially and was decolorized from all other cell organs. Any other stains used dyed also the pectins and the Actinomyces secretions (melanins) but with this iron tannate combination in one solution, the finest hyphae could be seen and photographed. Since hydrochloric acid was used in this stain, such Actinomyces inclusions must be very tannophylic; much more so than any animal intercellular inclusions so far described.     

Alcoholic Hydrochloric Acid-Carmine as a Stain for Chromosomes in Squash Preparations

A regressive bulk carmine staining schedule was adapted from a formula proposed by P. Mayer. The stain is made by boiling gently 4 gm of certified carmine in 15 ml of distilled water to which 1 ml of concentrated HC1 has been added. After cooling, 95 ml of 85% alcohol is added, and the solution filtered. Fixed tissue is soaked in the stain until thoroughly penetrated; squashes are then prepared as usual, but plain 45% acetic acid is used as the temporary mounting medium instead of aceto-carmine. The advantages of this method are: intense, precise staining of chromosomes coupled with a lightly stained cytoplasm; consistency and uniformity of results; simplicity of use.     

A Rapid Stain Method for Detecting Certain Plant Virus Inclusions

The trypan blue method for detecting certain virus crystallin inclusions is greatly facilitated by a pre-stain treatment with a diethyl-ether-ethyl-alcohol solution made up in physiological saline (5-ml. of diethyl ether and 10 ml. of ethyl alcohol diluted to 100 ml. with physiological saline solution). Maceration of the study material in trypan blue solution also gives good results.     

Smear Technique for Studying Lepidopteran Chromosomes

There is scant information on the chromosomes of Lepidoptera, the largest order of insects with more than 100,000 described species (Imms 1965), despite the abundance of the material and cosmopolitan distribution of many species. the lack of information on the chromosomes (the haploid number being known for about 1% of the total number of species) and detailed analysis of the karyotype in this order may be partly due to the small size and isodiametric nature of the chromosomes and partly due to the difficulty in obtaining satisfactory squash preparations of the adult testes, which are enclosed together in a thick-walled scrotum. This difficulty of the interference of the scrotum in obtaining stages satisfactory for study (fixation of the material worsens the situation, for the scrotal wall hardens on fixation) seems to have discouraged the cytological study of this group, especially in the adults. Some workers have tried to circumvent this difficulty by squashing larval testes, which are not enclosed in a common scrotum. This method, however, calls for rearing larvae through the pupal stages for correct identification of the adults.