Double Staining for Comparative Measurements in Squash Preparations

Comparative measurements of nuclei or chromosomes following different treatments are seldom made on squash preparations, since variations which arise during preparation of the slides may easily mask genuine treatment differences. This drawback may be overcome by making use of dyes which, when substituted for basic fuchsin in Schiff's reagent, will give a Feulgen-type reaction with chromatin. By selecting dyes of contrasting colours, it is possible to intermingle cells from different treatments in the same squash preparation, and to perform comparative measurements on adjacent cells.

Suitable dyes which contrast well with basic fuchsin are toluidine blue, or azure A (which stain chromatin blue) and chrysoidin yellow (which stains chromatin yellow). These dyes are made up and used in the same manner as ordinary Feulgen reagent.

Samples of cells from the two treatments to be compared are fixed, washed and hydrolysed in 1 N HCl at 60 C. One sample is stained in regular Feulgen reagent, the other in the contrast dye, then both are macerated and thoroughly mixed on the same slide in a single drop of 45% acetic acid. A coverslip is added, and the preparation flattened to the required amount and made permanent after dry-ice removal of the cover. This technique may also be utilised for comparative grain counts in autoradiography, provided that the contrast dye does not cause chemical fogging of the film.     

The Use of Haematoxylin for Squash Preparations of Chromosomes

A simplified propionic-iron alum-haematoxylin stain for rapid squash preparations of chromosomes requires only two stock solutions: (A) 2% haematoxylin and (B) 0.5% iron alum, both in 50% propionic acid. For use, suitable volumes of A and B are mixed. With unripened solution A, equal volumes should be used and the stain is ready for use 1 day after mixing. As the haematoxylin ripens, progressively smaller amounts of B are required and the mixture may be used immediately. The stain gives excellent results when used in the same way that orcein and carmine are currently employed, with a wide range of animal and plant (including fungal) chromosomes, and with good nucleolar staining. It may be used either following acetic alcohol (1:3) fixation or as joint fixative and stain on unfixed material. In fungal material, where Lu's BAC fixative is recommended, the centrioles are also stained.     

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.     

Double Fluorescent Staining for the Separate Demonstration of Chromosomes and Microtubules in Mitotic Cells In Vitro

A simple fluorescent method for double staining of mitotic cells using a rhodamine B indirect immunofluorescent method for tubulin and the DNA-specific fluorescent dye Hoechst 33258 for nuclei and chromosomes is described. This procedure enables one through the use of appropriate excitation filters to view at will either chromosomes and nuclei or tubulin within the same cell.     

A Feulgen-Carmine Technic for Staining Fungus Chromosomes

Apothecia of Pyronema confluens Tul. were stained by the Feulgen reaction and then squashed and mounted in propiono-carmine. Exceptionally clear differentiation of the chromosomes was obtained by this method. Snail stomach cytase was used as an aid in flattening the asci and a simple method for its extraction is described.     

Bacterial Maceration for Feulgen Squash Preparations of Plant Tissues

An active pectinolytic extract obtained from Bacterium aroideae is an effective macerating medium for making Feulgen squash preparations from materials which are not sufficiently macerated by Feulgen hydrolysis in normal hydrochloric acid. To obtain the extract, the bacterium is cultured in a suitable liquid medium for 4 days at about 25°C. The cultures are then bulked, toluene is added and the whole is shaken and left overnight. The liquid may then be cleared by centrifuging and stored ready for use in small volumes over dry ice. The tissue to be macerated is fixed, bleached if necessary, washed and soaked in the active extract 2-4 days before Feulgen staining. The organism is readily obtainable, is easy to culture and the strain remains constant. This, coupled with the fact that the extract, once prepared, retains its full activity for at least 6 mo makes it a convenient source of pectinolytic enzyme for the plant cytologist.     

A Staining Rack for Handling Cover-Glass Preparations

A porcelain staining rack (Fig. 1) has been devised for handling cover-glass preparations. The design is along the general lines of staining racks for slides commonly sold by commercial houses. It consists of three parallel rods—one at the bottom, two on the sides. These three rods are held together by two end pieces. Each of the rods has twelve slots, thus the rack holds twelve cover glasses. The slots have been arranged to hold the cover glasses some distance apart. Circular cover glasses having a diameter of 22 mm. are most desirable. Each of the two end pieces has a hole near the top for inserting one end of the wire tongs or handle (see Fig. 1) which is used for removing the rack from the stender dish. The staining rack is 40 mm. long and 35 mm. high and is designed, as shown in the figure, to fit the ordinary Stender dishes commonly used in many laboratories.     

A Staining Rack for Handling Cover-Glass Preparations

A porcelain staining rack (Fig. 1) has been devised for handling cover-glass preparations. The design is along the general lines of staining racks for slides commonly sold by commercial houses. It consists of three parallel rods—one at the bottom, two on the sides. These three rods are held together by two end pieces. Each of the rods has twelve slots, thus the rack holds twelve cover glasses. The slots have been arranged to hold the cover glasses some distance apart. Circular cover glasses having a diameter of 22 mm. are most desirable. Each of the two end pieces has a hole near the top for inserting one end of the wire tongs or handle (see Fig. 1) which is used for removing the rack from the stender dish. The staining rack is 40 mm. long and 35 mm. high and is designed, as shown in the figure, to fit the ordinary Stender dishes commonly used in many laboratories.     

Staining Air Dried Protoplasts for Study of Plant Chromosomes

The air drying technique used in mammalian cytology was applied to isolated plant protoplasts for study of chromosomes. For cultured celery cells, this technique resulted in good spreads of metaphase chromosomes with high resolution. Mitotic chromosomes of Brassica species are relatively small, poorly stained by common stains, and difficult to spread by the squash technique. In this study, however, the chromosomes of B. carinata in callus culture were spread well and stained clearly with Giemsa staining solution. The chromosome preparations by the present techniques should also be amenable to chromosome banding studies in plants.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

Silver Staining for Nucleolar Organizing Regions of Vertebrate Chromosomes

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.     

Aceto-Iron-Haematoxylin for Staining Chromosomes in Squashes of Plant Material

Haematoxylin can be used successfully in the acetic squash technic if adequate mordanting is provided, (a) in the stain—composed of 4% haematoxylin and 1% iron alum in 45% acetic acid—and (b) in a step that combines additional fixation, mordanting and maceration in a 1:1 HCl-alcohol mixture, to which is added chrome alum, iron alum and iodic acid: 0.1 gm of each to 6 ml of HCl-alcohol. The material is usually given a preliminary fixation in 1:3 acetic alcohol, then macerated, fixed and mordanted in the acidified alum-HIO₃ step for 10 min, transferred to Carney's fluid (6:3:1) for 10-20 min, squashed in a drop of stain and gently heated. In some species, the preliminary fixation may be omitted. The method yields intensely and selectively stained chromatin. To secure consistently good results, the stain can be diluted with 45% acetic acid, and the iodic acid omitted for some plant materials.     

A Technic for Differential Staining of Nucleoli and Chromosomes

A procedure is described which enables a stain to be definitely located in the substance of the nucleolus. Material is fixed in either Navashin or Levitsky; the chromatin is stained by means of the improved Feulgen technic introduced by de Tomasi, and preparations brought thru the washing solutions down to distilled water. From distilled water the material is transferred to a mordant solution, 5% sodium carbonate in water, in which it is left for at least one hour. After mordanting wash well with water then stain for ten minutes in light green solution (90% alcohol, 100 cc, light green SFY, 0.5 g, aniline oil, 2 drops, well shaken); differentiate in alcoholic sodium carbonate solution, (70% alcohol saturated with carbonate); treat with 95% alcohol, absolute alcohol, equal parts xylene and absolute alcohol, clear in pure dry xylene and mount in neutral balsam. Cytoplasm and karyolymph should be quite clear, with magenta chromatin and well defined green nucleoli. The light green does not behave like a simple counterstain as in previous technics but as a definite stain for nucleolar material.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

Quinacrine Fluorescence for Identifying Metaphase Chromosomes, with Special Reference to Photomicrography

Metaphase chromosomes of humans and other species can be identified by their quinacrine fluorescence patterns. For the study of human chromosomes, standard phytohemagglutinin-stimulated leukocytes are used. A 45-60 min interval of colchicine treatment is used to obtain a higher proportion of extended chromosomes with contiguous chromatids which have generally proved to be most informative. Slides are stained with 0.5% quinacrine for 6 min, rinsed in running tap water for 3 min, and rinsed and mounted in a Tris-maleate buffer at pH 5.6. For photomicrography, a microscope with an HBO 200 w high-pressure mercury vapor lamp, 3 mm BG 12 excitor filter, darkfield condenser, fluorite 95x objective, K510 barrier filter and a nonautomatic exposure microphotographic attachment with an 8x eyepiece is used. With this system, images of adequate brightness reach the film plane, thus making possible the use of fine-grain, high definition 35 mm films, such as Kodak Panatomic-X and H & W Control VTE panchromatic. Photographic prints of moderately high contrast are used for preparing karyotypes in which every human chromosome can usually be readily identified.