Chlorazol Black E As An Aceto-Carmine Auxiliary Stain

In making chromosome counts on plants and plant parts treated with colchicine it was found that in cases where aceto-carmine alone is not satisfactory—as in axillary buds of apple, pear, plum, peach, apricot, and cherry—the following method was effective : Dissect out the meristematic parts of the axillary bud under a binocular (or cut free-hand sections) and transfer the dissected tissue immediately to a solution of 3 volumes alcohol to 1 volume acetic acid for killing and fixing. Let the fixative act at least 10 minutes; a longer time, 12-24 hours, improves the staining quality. Wash in at least 3 changes of 70% alcohol to remove most of the acid. Stain for 5-25 minutes in 1% chlorazol black E² in 70% alcohol. Rinse in 3 changes of 70% alcohol to remove excess stain. Transfer the material to a slide, cover with a drop of aceto-carmine, and if necessary, dissect further under a binocular. Cover with cover glass, heat, flatten and seal, or run Zirkle's fluid under the cover for permanent mounting. For smears of sporocytes, chlorazol black E may also be employed alone, or in combination with aceto-carmine, if a dark purple nuclear stain is desired.     

Staining Root-Tip Smears with Aceto-Carmine

A number of schedules have been suggested for staining root-tip smears with aceto-carmine (Brown, 1937; Burrell, 1939; Ganeshaw, 1939; Howe, 1946; Smith, 1947; Warmke, 1935). The variation that exists between plant species has undoubtedly been responsible for many of these modifications. In addition, the nature of the information desired has also been a determining factor in the development of new technics (Aisima, 1941; Howe, 1943).     

The Aceto-Carmine Method for Fruit Material

It is not easy to make good aceto-carmine preparations of plants with small chromosomes at meiosis because the cytoplasm readily takes up the stain and this prevents a sharp differentiation. The staining reaction depends on the composition of the pre-fixative, the duration of fixation, strength of aceto-carmine and amount of iron used. These factors can be varied independently. Since not only species but their varieties differ markedly from one another in their behavior, the best results can be secured only after experiment with individual plants to discover the most suitable combination. Suitable combinations of these factors for some fruit plants are described. In general they demand (1) a weaker solution of aceto-carmine and more iron than has hitherto been used in the aceto-carmine technic, and (2) the introduction of iron and carmine into the pre-fixative. Iron acetate is added to a dilute solution of carmine in glacial acetic acid until the solution assumes a deep red color, without precipitation, and this solution is used as the acetic acid component of an acetic-alcohol pre-fixative. Anthers are colored purple by treatment with this fixative, but since it has only a mordanting effect they need to be smeared and stained in the ordinary way.     

Manipulation of Tissues for Improved Aceto-Carmine Staining

The importance of the study of the structural-mechanic properties of any particular material in order to improve the technic for aceto-carmine smears and to obtain better preparations of that material has not been, perhaps, sufficiently emphasized in the large number of papers on such cytological technic. The usefulness of such a study will be shown here in two cases met by the writer.     

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.     

A Smear Technic for Some Species of leguminosae

Carnoy's No. 2 fluid (absolute alcohol-glacial acetic acid-chloroform) modified by saturation with mercuric chloride was found to be an effective killing and fixing agent for microsporocytes of Trifolium pratense, T. incarnatum, T. repens, T. hybridum, and Glycine max. Microsporocytes so fixed were softened in a 0.1N HCl solution at 45°C. for approximately 10 minutes, then smeared by die standard aceto-carmine technic. The modifications enabled chromosome counts to be made successfully in microsporocyte smears, whereas satisfactory smears could not be made when standard technics of killing and fixing were used.     

The Venetian Turpentine Mounting Medium

As a mounting medium to follow aceto-carmine the following modification of Zirkle's is suggested: Venetian turpentine, 25 ml.; phenol, 50 ml.; propionic acid, 35 ml.; acetic acid, 10 ml.; water, 20 ml. The technic can be employed with either root-tip or pollen-mother smears, and has been used with quite a variety of plants. It is especially valuable where it is desired to make temporary mounts permanent. The method is simple, and with reasonable care no displacement of marked cells occurs.     

Iron-Alum Aceto-Carmine Staining for Chromosomes and Other Anatomical Features of Rhodophyceae

The chromosomes, certain intracellular structures and gross anatomical details of many red algae, which, as a class, have proved technically difficult material, can be demonstrated by staining with aceto-carmine after a mordant bath of iron alum. Acetic-alcohol mixtures are used as nuclear fixatives and formalin-acetic-alcohol and other similar fluids for preservation of anatomical features. The tougher more cartilaginous thalli of some species can be softened, if squashes are desired, by prolonging fixation (24-48 hr.) in acetic alcohol and subsequent washing. The fixatives are washed out of the material before the latter is transferred to 0.5-5.0% ferric ammonium sulphate, the concentration of which may be altered according to the material. Excess mordant is removed by washing and the material stained in Belling's aceto-carmine containing a trace of ferric acetate as a “ripener”. The degree of heating before covering is critical as it controls the quality of the staining. Squashing must be very thorough to spread the chromosomes which are usually very small but only slight controlled pressure is necessary when diffuse structures such as carposporophytes, nemathecia or medullary filaments are being demonstrated. Paraffin sections mounted on slides can also be stained by this method.     

Use of an aceto-carmine procedure to examine the excysted metacercariae of Echinostoma caproni and E. trivolvis.

A simple aceto-carmine procedure was developed to relax, fix, stain, and clear excysted metacercariae of Echinostoma caproni and E. trivolvis. This procedure allowed for morphologic details on whole metacercariae comparable to those seen with more elaborate staining procedures. Differences in dimensions and staining intensities between the two species of metacercariae are described.     

Some Synthetic Resins IN Combined Fixing, Staining and Mounting Media

Many of the recently devised plasticizers and resins can be utilized to advantage in cytological technics. Some of them have solubilities which enable us to incorporate them in such fixing and staining solutions as aceto-carmine and propiorric-carmine. They are non-volatile, do not alter the fixation images of the fluids with which they are mixed, and serve as mounting media as the volatile components evaporate. Thus it is possible to make a permanent slide in a single operation. These newer compounds are better adapted for this technic than are the natural balsams which have been used previously, as their greater tolerance for water provides a much greater margin of safety. Procedures are described for the utilization of (1) Rezyl 7020, a water-soluble resin (now, unfortunately, not available), which dries to form a water insoluble film, (2) Amberol 750 and (3) Bakelite BR-7160, two alcohol soluble resins, more miscible in solutions containing water than are the natural balsams. Formaldehyde can be included in the aceto-carmine and propionic-carmine fluids with the result that more nuclear detail is preserved. Lacto-gelatin has some valuable properties as a mounting medium and can be used when the specimen is stained with orcein. Carmine, which gives a permanent stain in Rezyl 7020, Amberol 750 and Bakelite BR-7160 fades in lacto-gelatin.     

Root-Tip Smear Method for Difficult Material

A technic is outlined for the preparation of difficult material for the study of chromosome number and morphology in root-tip smears. The chief objective is to obtain polar views of the metaphase plates rather than equatorial views. To achieve this end it is recommended that fresh root-tips be cut free-hand into thin cross sections. The important features are: thin freehand cross sectioning of the fresh root-tips; fixing in Belling's iron aceto-carmine solution; maceration for 2-5 minutes in 50% HCl in 95% alcohol; and mounting in “Diaphane.”     

Karyological Leaf Squashes in Grasses, Aided by Pectinase Digestion at 45 C

Longitudinal sections of rapidly growing leaf shoots were soaked for 2-4 hr in 0.002 M 8-hydroxyquinoline at 25 C, blotted, and fixed in 3:4:1 ethanol, chloroform, acetic acid. A 30 min maceration at 45 C in a pectinase solution (Pectinal 59-L; Rohm and Haas) softened the material for staining and squashing. Excess pectinase was removed and 1% aceto-carmine stain was applied. After locating and gently tapping the cover clip to disperse the cells, heavy pressure was applied with a No. 9 rubber stopper and the heel of the hand. By the use of this procedure, karyotypes could be constructed in several genera of forage grasses. The karyotype of Paspalum notatum Flugge is illustrated.     

An Aceto-Carmine Squash Technic for Mature Embryo Sacs

A method is described by which, whole embryo sacs of Nicotiana, Petunia and no doubt of certain other genera can be obtained readily in aceto-carmine ovule squashes. Although application of the technic to megagametogenesis and fertilization stages is stressed in this paper, use of the method allows development to be traced from the archespore up to the second or third division of endosperm nuclei. The success of the technic depends on four phases:-1) fixation in a medium that causes cell and nuclear structures to become pliable, yet rigid enough that their spatial relationships are not greatly distorted in squashing; 2) heat, which apparently increases the cohesion of cytoplasmic and nuclear constituents; 3) maceration to separate the embryo sac from surrounding cells; and 4) the use of a stain that differentiates the various nuclear structures as well as those of the cytoplasm. Staining of the cytoplasm, essential in some embryological investigations, is one advantage of the aceto-carmine squash method over the Feulgen procedure. In contrast to the Feulgen ovule squash method the aceto-carmine technic will probably be most useful in genera having numerous small ovules. Advantages and defects of the aceto-carmine procedure as compared with the paraffin technic are discussed, likewise the possible usefulness of the former in studies of sterility and in certain other special connections.     

A simple staining method for cryptosporidian oocysts and sporozoites

A useful staining method for detection of cryptosporidian oocysts and sporozoites was developed and described. The modified Kohn's one-step staining technique with an additional modification, i.e., longer staining time and higher staining temperature than those originally described, was tested on fecal smears from cats infected with Cryptosporidium sp. By this improved staining procedure, the oocyst appeared as a slightly oval body containing four internal sporozoites colored blue to blue-gray. The oocyst wall was stained dark green to black. The morphological feature of the oocyst was recognized much more clearly by this staining technique than by such currently used techniques as acid-fast and Giemsa staining. This staining procedure proved to be simple and less costly and to secure a good preservation.     

Smear Methods for the Study of Chromosomes in Ascomycetes

Several modifications of the aceto-carmine smear technic used by the author in studies of the meiotic chromosomes of Neurospora are outlined. The critical role of the staining solution is stressed and suggestions are given for preparing a satisfactory carmine solution. The ease and rapidity of this method suggests its use in cytologic work on other Ascomycetes.     

Procedure to Facilitate Chromosome Counts in Difficult Plant Material

Numerous mitotic plates of contracted and well-spread chromosomes may be obtained from root tips of plants set on melting ice or snow overnight at room temperature. After 1:3 acetic-alcohol fixation for 0.5 to 3 hours the material is mordanted in a mixture of 7 parts of alcohol plus 21/2 parts of 3% ferric ammonium sulfate for 3 hours to overnight. This solution may be used as storage fluid for flower buds. Deep chromosome coloration without precipitates is secured by staining in a few drops of aceto-carmine for 10-15 minutes after which the tissues are softened by heating in aceto-carmine diluted with 3 parts of 45% acetic acid.     

An immunogold-silver staining method for detection of cell surface antigens in cell smears

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.     

Trypan Blue as a Nuclear Stain for Plant Material

Although McWhorter¹ and later Rich² mentioned that trypan blue stained the nuclei of plant cells, their procedures were concerned with the demonstration of virus inclusions. The following method was developed and is presented with the emphasis on nuclear staining. The present author hopes that others will try it for comparison with the popular aceto-carmine and aceto-orcein methods.     

Traditional natural dyeing using crotal

Although McWhorter¹ and later Rich² mentioned that trypan blue stained the nuclei of plant cells, their procedures were concerned with the demonstration of virus inclusions. The following method was developed and is presented with the emphasis on nuclear staining. The present author hopes that others will try it for comparison with the popular aceto-carmine and aceto-orcein methods.