The Acetocarmine Smear Technic

The acetocannine smear technic is described primarily for beginners. An effort was made to bring together into one paper the work-a-day details to be followed in collecting specimens and making preparations of plant chromosomes at prophase, of condensed chromosomes at meiosis and mitosis (including mitosis in microspores), and of coiled chromonemata. A schedule for converting temporary slides into permanent mounts is given, and a technic for making smears of the chromosomes in the salivary glands of Drosophila is described.     

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.     

A New Hematological Stain: I. Constituents and Methods of Use

The stain proposed by the author is quickly and simply prepared by mixing equal parts of the following permanent stock solutions:

The mixed stain is usable, for at least eight months, and is applicable to practically all hematological purposes: blood smears, fixed sections, frozen sections, and touch preparations. In the technics utilized to produce its action on preparations treated in different ways, the only variants are the methods for treating the cells, while the stain itself remains totally unchanged for all purposes. For blood smears fixed with methyl alcohol, the technic consists merely of pouring the stain on the slide, leaving it for 5 to 7 minutes, and then washing it off. On sections, a further process of differentiation with acid acetone is rapidly carried out. The various types of granules, including megakaryocyte and platelet granules, are clearly demonstrated. For frozen sections, the technic is extremely rapid, yet yields excellent differentiation.     

A Flagella Staining Technic for Soil Bacteria

In an attempt to stain the flagella of soil bacteria, many of which have flagella so fine that they are hard to stain by most methods, a technic was developed which combines the best points of Hofer and Wilson's method with that of Bailey as developed by O'Toole. Satisfactory preparations have been obtained for organisms of the genera Pseudomonas, Phytomonas, Alcaligenes, Escherichia, Azotobacter, and Bacillus. This technic, therefore, is recommended as a rapid and constant method for routine flagella staining of all motile aerobic organisms; it combines Hofer and Wilson's method of cleaning the slides with O'Toole's technic of spreading the smears and with a modification of Bailey's mordant.     

A simple staining method for vaginal smears using red ink.

In adult rhesus monkeys each phase of 37 menstrual cycles was accurately classified by cellular identification of vaginal smears stained with red ink. This technic was simple, quick, and inexpensive, and produced well-stained slides in a few seconds.     

Treatment of Trillium Erectum Prior to and During Mass Production of Permanent Smear Preparations

Technics used in handling dormant plants of Trillium erectum L. prior to and during microsporogenesis are described. Normal meiosis occurs during storage of the plants at 4-6°C. Approximate times in days required for various stages of meiosis are presented. Schedules used for the mass production of aceto- or propiono-carmine smears of microsporocytes, microspores and pollen tubes are also given.     

Demonstrating Weakly Acid-Fast Tubercle Bacteria in Sputum

Acid-fastness of the tubercle bacteria has long been used as the common method of diagnosis in sputum. It has been suggested sometimes that tuberculosis could occur without demonstrable bacteria, as well as with acid-fast bacteria, non-acid-fast bacteria or granules. It is shown in this paper that some of the sputa which are negative to the standard staining technic will show rods, rods with round polar bodies, or similar bodies without the rod portion. It is also pointed out that the decolorization of the smears by acid alcohol be shortened to approximately 3 to 5 seconds and picric acid be used as a counterstain. These forms are apparently the varying stages of the loss of acid-fastness. It is essential that a counterstain be used which will not interfere, and yellow is indicated because it does not absorb the red rays. Sputa which are negative to the standard acid-fast staining technic but which come from persons with a variable intermittent fever should be stained by this modified technic before they are pronounced germ-free.     

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 Use of Picric Acid with the Gram Stain in Plant Cytology

The technic described involves the use of a saturated solution of picric acid in absolute alcohol in the process of dehydration following the gentian-violet-iodine stain as applied to plant cytological material. The method is suitable for both paraffin sections and smears of pollen mother cells fixed in Navashin's or Flemming's solutions. Differentiation in clove oil is very easy since cytoplasm destains immediately, while chromatic material destains very slowly following picric acid. Chromosomes are stained more distinctly than with the usual Gram stain and do not fade.     

A Modified Wright's Method for Staining Blood Smears

After the blood smear is treated for the proper length of time with Wright's stain, neutral distilled water is used for diluting the stain. After the slide has been treated with neutral distilled water until the smear becomes pinkish it is then treated with pure absolute methyl alcohol which destains the plasma. Neutral distilled water is again kept on the mount until the corpuscles are well stained. Then the slide is dehydrated with absolute ethyl alcohol, cleared with clove oil and completed in the usual way.

Blood smears of different groups of vertebrates were uniformly brilliantly stained with the above technic.

Several lots of Wright's dry stain have been tested with the modified technic and no difficulties have been encountered in its application.     

A Smear Technic for Demonstrating Cell Inclusions with Characteristics of Both Mitochondria and Bacteria

Cytoplasmic inclusions of various types of cells have been investigated by macerating or smearing and fixing and staining by different mitochondrial methods of technic. The results obtained as regards granular, rod-like, filamentous and globular forms immediately suggest a relation between these and similar cell inclusions which have in the past been described as mitochondria in certain cases of this material. While mechanical disturbance and drying before fixation apparently do not alter the staining properties of these forms, alcohol produces somewhat variable results depending upon the kind of material being investigated. Results indicate the presence in these smears of numerous intracellular bacteria, readily misinterpreted as mitochondria. In addition, there occur in certain cells, both in smears and sections, inclusions of indeterminate nature.     

The Effect of Organic Solvents on the Appearance of Bacterial Nuclei

Treatment of bacterial smears with organic solvents was found to produce a typical appearing nuclei which could lead to false interpretation of nuclear events. The crystal violet nuclear stain, which does not utilize organic solvents or acid hydrolysis, was used as a basis of comparison. Acid hydrolysis also was observed to affect the appearance of the nucleus. It is concluded that caution should be used in interpreting nuclear structure and activity in bacterial cells, especially when organic solvents or acid hydrolysis are involved in the staining technic.     

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.     

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.”     

A New Fixative for Vaginal Smears

Two vaginal smear fixatives have been presented for use in cytologic studies by the Papanicolaou technic for the diagnosis of cancer of the genital tract. They are to be used in lieu of equal parts of ethyl alcohol and ether, because of the volatility, waste through evaporation, fire hazard, and expense. They are (a) tertiary butyl alcohol, 75% and ethyl alcohol, 25% (b) tertiary butyl alcohol, 75% and ethyl phosphate, 25% (by volume). The cytologic details and staining qualities of vaginal smears have been maintained or improved.     

Identification and Counting of Basophil Leucocytes

A 1% alcoholic solution of toluidine blue is used and recommended for specific staining of the basophil leucocytes in blood smears as well as in tissue sections. The cytoplasmic granules show a metachromatic (purple) stain in contrast to other elements taking an orthochromatic (blue) stain. For direct counting a 0.05% alcoholic solution of toluidine blue adjusted to pH 7.7, and containing an optimal concentration of saponin as a hemolyzing agent, has been successfully used for identifying and counting basophils, eosinophils and all other leucocytes, simultaneously in the same chamber. Blood dilution of 1:10 by means of micropipettes is preferable to that made by the ordinary leucocyte diluting pipettes. The technic described is recommended for routine use in hematological laboratories.     

The Papanicolaou Stain for Negri Bodies in Paraffin Sections

Paraffin sections ot the hippocampus (Amnion's horn) from brains of dogs and cows, fixed in sublimate-alcohol (HgCl₂, sat. aq., 1 vol.; absolute alcohol, 2 vol.) were stained by Papanicolaou's (1942) method for vaginal smears. Negri bodies were stained a bright rose color, with nucleoli dark blue. Even though the orange G were omitted, good staining of Negri bodies was obtained. Eliminating this dye simplifies the technic without impairing its effectiveness in the diagnosis of rabies, but the complete staining gives a somewhat more colorful and brighter histologic picture.     

Identification and Counting of Basophil Leucocytes

A 1% alcoholic solution of toluidine blue is used and recommended for specific staining of the basophil leucocytes in blood smears as well as in tissue sections. The cytoplasmic granules show a metachromatic (purple) stain in contrast to other elements taking an orthochromatic (blue) stain. For direct counting a 0.05% alcoholic solution of toluidine blue adjusted to pH 7.7, and containing an optimal concentration of saponin as a hemolyzing agent, has been successfully used for identifying and counting basophils, eosinophils and all other leucocytes, simultaneously in the same chamber. Blood dilution of 1:10 by means of micropipettes is preferable to that made by the ordinary leucocyte diluting pipettes. The technic described is recommended for routine use in hematological laboratories.     

A study of the kinetics of microsporogenesis in Campelia zanonia (L.) H.B.K.

During maturation, microspores pass through a series of morphologically distinguishable stages or compartments. A study has been made of the systematic fluctuations in the frequency of microspores in these compartments, when plants are grown under rigidly controlled conditions. A new approach to the construction of cumulative flux rate curves is described; these give the number of cells passing the compartment boundaries per unit time. The curves obtained indicate that simple models, which assume constant flux rates and compartment transit times, will not explain the observations. It is evident that not only do microspores mature at different rates, but that the maturation rate of individual microspores varies during the developmental sequence. The overall process may be controlled by the intimate relationship which exists between the microspores and the tapetal periplasmodium in the Tradescantiae.