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.     

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 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.     

Acid Thionin Stain for Nissl Bodies on Frozen Sections

Using a buffered acid thionin stain with carbolxylene as a clearing agent, a reliable stain for Nissl bodies may be performed on frozen sections of fresh or old formalin-fixed material in a relatively short period of time. The technic is simple: the buffering of thionin makes regressive differentiation unnecessary.     

Some monoclonal anti-human lymphocyte and class II MHC antigen antibodies do not stain snap frozen Macaca fascicularis lymphocytes or tissue sections.

Experiments were designed to stain snap frozen and fixed Macaca fascicularis peripheral blood mononuclear cell (PBMC) preparations and colon sections assuming that monoclonal mouse anti-human lymphocyte and class II major histocompatibility complex antigen antibodies would cross-react with the monkey counterparts to the human antigens. Most of the monoclonals used in this study did not stain the monkey frozen and fixed tissues in immunoenzymatic protocols despite adequate staining of control human tissues. The fact that snap frozen Macaca fascicularis PBMCs and tissue sections were not always stained is of practical importance for experimental design in Macaca fascicularis using anti-human reagents.     

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.     

Recent Advances in Microtechnic. I. Methods of Studying the Development of the Male Gametophyte in Angiosperms

Among methods used for a study of nuclear details in the development of pollen grains, the following were found to be very satisfactory: (1) warming the entire grains in aceto-carmine and then clearing with chloral hydrate; (2) making smear preparations stained with crystal-violet-iodine or iron alum hematoxylin. For paraffin sections, a counterstain with dilute alcoholic erythrosin is often very useful after the usual iron hematoxylin technic.

A method of making cultures of pollen tubes on slides coated with thin films of sugar agar is described in detail. The tubes can be fixed by immersing the slide in formol-acetic-alcohol and then stained by any desired schedule. Iron alum hematoxylin was found to be the most satisfactory, but the Feulgen reaction is very valuable in such cases where the nuclei are obscured by the density of the pollen tube cytoplasm. Living pollen tubes can be kept under observation by dissolving a small quantity of neutral red or other vital stain in the sugar agar before it is spread on the slide.

For studying stages in fertilization or gametogenesis, styles should be fixed and sectioned only after a preliminary study with iodine-chloral-hydrate or safranin-anilin-blue or aceto-carmine. Once the extent to which pollen tubes grow in a given time in the stylar tissues has been determined, it is possible to fix material with some knowledge of what it is going to show.

Some other methods, that have not been tried by the authors but appear to be valuable, are also briefly described.     

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.     

Marchi's Staining Method: II. Fixation

Following experimental lesions, spinal cords of cats and rabbits were fixed with acid, neutral, and alkaline solutions. Staining was limited to a chromate-osmic (Marchi's) solution and a chlorate-osmic solution. The following conclusions were drawn:

The presence of an acid in the fixative caused normal myelin sheaths to stain. This effect was reduced by washing tissues before staining, by adding acetic acid to the stain, or by employing a non-formalin fixative. It was, however, at no time entirely obviated.

A study was made of the granular deposits which occur in nearly all Marchi preparations and which are especially confusing if very light backgrounds are obtained.

The staining reactions of the granular deposits were very similar to those of degenerating myelin but some suppression of the granules was obtained by adding KCIO₃ to the formalin fixative.     

Cytodiagnosis of central neurocytoma in intraoperative preparations

OBJECTIVE: To assess the cytologic features in smear preparations of 3 central neurocytomas. STUDY DESIGN: Three patients with central neurocytoma underwent intraoperative frozen section diagnoses, and the cytologic evaluations are presented. RESULTS: The smears typically showed cellular tumors composed of isomorphous, round cells. The tumor cells showed ill-defined cytoplasm oval nuclei with finely granular chromatin and micronucleoli. A fibrillary matrix in the background was noted in all cases. The tumor in the 20-year-old patient exhibited numerous giant cells with phyagocytosed hemosiderin granules between small, round tumor cells. Permanent sections, immunohistochemistry and electron microscopy confirmed that all cases were central neurocytomas. CONCLUSION: Central neurocytomas can be diagnosed reliably using combined cytologic preparations and frozen sections. The appearance of numerous macrophages that phagocytose hemosiderin between neoplastic cells should also be considered characteristic of the cytomorphology of central neurocytomas.     

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.     

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.     

Cytochemical method for staining fish basophils

The occurrence of basophils in peripheral blood of 15 freshwater teleosts was examined using the metachromatic stain, toluidine blue, on blood smears fixed with lead subacetate. Metachromasia was more identifiable through better preservation of the granules. The occurrence of basophils in teleost blood was confirmed as very rare and was not caused by failure to preserve the basophil granules.     

Combined Carbowax-Paraffin Technic for Microsectioning of Fixed Tissues

A combined Carbowax-paraffin technic for microsectioning fixed tissues gave ribbon sections as do paraffin infiltrated and embedded tissues. Blocks of formalin or alcohol fixed tissues 2 mm. thick were infiltrated with H.E.M. (Polyethlene Glycol: Carbowax, Hartman-Leddon Co.) or with one of the following polyethylene glycol ester waxes (Glycol Products Co., Inc.) for 4 hours at (61°C.): Polyethylene Glycol 600(Di) Stearate; Carbowax 1000-(Mono) Stearate; Carbowax 4000 (Mono) Stearate; Carbowax 4000 (Mono) Laurate; Carbowax 6000 (Mono) Oleate. The Carbowax infiltrated tissues were placed for 10 minutes in xylene (61° C.), into paraffin (61° C.) for 30 minutes, then into molten paraffin contained in separate molds. (The xylene passage can be excluded for preparations which preclude its use). The blocks were hardened rapidly by submerging in ice water and were fastened to carriers as in the usual paraffin technic. Tissues were cut 6 µ thick. Segments of ribbon were spread on a water bath and mounted on slides. After drying, tissues were stained directly with hematoxylin-eosin or were carried through xylene and alcohols as in routine paraffin preparations prior to staining. The Sudan III fat stain and Best's carmine stain for glycogen were applied as in usual technics. Cellular detail was well preserved and structures did not show the extent of distortion and shrinkage encountered in ordinary paraffin technic preparations.     

A Rapid Histological Technic for Staining Latex in Roots of Taraxacum Kok-Saghyz

The freezing technic described in this paper provides permanent slides of sections of the root of Taraxacum kok-saghyz with latex preserved in place. The following schedule is used: (1) Prefreeze the piece to be sectioned before removal from the root. (2) Mount in ice and section with chilled microtome knife. (3) Plunge frozen sections into the combination coagulant and stain prepared from Calco oil blue N. A., acetic acid and ethyl alcohol. (4) Wash in water. Aspirate if necessary. (5) Mount on a slide using Karo.

This technic is rapid and simple. The sections are well adapted to making counts and measurements of latex tubes since there has been a minimum of latex loss. Latex is retained in place by keeping tissues frozen until introduced into the coagulant.     

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.     

Distribution of acid phosphatase, beta-glucuronidase, n-acetyl-beta-d-glucosaminidase and beta-galactosidase in cornea of albino rabbit.

Activities of acid phosphatase, beta-glucuronidase, N-acethyl-beta-D-glucosaminidase and acid beta-galactosidase were investigated histochemically in rabbit corneas. Frozen sections after block fixation in cold 4% formaldehyde with 1% CaCl2 followed by washing in cold physiological saline as well as cold microtome sections of corneas quenched in petroleter chilled with acetone-dry ice mixture, transferred to nonprecooled slides or semipermeable membranes were used. Standard aqueous media were employed in the case of free-floating frozen sections of fixed corneas as well as of cold mictrotome sections (postfixed in cold 4% formaldehyde). Agar media were used in connection with the technic of semipermeable membranes. Gomori method (in the case of acid phosphatase), simultaneous azocoupling methods (substrates derivated of naphthol-AS-BI with hexazonium-p-rosanilin) in the case of acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase and the indigogenic method in the case of acid beta-galactosidase were applied. Enzyme activities in sections of fixed corneas were minimal in comparison with those in cold microtome sections of unfixed material revealed particularly with the technic of semipermeable membranes which is to be preferred. This technic is recommended in studies concerned with lysosomal enzymes in the cornea, particularly in keratocytes. All enzymes investigated were present in corneal epithelium, keratocytes and endothelium. Acid phosphatase displayed the highest activity followed by beta-glucuronidase and acetyl-beta-D-glucosaminidase. The activity of beta-galactosidase was the lowest. For the demonstration of activities in keratocytes sections parallel to the surface are very suitable. In these sections enzyme activities were demonstrated in small granules (apparently lysosomes) present in the central part of their cytoplasm as well as in projections. Diffuse staining was also seen, being the highest in the case of acid phosphatase.     

A Note on the Gram Stain

A modification of the Gram stain in which iodine-alcohol is substituted for 95% alcohol as a decolorizing agent has been found particularly useful in staining Gram-positive organisms in tissues and also for smears. The technic for tissue sections follows:

1. Apply nuclear stain.
2. Wash.
3. Stain in Hucker's gentian violet 2 to 3 minutes (i. e. 1 part Sat. Alc. Sol. crystal violet to 4 parts 1% Aqu. Sol. ammonium oxalte).
4. Wash in water.
5. Stain in Gram's iodine 5 minutes.
6. Wash in water.
7. Decolorize in 95% alcohol to which enough tincture of iodine has been added to give a mahogany color.
8. Counterstain.
9. Dehydrate and mount.

     

Microwave-assisted staining procedures in routine histopathology

Summary The use and practicability of microwave-assisted staining procedures in routine histopathology over more than three years has been evaluated. A domestic microwave oven was used to speed up the following staining procedures: Haematoxylin-Eosin (for frozen sections), Romanowsky-Giemsa, Periodic acid-Schiff (PAS), Ziehl-Neelson, Papanicolaou, Feulgen and Grocott — stain on buffered formalin fixed sections or cytologic smears. These staining procedures can be made highly reproducible providing; (1) Staining vessels are placed in the same position inside the oven; (2) Accurate timing in seconds is observed. Microwave-assisted staining procedures are equal to or even superior to those of the standard methods. Staining times can be reduced to 2%–10% of the conventional staining procedures. The basic staining protocols are presented.     

Comparison of ThinPrep and conventional preparations on fine needle aspiration cytology material

OBJECTIVE: To compare the various cytologic features on ThinPrep 2000 (TP) (Cytyc Corporation, Marlborough, Massachusetts, U.S.A.) and conventional preparation (CP) specimens from fine needle aspiration cytology (FNAC) material by a semiquantitative scoring system. STUDY DESIGN: In this prospective study a total of 71 consecutive cases were included. In each case, two passes were performed. The first pass was used for conventional preparations, with direct smears made and fixed immediately in 95% alcohol for Papanicolaou stain. For TP preparation a second pass produced material for processing in the ThinPrep 2000. The TP and CP slides were studied independently by two observers and representative slides of CP and TP compared for cellularity, background blood and necrotic cell debris, cell architecture, informative background, presence of monolayer cells, and nuclear and cytoplasmic details by a semiquantitative scoring system. Statistical analysis was performed by Wilcoxon's signed rank test on an SPSS program (Chicago, Illinois, U.S.A.). RESULTS: TP preparations contained adequate diagnostic cells in all cases and were tangibly superior to CP preparations concerning monolayer cells, absence of blood and necrosis, and preservation of nuclear and cytoplasmic detail (statistically significant, Wilcoxon's signed rank test, P < .000). CONCLUSION: TP preparations are superior to conventional preparations with regard to clear background, monolayer cell preparation and cell preservation. It is easier and less time consuming to screen and interpret TP preparations because the cells are limited to smaller areas on clear backgrounds, with excellent cellular preservation. However, TP preparations are more expensive than CP and require some experience for interpretation.