Identification of Spermatozoa in Criminological Investigations

Materials used for study were viral smears or ultra-thin sections containing viral cell inclusions. They were stained with the Feulgen reaction and other cytochemical procedures. Stained preparations were dried and then shadow-cast with metallic chromium for 30 seconds in a bell jar with a vacuum of at least 0.1 µ (10^?4mm.) of mercury, and placed at a shadowing angle of 10–12°. Shadow-cast preparations were cleared with xylene and mounted in Canada balsam. Dried smears or deparaffinized sections without staining were suited to this method also. A virus which stained indistinctly with cytochemical procedures alone could be adapted to visible light microscopy by shadowing, and in addition, used for observations on its chemical composition.     

Freeze-Dehydration for Permanent Mounts after Aceto-Orcein Stain

Fresh smears of mammalian material were stained 5 min (only) by La Cour's (1941) aceto-orcein method at 60°C, briefly rinsed and drained, and then dehydrated in a prechilled equal-parts mixture of absolute alcohol and anhydrous acetone held 2 hr or more at — 20°C or lower by means of dry ice. The preparations were differentiated 30 sec at room temperature in 95% alcohol containing 1% HCl, counterstained (optional) in a 0.01% solution of fast green FCF in absolute alcohol for 10 sec, passed through 2 changes of xylene and mounted in HSR or other synthetic resin.     

The Feulgen Reaction After Hydrolysis at Room Temperature

Among the cytochemical methods for demonstrating desoxyribonucleic acid, the hydrochloric-acid-Schiif reaction has been valuable since Feulgen reported it in 1924. A difficulty in that technic is that the section may come loose from the slide; this is caused by hydrolysis at 60° C. When sections were hydrolyzed by 1, 3, 5 or 6 N HCl at room temperature for 15 minutes, adequate hydrolysis and the strong development of color occurred with 5 N HCl. Similarly successful results were obtained with 5 N nitric acid hydrolysis for 10 minutes. Both procedures appear to be as practical as hydrolysis in 1 N HCl at 60° for 4-6 minutes.     

Modifications of Kohn's chlorazol black E staining and Wheatley's trichrome staining for temporary wet mount and permanent preparation of Entamoeba histolytica.

Preparation of stained smears of Entamoeba histolytica has several drawbacks. We therefore tried to simplify the staining procedures by modifing Kohn's chlorazol black E staining and Wheatley's trichrome staining techniques. Trophozoites and cysts of axenically cultured E. histolytica and Entamoeba invadens, respectively, and trophozoites and cysts of E. histolytica in stools of patients were used. Karyosomes and peripheral chromatin of nuclei and chromatoid bodies became distinctly visible after amoebae were suspended in the basic solution of Kohn's stain. Amoebae fixed in suspension with either basic solution or Bouin's fixative were clearly stained with Kohn's and trichrome preparations, both as wet mounts directly and as permanent slides after processing for mounting. These procedures were easier when the basic solution was used as a fixative and trichrome stain was employed. Erythrocytes ingested by trophozoites, however, were not stained with either of these preparations after fixation in the basic solution but were clearly stained when Bouin's fixative was used. Cysts of E. histolytica in stools concentrated using basic solution (instead of formalin) and ether were also stained with these stains. Consequently, without employing highly toxic mercuric chloride, wet mounts and permanent smears can be prepared with permanent stains, and preserved cysts can be stained after concentration.     

Microfluorometry of pectic materials in the dehiscence zone of almond (Prunus dulcis [Mill.] DA Webb) fruits

We examined the middle lamella and the primary and secondary walls in almond pericarp dehiscence zone cells using a fluorescent cytochemical method which permitted specific, quantitative detection of pectic cell wall materials. Glycol methacrylate-embedded sections were stained with coriphosphine and pectin-specific fluorescent emissions at 630 nm were quantified using green excitation (546 nm). Examination of sectioned material extracted with purified pecto-lytic enzyme preparations was used to demonstrate the relative specificity of the staining reaction for pectic substances.     

Gomori's Hematoxylin as a cHromosome Stain

The chromic hematoxylin of Gomori (1941) can be used as an excellent chromosome stain after hydrolysis of the tissue in warm 1-N hydrochloric acid. The hydrolysis must be accurately timed for different material as in the case of the Feulgen reaction. The staining of sections can be performed at room temperature and requires about 15 minutes. For pieces of tissue and whole preparations, it is recommended to stain at 60°C. for 40 minutes. Sections stained at room temperature can be differentiated in 1% hydrochloric acid alcohol for one minute and can be counterstained with phloxine according to Gomori's formula. Whole preparations or sections stained at 60°C. must be differentiated in 45% acetic acid for half an hour or more. Tissue pieces may, after staining, be squashed and examined in the acetic acid, but the preparations can also be made permanent. The blue-black stain is very selective and has the advantage of giving high contrast, and it is nonfading, and insoluble in water and other common reagents. It proved definitely superior to other chromosome stains for difficult material such as planarians, rabbit blastocysts, and cleavage stages of sea urchins. Though both the procedure and the result of this method show some similarity to the Feulgen reaction nothing can be said with certainty about its chemical basis.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

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.     

Standard Specimens for Stain Calibration: Application to Romanowsky-Giemsa Staining

Standardized specimens with reprodcible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied ±5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as ±5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.     

Comparative Ploidy Studies Using Cytological and Paraffin Section Preparations

The ploidy profiles of benign and malignant tumours can be obtained using image analysis. However, the results of ploidy studies have varied according to the type of specimen used. We compared the ploidy profiles of paraffin embedded thin sections, cytospin preparations of disaggregated cells, and cytological smears from the same specimen as defined by image analysis. Ten benign breast lesions, 10 breast carcinomas and 10 malignant melanomas were investigated in this way. Preparations stained by the Feulgen technique were examined using the MD20 video image analysis densitometry system. Ploidy profiles were obtained by measuring the integrated optical density of at least 200 nuclei. By paying proper attention to the quality of fixation and presence or absence of cytoplasm around cells, comparable results were found for all preparations in each case. We therefore conclude that if careful attention is paid to the technical quality of the material, reliable ploidy results can be obtained by image analysis.     

Filipin as a cholesterol probe. II. Filipin-cholesterol interaction in red blood cell membranes

Filipin, a mixture of polyene antibiotics which form complexes with cholesterol, perturbs membrane lipid organization, and causes hemolysis of erythrocytes, is increasingly used as a cytochemical probe for the distribution of cholesterol in cell membranes. We used light (phase-contrast, dark-field and fluorescence) and electron microscopical techniques (whole-mount shadowing, negative staining, and freeze-fracture) to study the interaction of filipin with unfixed and glutaraldehyde-fixed human red blood cell (RBC) membranes. Lysis time and extent depended upon the cholesterol:filipin (C:F) ratio. Lysis was prevented by osmotic protection with high MW dextran. Filipin treated cells fluoresced, but variation in fluorescence intensity among unfixed as well as among fixed cells was evident both at low and high C:F ratios. Negatively stained preparations of unfixed cells lysed on grids or in suspension revealed ring- or C-shaped filipin-induced lesions (FIL) equipped with a veil-like appendage; single FIL, and FIL fused by their veils into aggregates, were shed from membranes. FIL at the surface proper of shadowed whole-mounts and of freeze-etched preparations of prefixed cells appeared as single, dispersed or aggregated cylinders protruding to variable heights above the membrane's plane; aggregated FIL were shed from cells. The freeze-fracture appearance of FIL differed in membranes fixed before or after filipin treatment. E- and P-faces of post-fixed membranes exhibited cylindrical protrusions and depressions, respectively; in essence, the reverse was found in pre-fixed RBC. Both pre- and post-fixed membranes showed considerable variation in the number of FIL on individual cells whether incubated at high (1:1) or low (1:5) C:F ratios, or for a short (10 min) or a long (80-180 min) time. Aggregation and shedding of FIL was evident in all preparations. Thin layer chromatography of the incubation fluid after sedimentation of cells showed that membrane cholesterol was shed from incubated cells. The presented data question the feasibility of filipin as a probe for the topographical distribution of cholesterol in cell membranes.     

Tissue Shrinkage Caused by Attachment of Paraffin Sections to Slides: its Effects on Staining

Sections were cut from a wide variety of tissues, and those from each block were divided into four groups before attaching and drying on slides. Four commonly accepted sources of heat were used for drying: (a) gas hotplate set at 65° C; (b) incubator, 37°; (c) oven, 56°; and (d) room temperature, 20°. After drying, the sections were stained, then examined for intensity of staining and for distortion caused by shrinkage. With both soft and decalcified tissue stained by haematoxylin and eosin, the best results occurred in the sections dried at 20° C; the next best at 37°. When stained by Van Gieson's method, both types of tissues were best after 20° drying, but the second-best group showed differences in favour of 56° for soft tissues and 37° for decalcified. After drying decalcified tissue at 65°, the staining of collagen by acid fuchsin was almost completely absent. When impregnated with silver, for reticulin, the best results for soft tissues were after 56° drying; second best, 20°; but decalcified tissues showed a reversal of this order. After PAS, there was an increasing intensity of staining from 20° to 65°, with soft tissue; evidence that histochemical interpretation could be strongly influenced by drying temperature.     

Identification of Campylobacter pylori in gastric biopsy smears

The use of gastric biopsy imprint smears to diagnose Campylobacter pylori was compared with the use of tissue sections and cultures. Multiple gastric biopsies were taken from the mucosa of 42 patients during endoscopy. Imprint smears were prepared from the samples used to make tissue sections; other samples were used for microbiologic culture. There was a good concordance (93%) between the morphologic diagnosis of C pylori in the air-dried, Giemsa-stained smears and the tissue sections; the cytologic preparations were clearly positive in six cases (14%) whose sections contained low numbers of the organisms. There was a concordance of 83% between the combined morphologic techniques and the bacteriologic culture. Six positive cases were detected only by the morphologic techniques while one positive case was detected only by bacteriologic culture. C pylori was identified in one or more preparations of the antral biopsy specimens in 23 (55%) of the 42 cases, including 23 (74%) of the 31 cases with a final diagnosis of gastritis or ulcer. These results show the usefulness of the cytologic study of gastric biopsy smears in diagnosing C pylori infections.     

Immunogold labeling of metaphase cells

A method is presented for the phenotypic identification of metaphase cells stained for chromosome aberration and SCE analysis. The cells are labeled in suspension with antibodies conjugated with colloidal gold, and then chromosome preparations are made using a cytocentrifuge. A silver development (IGSS) procedure is used to enhance the gold labeling for light microscopy. A variety of fixatives may be employed, permitting various cytogenetic and cytochemical staining procedures to be used.     

Needle aspiration cytodiagnosis of the breast with abundant acellular calcific material mimicking soft tissue calcinosis

OBJECTIVE: To investigate calcific deposits in breast tissue by needle aspiration cytology (NAC). STUDY DESIGN: The aspirate was obtained using a disposable 10-mL syringe and 22-gauge needle. No smears were made, and material was collected as needle and syringe washings in a cytology container in which 30% ethyl alcohol in physiologic saline was present. From about half of this material, filter preparations were made on 3-microm Schleicher and Schuell filters and stained by the Papanicolaou method; the remainder of the aspirate was spun, and a cell block was made from the sediment and sections cut and stained with hematoxylin-eosin (H-E) stain. RESULTS: The filter preparations and sections of cell block in all the cases showed numerous amorphous, basophilic, deep-stained fragments of refractile material and some fragmented bod ies and concentric lamellations, which were diagnosed as calcium. No breast elements or malignant cells were found. The patients had no further treatment and on follow-up were clinically well, with no changes. CONCLUSION: The 9 cases described are interesting because the cytohistologic findings in the NAC sample mimicked soft tissue calcinosis.     

Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

U.S. Choi and D.Y. Kim Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours Objective: To investigate whether Diff‐Quik stained fine needle aspirate smears can be used to evaluate Ki‐67 expression by immunocytochemistry. Methods: Both cytological and histological samples were obtained from 24 dogs with spontaneously developed mammary gland tumours. The cytological and histological specimens were examined by Diff‐Quik and H&E stains, respectively. After examination, both samples were immunostained using the same Ki‐67 antibody. The % Ki‐67 values were calculated based on the percentage of positively stained tumour cells per 500 and 1000 tumour cells in cytology and histology specimens, respectively. Results: Ki‐67 staining was successful in 17/24 smears (71%) and 19/23 sections (83%). The correlation coefficient between the percentage of Ki‐67‐positive cells in cytological smears and in the histological sections was 0.677 (P < 0.01). These values were significantly different between histologically benign and malignant tumour groups both in cytology and histology samples (P < 0.001). The threshold value of the percentage of Ki‐67‐positive cells for distinguishing benign from malignant tumours was set at 4.85% with 90.9% sensitivity and 92.3% specificity by Receiver Operating Characteristic (ROC) curve using histopathology as the gold standard. Conclusion: Diff‐Quik‐stained cytology smears can be used to detect the presence of Ki‐67 antigen when histology sections are not available.     

Cytochemistry and biochemistry of acid phosphatases. III. Inhibition experiments of lysosomal and secretory acid phosphatases of the rat ventral prostate

Biochemical and cytochemical inhibition experiments of rat prostatic acid phosphatase were performed using enzymes separated on isoelectric focusing (IEF) gels, and thin sections of the rat ventral prostate. Various inhibitors, including L (+) tartrate, mercuric ions and sodium fluoride were applied to electrofocused enzymes which were subsequently stained for acid phosphatase activity. Enzymes focused on IEF gels at pH 7.9 and 8.1, respectively, were inhibited with 1.8 x 10-3 M tartrate, while the enzyme activities with isoelectric points (pl) of 5.6 and 7.15, respectively, were only slightly inhibited by this compound. Using 10-3M mercuric ions, enzymes with pl of 5.6 and 7.15 were inhibited while the enzymes with pl of 7.9 and 8.1 were still active. The biochemical procedures were adapted to chopper sections of perfused-fixed ventral prostate of the rat. Preincubation of the sections with 2.4 x 10-3M mercuric chloride blocked the secretory enzyme and most of the lysosomal enzyme and resulted in an artificial staining of the Golgi apparatus and other cytoplasmic organelles. Nuclear precipitates however were prevented. L (+) tartrate could not be used at the ultrastructural level since it developed false positive results by the formation of lead tartrate. The results indicate that no selective inhibition of either secretory or lysosomal acid phosphatase can be achieved at the ultrastructural level using metal salts or tartrate, respectively.     

Ninhydrin Staining of Tissue Sections

This method represents a considerable improvement over earlier ninhydrin procedures. Celloidin sections were stained after mounting in a medium which clears with incubation at 55 °C. There appears to be no reason why paraffin section cannot be used. The sections were not placed in a large volume of ninhydrin (0.25% triketohydrindene hydrate in n-butanol) but only a small volume was sprayed onto the slide. Distortion resulting from heating in boiling water to develop the color was avoided by a slower treatment of 3 days' incubation at 55 °C. The use of water as a solvent in staining is also avoided, thus minimizing the possibility of color migration and insuring against the development of the intensely colored products of the ninhydrin reaction that occur in aqueous solution. Slides need not be observed upon the day of preparation, since the color was stable for about a week after its formation.     

Staining Streptomyces Scabies in Lesions of Common Scab of Potato

Toluidine blue can be used to stain Streptotnyces scabies distinctively in slide cultures or in the lesions of common potato scab. This staining method is based on the metachromaticism of volutin, a constant constituent of the spores and mycelium of S. scabies. Either sections or smears are fixed in FPA (formalin, 5 parts; propionic acid, 7.5; 50% alcohol, 87.5), stained in a 1:100 dilution of saturated aqueous toluidine blue from 20 minutes to 24 hours, dehydrated in an acetone-xylene series and mounted. Cellular constituents of the potato tuber stain blue or are colorless whereas the mycelium of Streptomyces appears as a series of red volutin spheres in the blue stained cytoplasm. The criteria of volutin and cytoplasmic staining along with the 1 µ diameter of the mycelium make it possible to distinguish Streptomyces from the other microorganisms and cells in the lesion region.     

Sputum cytology in the diagnosis of pulmonary paracoccidioidomycosis

The presence of Paracoccidioides brasiliensis was determined in sputum samples from 50 patients with paracoccidioidomycosis using four different techniques: (a) cell-block preparations stained with silver methenamine, (b) direct microbiologie examination, (c) smears stained with Shorr, and (d) smears stained with silver methenamine. Overall, cell-block preparations and smears stained with silver methenamine proved to be the most sensitive techniques, followed by smears stained with Shorr and direct microbiologic examination in decreasing order of sensitivity. Sputum cytology tended to be less positive in patients with interstitial pulmonary lesions as determined by chest X-ray than in patients with alveolar lesions. In addition to its high sensitivity, cell-block preparation technique allows storage of blocks and slides for further studies.