Collodion Fixation and Protection of Cytological Smears for Transportation and for Staining by Papanicolaou's Method

A 1% collodion solution in a 1:1 mixture of 95% alcohol and ether can be used to fix smears and to form a protective coating on them. Such coating permits transportation of dry slides over long distances to a laboratory for staining and diagnosis. The collodion solution can be used several times provided that it is filtered after each specimen is coated. With urine sediments, the application of the coating keeps a large number of cells on the slide, hence facilitates a thorough examination and more reliable diagnosis. Papanicolaou's method is recommended for staining the coated specimens, especially those for the diagnosis of cancer.     

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.     

Rapid Removal of Tissue Culture Monolayers by Collodion for Subsequent Staining

Removing cultures from roller tubes before staining eliminates the destaining which often occurs when the cells are first stained and then removed by embedding in collodion. The cells are fixed in situ, dehydrated, and covered with collodion (Merk's flexible) for 10 min. The collodion is poured off, the fluid residue lining the tube allowed to dry for 10 min, and the tube is filled with tap water. The collodion cast containing the cells is loosened and removed, cut into strips, placed on slides and blotted into firm contact. The collodion is then dehydrated and dissolved with absolute alcohol followed by a 1:1 mixture of alcohol and ether. The slides can then be rehydrated and stained by conventional methods.     

An Apparatus for Fixation and Supravital Staining of Tissues by the Perfusion Method

An apparatus is described for the perfusion of the circulation which permits accurate control of the temperature, pressure and rate of flow of the perfusing fluids. Using this apparatus, the tissues can be perfused initially with saline and subsequently with a fixing solution or supravital stain without changing the cannula. The apparatus can be pre-set to any given requirement, and will thus give reproducible results. It is not suitable for the application of warm volatile fixatives, but these can be perfused cold after preliminary perfusion with warm saline.

Perfusion is improved by the subcutaneous injection of heparin before death, and by the administration of amyl nitrite either in the perfusing fluid or as a vapor during anaesthesia.     

The History of Staining the Development of Cytological Staining

Perhaps no other period has contributed more to our knowledge of the cell than the period 1875-1895. During these years most fundamental cytological phenomena were seen and described. Mitosis, maturation and fertilization, the great cornerstones of cytology, were firmly laid by the remarkable researches of Flemming, Strasburger, Van Beneden, Oscar and Richard Hertwig, Boveri and many others. Upon these researches experimental cytology developed and the significance of the morphological phenomena to inheritance and development was pointed out by such masters as W. Roux, Weismann, O. Hertwig, Boveri and E. B. Wilson.     

Fixation and Staining of Planaria for Histological Study

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acld-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.     

The Cytological Recognition of African Siderosis On Bone Smears

Eight cases of African siderosis primarily recognized on cytological smears are presented and discussed. All the smears were obtained from Jamshidi needle biopsies of vertebral bodies. Six cases showed siderosis only, while a seventh showed two pathological processes on one slide, namely metastatic keratinizing squamous carcinoma and siderosis. The remaining case showed cytological evidence of tuberculosis and siderosis. All cases were histologically confirmed, an additional feature in two cases being osseous tuberculosis which was not evident on the cytological smears. A search of the literature failed to reveal any report on the cytological recognition of this disease, or its association with tuberculosis.     

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.     

A Method for Making Aceto-Carmin Smears Permanent

Anthers are collected and placed in a solution of 1 part acetic acid to 3 parts of absolute alcohol. The contents of the anther are squeezed out on a slide in a drop of Belling's iron-aceto-carmin solution and a cover glass placed over the drop. Care should be taken to remove all anther walls and flower parts. Heat the slide over an alcohol flame for a second, repeating 4 or 5 times. Place the slide in a petri dish filled with a 10% solution of acetic acid. When the cover glass has risen away from the slide gently remove the cover glass and place in a Coplin jar containing equal parts of alcohol and acetic acid. Likewise, place the slide in this solution. Run both cover and slide thru the following solutions: 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic acid to 9 parts absolute alcohol, absolute alcohol and finally equal parts of absolute alcohol and xylol. Recombine the cover and slide in xylol-balsam directly from this solution.     

A Method for Making Aceto-Carmin Smears Permanent

Anthers are collected and placed in a solution of 1 part acetic acid to 3 parts of absolute alcohol. The contents of the anther are squeezed out on a slide in a drop of Belling's iron-aceto-carmin solution and a cover glass placed over the drop. Care should be taken to remove all anther walls and flower parts. Heat the slide over an alcohol flame for a second, repeating 4 or 5 times. Place the slide in a petri dish filled with a 10% solution of acetic acid. When the cover glass has risen away from the slide gently remove the cover glass and place in a Coplin jar containing equal parts of alcohol and acetic acid. Likewise, place the slide in this solution. Run both cover and slide thru the following solutions: 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic acid to 9 parts absolute alcohol, absolute alcohol and finally equal parts of absolute alcohol and xylol. Recombine the cover and slide in xylol-balsam directly from this solution.     

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.     

Controlled-Heat Fixation of Smears and Thin Tissue Slices in Liquid Mercury

Fresh tissue slices 2 mm thick and blood films have been fixed by immersion in mercury maintained at 55 C. Fixation is effected in 1.5 min without an excessive temperature rise—the main disadvantage of flaming or hot air used for dry heat fixation. For haematology, in particular, the method is convenient to use as a standard procedure for a fixation that is compatible with many histochemical and most of the usual histological techniques.     

Relation of Abnormal Cytological Smears and Carcinoma of Cervix Uteri to Husband's Occupation

An analysis of the cytological records of almost 300,000 women in the Manchester area shows that the rates of positive/suspicious findings from population screening are highly correlated with the rates of mortality from cancer of the cervix when both are distributed according to the occupation of the husband. The correlation holds for various occupational groupings and for all the individual occupation units in which there are more than 1,000 women. This evidence strengthens the case for believing that the condition revealed by a positive smear is a stage in the development of invasive cancer of the uterine cervix.     

Foot's Silver and Acridine-Orange Fluorescence Staining of Pneumocystis Carinii in Smears and in Sections

Pneumocystis carinii was stained intensely by the modification of Hortega's silver carbonate method for reticulum. Paraffin sections and smears were fixed in formalin, then processed through all the steps of the staining technique as given in McClung's Handbook (Jones 1950), p. 258. Fluorescence microscopy was also very helpful in disclosing the parasite in air-dried smears. These, were processed through descending grades of ethanol and then stained in 0.01% Acridine orange in phosphate buffer, pH 6.0, for 2 min. The microorganism appeared in a brownish to red range of fluorescence, whereas disintegrated cellular nuclei ranged from green to yellow. A standard binocular microscope equipped with a high-pressure 200-watt mercury vapor burner (HBO 200, Osram) and the necessary filters (BG 12 as exciting and OG 5 or OG 4 as barrier filters) were used.     

Method for Staining Bacterial Flagella

The following method of staining bacterial flagella is ecommended for use on smears made from suspensions of 10 to 16-tour agar slant cultures, incubated 30 minutes at 37°C before spreadng on thoroly cleaned and named slides:

Cover with fixative (100 cc. of 1/4 sat. aqu. solution picric acid, with 5 g. tannic acid and 7.5 g. ferrous sulfate).

Wash with tap water, dry and cover with Fontana spirochaete stain; heat to steaming and allow to act for 1 to 2 minutes. Wash in ap water. The stain is prepared as follows: To 25 cc. 2% AgNO₃ add dilute ammonia till the precipitate which forms redissolves; then add more AgNO₃ till a faint turbidity results. A clear solution is useess.     

Staining of Mitochondria with Aniline-Acid Fuchsin After Zenker-Formol Fixation

It was observed that mitochondria are well-demonstrated by aniline-acid fuchsin staining after Zenker-formol fixation if the sections are not de-Zenkerized. Tests showed that after mordanting in HgCl₂, K₂Cr₂O₇, FeCl₃, or FeSO₄, mitochondria in sections from tissues fixed in neutral buffered formalin could be stained fairly intensely by the same method. Salts of Ag, Ba, Ca, Cd, Co, Cu, Mg, Mn, and Zn were ineffective. If the presence of occasional mercury crystals in the sections is not objectionable, demonstration of mitochondria in Zenkerformol fixed tissues offer speed and additional flexibility in the subsequent use of the blocks as advantages over usual methods.     

Neisseria gonorrhoeae Identification in Direct Smears by a Fluorescent Antibody-Counterstain Method

Direct smears from female patients have been considered unreliable for the detection of Neisseria gonorrhoeae by fluorescent-antibody (FA) methods because of inadequate background contrast of the fluorescent-stained smears and a scarcity of organisms on the smear. Evans blue dye employed as a counterstain eliminated the nonspecific background staining and increased the reliability of the direct FA procedure. Direct smears demonstrating positive fluorescence were obtained from 86% of a group of culturally positive named female contacts. The FA-counterstain technique is as sensitive as the presently recommended cultural procedures.