A Trichrome Stain for Insect Material

Deparaffinized insect sections are brought down to water and overstained in a 0.1% solution of azocarmine G in 1% acetic acid. They are then destained in a saturated solution of orange G until the azocarmine G is removed from the endocuticle and the latter is colored pale yellow. After washing, the sections are transferred to a 5.0 % solution of phosphotungstic acid in water for 3 min. They are then rinsed in distilled water and stained in a 0.1% solution of methyl green in 1% acetic acid until the endocuticle is green. Differentiation is done in 2 changes of 95% alcohol. The sections are then dehydrated either in absolute alcohol or dioxane, cleared in a mixture of “camsal”, eucalyptol, dioxane, and paraldehyde (1:2:2:1), and mounted in Mohr and Wehrle's medium, a mountant of the Euparal type.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

桂枝茯苓胶囊对卵巢囊肿患者囊肿核摘除术后血清雌激素、AMH及LSA水平的影响

目的:探究桂枝茯苓胶囊对卵巢囊肿患者囊肿核摘除术后血清雌激素、抗苗勒氏管激素(AMH)及脂质结合唾液酸(LSA)水平的影响。方法:选择我院收治的已行囊肿核摘除术的卵巢囊肿患者39例,将其随机分为实验组及对照组。对照组18例予甲硝唑联合青霉素注射治疗;实验组21例予桂枝茯苓胶囊治疗。治疗后,对比两组的临床疗效及治疗前后血清雌激素、AMH及LSA的水平。结果:治疗后,实验组总有效率(95.2%)显著高于对照组(72.2%),差异具有统计学意义(P0.05);治疗后,两组血清雌激素、AMH水平均明显升高,血清LSA水平显著降低,差异均具有统计学意义(P0.05);与对照组比较,实验组治疗后血清雌激素、AMH较高,而LSA水平较低,差异具有统计学意义(P0.05)。结论:桂枝茯苓胶囊可以有效提高行囊肿核摘除术卵巢囊肿患者的临床疗效,这可能与其提高血清雌激素及AMH水平并降低血清LSA水平有关。     

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.     

Staining Plant Tissues with Chlorazol Black E and Pianese III-B

The staining schedule was developed for a study of the mycorrhizae of red pine, Pinus resinosa Ait. From 70% alcohol, sections are stained in a saturated solution of chlorazol black E in 70% alcohol, 10-30 min; free dye removed by washing in 95% alcohol; stained 18-24 hr in Pianese III-b; rinsed in 95% alcohol, acidified by the addition of 2 ml of saturated aqueous picric acid per 100 ml, 3-4 changes or until the last change is pale yellow or light green; and rinsing in 95% alcohol to remove the acid. If the acid fuchsin is too intense, a cautious differentiation with 95% alcohol containing 1-3% of a 0.1 N solution of NaOH is made. If too much chlorazol black is removed, the effect can be compensated by overstaining with this dye at the beginning of the process. Sections are dehydrated, cleared, and covered in the usual manner. This stain has applications to plant tissues generally, and is particularly effective for meristematic tissues. It shows details of cytoplasmic structures and gives sharp delineation of primary cell walls.     

正常新西兰白兔阴茎组织超声成像特征的实验研究

目的:明确正常新西兰白兔阴茎组织超声成像特征。方法:性成熟健康新西兰白兔3只(月龄4-5月),猝死后将阴茎切除放入4%中性福尔马林中固定24小时。将阴茎标本置入纯净水中,进行超声成像,扫查切面选择阴茎横截面。将阴茎标本横断面制成病理切片,进行HE染色观察。结果:超声成像清晰显示:阴茎包皮及皮下软组织、阴茎海绵体白膜、阴茎海绵体、尿道海绵体,这些结构的空间位置关系与阴茎标本和组织病理切片完全一致;同时,利用二维超声图像显示的白膜边界进行阴茎海绵体内径的测量,测值与组织病理切片基本一致。结论:利用二维超声可以观察和测量新西兰白兔阴茎组织结构,超声成像可以作为新西兰白兔阴茎疾病模型研究的一项影像学检测方法。     

The New Domestic Cresyl Echt Violet

Specimens of both vertebrate and invertebrate nerve-containing tissues were fixed 2-3 days in Bouin's fluid, soaked 2 days in alcohol containing 2% strong ammonia water, dehydrated and embedded in paraffin. The sections were mounted with gelatin adhesive according to Masson's procedure, dewaxed, passed through graded alcohols to water, then back to 2% ammoniated 80% alcohol for 12-24 hours. The slides were rinsed 3-5 seconds in distilled water, impregnated about one and a half hours in 40% AgNO₃ at increasing temperature up to 45°C. The slides were flooded with 62.5% formalin and this solution allowed to remain 3-5 minutes; they were then blotted with filter paper. A second impregnation in ammoniated silver carbonate, controlled under the microscope, was followed by a 10-minute treatment with 10% aqueous acetic acid, toning with gold chloride, then thiosulfate and finally washing. Counterstaining with ponceau red or acid fuchsin, eventually followed by aniline blue or fast green, dehydration and covering, completed the process.     

Enhancement of the periodic acid — Schiff (PAS) and periodic acid — thiocarbohydrazide — silver proteinate (PA-TCH-SP) reaction in LR White sections

Summary LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination:1) The PAS reaction in semithin sections turns out stronger afterpartial (70% ethanol) than complete (100% ethanol)dehydration of the tissue before its transfer to 100% LR White.2)Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end.3) The use ofhot silver proteinate (50° C) plus strong silver enhancement (15–20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.-Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins

Summary A new general method has been developed for the specific histochemical identification ofO-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose andN-acetylhexosamine with an ester substituenent(s) located on a potentialvicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-Bh*-Bh-PAS) method] the initial PA-Bh treatment rendersvicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from bothO-acyl sugars andO-acyl sialic acids by saponification (KOH), sialic acidvicinal diols are selectively removed by the PA^*-Bh sequence andO-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections.Using the PA-Bh-KOH-PA^*-Bh-PAS method we have demonstrated, for the first time, thatO-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence ofO-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA^*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars andO-acyl sialic acids are selected.     

Suggested Counterstains for Davenport Reduced Silver Preparations of Peripheral Nerves

—Peripheral nerves which have been fixed in a mixture of formaldehyde and acetic acid and stained according to the method of Davenport can be successfully counterstained for demonstration of myelin sheaths and stroma. After mounted sections have been silvered, reduced and toned, the coating of nitrocellulose is removed by passing thru two changes of acetone. Following brief washes in 100,95,85 and 75% alcohols they are stained in an acidified aqueous solution of azo carmine for 30 to 60 minutes. Excess azo carmine is extracted with anilin alcohol followed by acetic alcohol after which the sections are mordanted for 15 to 60 minutes in a 5% aqueous solution of phosphotungstic acid. Without washing they are transferred to a stain mixture of either anilin blue and orange G (acidified) or light green and orange G (acidified) where they remain from 1 to 5 hours. After destaining in 95% alcohol and dehydration in absolute alcohol the sections are mounted in dammar. Result: axons stain black; sheath and fibroblast nuclei, red; myelin sheaths, orange; and connective tissue, blue or green. When the counterstains are applied to ganglia, cytological details of individual cells are demonstrated.     

Consumer demand for caesarean sections in Brazil: informed decision making,patient choice,or social inequality? A population based birth cohort study linking ethnographic and epidemiological methods

ObjectivesTo investigate why some women prefer caesarean sections and how decisions to medicalise birthing are influenced by patients, doctors, and the sociomedical environment.DesignPopulation based birth cohort study, using ethnographic and epidemiological methods.SettingEpidemiological study: women living in the urban area of Pelotas, Brazil who gave birth in hospital during the study. Ethnographic study: subsample of 80 women selected at random from the birth cohort. Nineteen medical staff were interviewed.Participants5304 women who gave birth in any of the city''s hospitals in 1993.ResultsIn both samples women from families with higher incomes and higher levels of education had caesarean sections more often than other women. Many lower to middle class women sought caesarean sections to avoid what they considered poor quality care and medical neglect, resulting from social prejudice. These women used medicalised prenatal and birthing health care to increase their chance of acquiring a caesarean section, particularly if they had social power in the home. Both social power and women''s behaviour towards seeking medicalised health care remained significantly associated with type of birth after controlling for family income and maternal education.ConclusionsFear of substandard care is behind many poor women''s preferences for a caesarean section. Variables pertaining to women''s role in the process of redefining and negotiating medical risks were much stronger correlates of caesarean section rates than income or education. The unequal distribution of medical technology has altered concepts of good and normal birthing. Arguments supporting interventionist birthing for all on the basis of equal access to health care must be reviewed.

What is already known on this topic

Women''s preferences for caesarean sections are understood to result from lack of knowledge and psychological aptitude to handle vaginal delivery and its consequencesEfforts to reduce the demand for caesarean sections have focused on providing consumers with correct information on the relative risks associated with vaginal and operative deliveries

What this study adds

In Brazil, many women prefer caesarean sections because they consider it good quality careRich women are more likely to have caesarean sections, supporting the notion that medical intervention represents superior carePoor women may implement a series of medicalised practices that justifies the need for greater medical intervention during birthInterventions for reducing caesarean sections by educating physicians and patients about risk factors associated with birthing procedures are not sufficient     

Embedding with Low Viscosity Nitrocellulose

The gelation and cutting of embedding masses of low viscosity nitrocellulose (L. V. N.) and of celloidin were compared. L. V. N. forms a firmer mass which can be cut into thinner sections than celloidin. It tolerates considerable water (up to 6%) in a solvent system of alcohol-water-ether, thereby permitting the use of 95% alcohol instead of absolute for making up the solution. The fluidity of its solutions permits transfer of tissue directly from alcohol-ether to a 20% embedding solution. Faults of L. V. N. are: the nitrated cotton lint contains some grit (hence its solution should be allowed to settle), the sections cut from it are somewhat more easily torn than celloidin sections, and it is sufficiently soluble in absolute alcohol to preclude the use of this fluid in handling sections.     

L-Xylo-3-hexulose,a new rare sugar produced by the action of acetic acid bacteria on galactitol,an exception to Bertrand Hudson's rule

BackgroundIn acetic acid bacteria such as Gluconobacter oxydans or Gluconobacter cerinus, pyrroloquinoline quinone (PQQ) in the periplasm serves as the redox cofactor for several membrane-bound dehydrogenases that oxidize polyhydric alcohols to rare sugars, which can be used as a healthy alternative for traditional sugars and sweeteners. These oxidation reactions obey the generally accepted Bertrand Hudson's rule, in which only the polyhydric alcohols that possess cis d-erythro hydroxyl groups can be oxidized to 2-ketoses using PQQ as a cofactor, while the polyhydric alcohols excluding cis d-erythro hydroxyl groups ruled out oxidation by PQQ-dependent membrane-bound dehydrogenases.MethodsMembrane fractions of G. oxydans were prepared and used as a cell-free catalyst to oxidize galactitol, with or without PQQ as a cofactor.ResultsIn this study, we reported an interesting oxidation reaction that the polyhydric alcohols galactitol (dulcitol), which do not possess cis d-erythro hydroxyl groups, can be oxidized by PQQ-dependent membrane-bound dehydrogenase(s) of acetic acid bacteria at the C-3 and C-5 hydroxyl groups to produce rare sugars l-xylo-3-hexulose and d-tagatose.ConclusionsThis reaction may represent an exception to Bertrand Hudson's rule.General significanceBertrand Hudson's rule is a well-known theory in polyhydric alcohols oxidation by PQQ-dependent membrane-bound dehydrogenase in acetic acid bacteria. In this study, galactitol oxidation by a PQQ-dependent membrane-bound dehydrogenase represents an exception to the Bertrand Hudson's rule. Further identification of the associated enzymes and deciphering the explicit enzymatic mechanism will prove this theory.     

New Fuchsin-Hematoxylin-Eosin; A Stain for Acid-Fast Bacilli and Surrounding Tissue

This is a staining technique for histopathologic evaluation of tissue reaction in the environs of acid-fast tubercle bacilli (avian and bovine) in sections. Fresh tissue is fixed in 10% neutral formalin and processed in the usual manner for embedding in paraffin. Sections are cut approximately 6 μ. thick, dewaxed, hydrated, and stained with Harris' hematoxylin. They are rinsed in tap water, differentiated in add alcohol, washed in tap water, given a distilled water rinse and stained at 20-30° C in a 1% solution of new fuchsin in 5% phenol. Each slide is then handled individually by placing it directly into a saturated aqueous solution of Li₂CO₃ and agitated gently for a few seconds. This is followed by differentiation with 5% glacial acetic acid in absolute or 95% ethyl alcohol until the color stops running. Two rinses in absolute or 95% ethyl alcohol follow. The sections are then counterstained in the color add of eosin Y prepared according to the method of Schleicher (Stain Techn., 28, 119-23, 1953) and used as an 0.025% solution in absolute alcohol. Following passage through 2 changes of absolute alcohol, the sections are cleared in xylene, then mounted in Permount or similar synthetic resin. The add-fast barilli are emphasized by their bright retractile red color within a contrasting background of hematoxylin and eosin.     

The Staining of Brunner's Gland and Other Neutral Mucins by Carmine,Hematoxylin and Orcein in Alkaline Solutions

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

Reduced Methylene Blue as a Stain for Crustacean Nerves

Effective in situ staining of crustacean nerves was achieved with leuco methylene blue reduced with either ascorbic acid or sodium hydrosulfite (Na₂S₂O₄). A stock solution of methylene blue, 0.4% (ca. 0.001 M), and the reductants, ascorbic acid or sodium hydrosulfite (0.01 M), were prepared in van Harreveld's crayfish physiological solution. Methylene blue stock solution was mixed with either of the reductants in the approximate ratio of 1:10, v/v, and titrated to the end point. Ascorbic acid reduction is light catalyzed and requires intense illumination during titration. The cleared or leucomethylene blue stock solution is suitable for immediate use as a working nerve stain. With either reductant, the working solution oxidizes on standing in air, but can be titrated repeatedly without loss of staining properties. Dissected nerve trunks or tissue were immersed in the working stain for 20 min at room temperature and the staining process observed until suitable contrast developed. Excess dye was decanted and the tissues flooded with crayfish physiological solution. Contrast could sometimes be enhanced by flooding the stained area with 1% hydrogen peroxide in van Harreveld's solution. When permanent mounts were prepared, tissues were dehydrated with tertiary butyl alcohol in preference to ethyl alcohol series. For anatomical and neurophysiological studies of nerve distribution in crustaceans, the alternative use of either ascorbic acid or sodium hydrosulfite, as reductants for methylene blue, was preferable to the more complicated rongalit-technique and characterization of neural elements was fully as satisfactory.     

Detection of Para-Aminosalicylic Acid (PAS) in Urine: A Simple Spot Test

A simple spot test for the detection of PAS in urine has been described and its sensitivity compared with that of other methods such as the Ehrlich''s reagent and the ferric chloride tests. In patients receiving 4 g. PAS the three methods gave similar results in urine specimens collected within eight hours. The new test is an inexpensive micro method which can easily be performed on a large scale. There is no reaction with sulfonamide or salicylic acid derivatives.     

Infección múltiple fúngica en un paciente diabético

BackgroundMixed fungal infections although undervalued, are more common than mentioned in the scientific literature. These infections have a poor prognosis for the patient.ObjectivesWe present an unusual case of a 61-year-old diabetic male who had a rhino-orbito-sinusal zygomycosis in 2001. After surgical debridement of the infected parts, along with antifungal therapy with liposomal amphotericin B, the patient started improving. Several years later the patient was hospitalized due to a similar problem and was diagnosed of rhino-orbito-cerebral zygomycosis.MethodsIn both episodes, a histopathological examination and cultures were performed on the sinus lesions. Tissue sections were stained with haematoxylin and eosin, Giemsa, periodic acid-Schiff (PAS) and Grocott's methenamine silver, and cultures specific for fungi were performed.ResultsThe histopathology studies revealed the presence of bacteria, actinomyces and a mixed infection by at least four different fungi, all of them well differentiated by their morphology. Despite the rapid diagnosis the patient died due to spreading to the central nervous system.ConclusionsMixed infections by fungi are rare, but due to the high incidence of immunodeficiencies they could occur more often than reported. We would like to alert on the possibility of acquired mixed infection by fungi which have shown to be high aggressive and have a worse prognostic in patients with underlying diseases.     

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.     

Improvements In The Paraffin Method

Dilute hydrofluoric acid alone and in conjunction with glycerin and ethyl alcohol was employed successfully to soften various types of refractory plant materials embedded in paraffin. Serial sections were cut at 6-8 μ and no appreciable deleterious effects on cell walls, cell contents, or staining procedures occurred. “Tannins” and “phlobaphene compounds” can be removed from tissues softened by this method by treating the sections for 12-48 hours with an aqueous solution of chromic acid, potassium bichromate and glacial acetic acid prepared according to the formula given by Johansen (1940).