New Grocott Stain without Using Chromic Acid

We established a new “ecological” Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide.     

Yellow-brown (Hamazaki-Wesenberg) bodies mimicking fungal yeasts

We describe herein two cases in which yellow-brown (Y-B) bodies, also known as Hamazaki-Wesenberg bodies, were encountered in lymph nodes and initially misinterpreted as fungal organisms. Because of occasional "budding" forms and positive staining with "screening" fungal stains (Grocott methenamine silver and periodic acid-Schiff), Y-B bodies may closely resemble budding yeast fungi. We describe the characteristic light microscopic and ultrastructural morphologic features of Y-B bodies, and their histochemical staining reactions, and emphasize the value of the Fontana-Masson silver stain for recognition and identification of these bodies.     

Misdiagnosis caused by fungal contaminant in a histological stain solution.

Silver stains on tissue and cytology specimens are important in the evaluation of patients with suspected fungal infections. Care must be taken, however, to prevent misinterpretation of contamination artifacts. Two cases presenting such a problem are reported. The first patient had granulomatous leg lesions that microscopically showed characteristics of erythema induratum but with budding yeastlike organisms demonstrated by Grocott methenamine silver stain. Cultures and subsequent biopsies were negative for fungi. The second patient had a steroid-dependent chronic obstructive lung disease, and during evaluation for possible Pneumocystis carinii pneumonia, the Grocott methenamine silver stain on expectorated sputum showed budding yeastlike organisms. Sputum cultures were negative for fungi. Examination of the two Grocott light-green counterstain solutions demonstrated black, budding yeast cells similar to those seen in the specimens from the patients. Culture of the counterstain grew Exophiala (Phialophora) jeanselmei. Further studies revealed that this cause of misdiagnosis could be prevented by either filtering or adding thymol to the counterstain solution. Care regarding contamination of histological stain solutions is emphasized.     

原发性肺隐球菌病的病理诊断和超微结构观察

目的探讨原发性肺隐球菌病(PC)的病理诊断和超微结构特点。方法对27例PC的临床病理资料进行回顾性分析、组织化学染色和光镜观察,12例进行透射电镜观察。结果27例中,2例肺穿刺活检明确诊断,25例开胸探查,胶样病变2例,非干酪性肉芽肿病变25例,均检出新型隐球菌(CN)。黏液卡红(MC)、过碘酸雪夫染色(PAS)、奥尔辛蓝(AB)和Grocott六胺银(GMS)染色隐球菌的检出率分别为87．0％(20／23)、100％(27／27)、66．7％(18／27)和100％(23／23)。透射电镜观察,CN均有荚膜形成,大部分结构简单,细胞器不发达,细胞退变;部分隐球菌有细胞核、核仁、线粒体和空泡;CN的检出率为91．7％(11／12)。结论PC的临床表现和影像学缺乏特征性,穿刺或开胸肺活检仍然是PC诊断的主要手段。光镜结合电镜观察以及MC、GMS和PAS组织化学染色,能对PC做出明确诊断及鉴别诊断。     

PAS联合六胺银染色诊断局限性马尔尼菲青霉菌感染的应用

目的探讨建立快速诊断局限性马尔尼菲青霉菌感染的方法,以期早期诊断,降低死亡率。方法采用高碘酸一无色品红（PAS）染色联合六胺银染色对4例非AIDS马尔尼菲青霉菌感染者活检组织进行马尔尼菲青霉菌的特殊染色,分析感染者的临床表现和病理组织学诊断特点。结果患者均为广东本地居民,属局限性马尔尼菲青霉菌感染。除1例有发热外,临床上未见以发热、失重、贫血、白细胞增高为主的马尔尼菲青霉菌感染症状以及多器官损害症状。HE染色显示病理组织学类型为肉芽肿型和化脓型。病变组织经PAS染色与六胺银染色后,在油镜下增生的泡沫状巨噬细胞胞浆内,呈现大量红色或黑色的卵圆形及腊肠状酵母样青霉菌孢子。无荚膜,在腊肠状菌体中可见马尔尼菲青霉菌特有的裂殖时出现的横隔膜。结论PAS联合六胺银染色清晰显示组织中马尔尼菲青霉菌的特征性结构和形态,结合病理组织学特点、中国南方地区生活或旅游史,可以快速诊断局限性马尔尼菲青霉菌感染。     

Silver Acetate Autometallography: An Alternative Enhancement Technique for Immunogold-Silver Staining (IGSS) and Silver Amplification of Gold,Silver, Mercury and Zinc in Tissues

Abstract

Silver lactate autometallography in immunogold silver staining (IGSS) usually requires development in darkness to avoid excessive background staining. Our alternative method of silver enhancement of IGSS on paraffin sections from Bouin's or formalin fixed pancreas uses silver acetate in combination with hydroquinone in low pH buffer. The modification was tested with a range of antibodies in normal and diseased tissues (all routinely fixed and paraffin embedded), in acetone postfixed cryostat sections, and in semithin sections of glutaraldehyde fixed and resin embedded tissues. Silver acetate autometallography was also tested in various systems for the visualization of tissue metals like sulfides and selenides of mercury and zinc, silver, and gold. Comparisons between sections exposed to silver lactate and the silver acetate developer showed no significant difference in the number of structures stained. The degree of background staining was often lower when silver acetate was to used as the ion donor, especially with IGSS. The advantage the technique described here is that the development process can be controlled, using normal bright field light microscopy. (The J Histotechnol 11:213, 1988.)     

Differential diagnosis of trichosporonosis using conventional histopathological stains and electron microscopy

Obana Y, Sano M, Jike T, Homma T & Nemoto N
(2010) Histopathology 56, 372–383 Differential diagnosis of trichosporonosis using conventional histopathological stains and electron microscopy Aims: Although Trichosporon is a causative pathogen of white piedra and summer‐type hypersensitivity pneumonitis, fatal disseminated trichosporonosis cases have recently been increasing. However, Trichosporon is often confused with other fungi, especially Candida, in pathological specimens. The aim was to determine the utility of histopathological stains and electron microscopy for diagnosing trichosporonosis. Methods and results: Autopsy cases of trichosporonosis, candidiasis, aspergillosis and cryptococcosis were investigated using histopathological stains and electron microscopy. Using Grocott’s method, Trichosporon was weakly detected compared with other fungi. In contrast, diluted periodic acid methenamine silver (PAM) stain clearly enhanced the intensity of staining of Trichosporon compared with Candida. Furthermore, Alcian blue and colloidal iron stains predominantly detected Trichosporon. Electron microscopy after staining with diluted PAM demonstrated that Trichosporon has a variety of hyphal sizes and laminar deposition of rough silver granules, whereas Candida has uniform pseudohyphae and fine granules. The average diameter and population area of the granules were significantly higher in Trichosporon compared with Candida (P < 0.01). Meanwhile, the laminar structure was preserved in the cell walls of Trichosporon without silver stains, whereas a low‐density structure was observed in Candida. Conclusions: Histopathological staining patterns and electron microscopic findings can facilitate the diagnosis of trichosporonosis.     

蛋白多糖在大鼠小肠集合淋巴小结高内皮微静脉中的分布

目的 研究蛋白多糖(PG)在大鼠小肠集合淋巴小结高内皮微静脉(HEVs)中的分布，探讨PG对淋巴细胞归巢的调节作用。方法 阳性胶体铁染色-酶连续阻断法，光镜和电镜观察PG在HEVs内的淋巴细胞、内皮细胞、基膜上的分布。结果 HEVs的基膜和邻接基膜的淋巴细胞的细胞膜呈强阳性着色，能被透明质酸酶、肝素酶、硫酸软骨素酶ABC阻断；电镜显示，胶体颗粒主要排列于基膜的内、外侧及穿越基膜的淋巴细胞的细胞膜上，内皮细胞、穿内皮细胞的淋巴细胞和腔内的淋巴细胞不着色。结论 分布在HEVs的基膜和穿基膜的淋巴细胞的细胞膜上的PG，主要是硫酸乙酰肝素蛋白多糖、硫酸软骨素蛋白多糖和透明质酸，可能与归巢淋巴细胞穿越基膜密切相关。     

Silver impregnation of the glomerular basement membranes

A method based on the periodic acid-produced (secondary) argentaffinity of the basement membranes is described. Sodium tetraborate is used for alkalizing of the silver nitrate solution.     

A study of glomerular basement membrane anionic sites and glomerular visceral epithelial cell coat in protein overload proteinuria in the rat

The presence and distribution of anionic sites in the glomerular basement membrane and visceral epithelial cell coat has been demonstrated. No definite decrease in intensity or periodicity of staining of basement membrane particulate sites was seen in protein overload proteinuric animals and only one staining technique employed for electron microscopy (alcian blue 8GX) demonstrated a focal decrease in visceral epithelial cell coat staining in severely damaged glomeruli. A decrease in overall glomerular staining was also demonstrated by quantitative analysis of colloidal iron staining by light microscopy. The findings differ from those described in puromycin aminonucleoside nephropathy and nephrotoxic nephritis. Staining was demonstrated also in other basement membranes, in Bowman's capsule and along interstitial collagen fibres.     

PAS-lead hematoxylin as a stain for small-granule endocrine cell populations in the lungs,other pharyngeal derivatives and the gut

Epithelial cells of the several subtypes that comprise the small-granule cell population of the respiratory system are little studied, partly because adequate silver, monoamine fluorescence and other specific light microscopical preparations have been more difficult to obtain than in the gut and other organs possessing diffuse endocrine systems. Periodic acid-Schiff (PAS) in combination with MacConaill-Solcia's lead hematoxylin has in our hands proven dependable for routine staining of serial 2-m?m glycol methacrylate sections used in mapping the distributions of these cells along the airway. In lungs of mice, hamsters, kittens, and fetal rabbits, typical small-granule cells stain weakly or not at all with lead hematoxylin alone, hence are easily overlooked. PAS adds to the cytoplasm a diffuse magenta coloration; and because it is diastase-resistant, less brilliant than that of mucus but more so than bronchiolar cell secretions, and finer textured than lysosomal staining of other cells present, the effect is to highlight small-granule cells whether solitary or in clusters. Additional PAS staining of basement membranes and lead hematoxylin staining of cilia enhance the combined stain's resolving power. In thyroid gland, parafollicular cells stand out boldly against follicular elements; in small intestine, hematoxylin-positive endocrine cells are well differentiated from absorptive, mucous, and Paneth cells that surround them. Using a complementary monoamine fluorescence technique on plastic sections of lungs from control and 5-hydroxytryptophan-pretreated animals prior to staining, we can show that fluorescent epithelial cells are identical with those stained by PAS-lead hema-toxylin.     

Detection of Pneumocystis carinii. Comparative study of monoclonal antibody and silver staining.

The sensitivity of Pneumocystis carinii detection using silver stain (Grocott method) was compared to that using the avidin-biotin-complex immunoperoxidase (IP) staining method with anti-P. carinii monoclonal antibody. Silver stain detected only cyst wall, whereas IP stained both cyst wall and trophozoites. Serial sections of formalin-fixed, paraffin-embedded autopsy lung specimens from 41 acquired immune deficiency syndrome patients in three disease categories were stained: I--premortem or autopsy diagnosis of P. carinii pneumonia (13 cases); II--history of treated P. carinii pneumonia but no P. carinii detected in autopsy tissue specimens (15 cases); and III--no clinical or autopsy evidence of P. carinii pneumonia (13 cases). Smears from 7 bronchoalveolar lavages (3 positive) and 11 induced sputa (1 positive) also were stained. All cases of P. carinii in category I were detected with equal sensitivity. P. carinii undetected by silver stain in category II and III cases and in bronchoalveolar lavages and induced sputa were not revealed by IP. Detection of trophozoites by IP did not improve sensitivity because the staining pattern was amorphous or focally granular, and thus easily confused with nonspecific staining of mucin or intracellular or free particulate material. Reliable identification of trophozoites could be made only with coexisting cyst structures. Silver staining was more advantageous because it also identified fungal infections and was faster and more cost effective than IP.     

Antimicrobial Potentials of Silver Colloidal (Nanorods) on Clinical Isolates in Bayelsa State,Nigeria

Antimicrobial resistance in developing countries has long been an issue of major concern. Nanotechnology has become an eye opener for the intervention on multiple drug resistance organisms. In this study we investigated the antimicrobial potentials of Silver Nitrate (nanorods) solution used in managing infectious diseases, the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the product against microbial isolates were determined using standard microbiological techniques. The mean MIC and MBC of silver nitrate solution on fungi (0.16 µg/ml and 0.29 µg/ml respectively) was significantly lower than that of Gram positive organisms (2.35µg/ml and 2.62µg/ml) and Gram negative organisms (2.05 µg/ml and 2.10 µg/ml). Of all the Gram positive organisms, Staphylococcus spp recorded the lowest mean MIC and MBC while in the Gram negative organisms group, E. coli isolates showed the lowest mean MIC and MBC of the silver nitrate solution, though not significantly different from the other isolates. In conclusion, results from this study revealed that Silver Nitrate(nanorods) may have be broad spectrum in activity, but with higher antifungal potentials.     

Looks like fungi but it isn’t – identifying pseudo-fungi*

Pseudo-fungi are structures that look like true fungi. They often give a positive periodic acid-Schiff and a negative Grocott’s methenamine silver reaction. From a therapeutic point of view, it is important to correctly characterize these structures as one or the other. This review presents guidelines for their accurate histochemical diagnosis.     

Special Stains in the Microwave Oven

Abstract

We describe the modification of a number of stains for the microwave oven: Grocott's methenamine silver, acid fast bacteria, Giemsa, Lillie's melanin, methyl green pyronin, Masson's trichrome, and glycogen double digestion. Their adaptation has markedly reduced the time needed for staining. The staining procedures described here can be used on paraffin sections, frozen sections on coated slides, touch preparations, and cytology specimens.     

干燥综合症合并肺孢子菌肺炎一例并文献复习

目的 探讨干燥综合症合并肺孢子菌肺炎（PCP）的早期诊断方法。方法 对1例干燥综合症合并肺孢子菌肺炎患者的临床资料进行回顾性分析。结果 患者因干燥综合症接受免疫抑制剂治疗两月后出现不明原因发热、干咳,活动憋气。依据患者临床表现、胸部X线和CT征像、痰肺孢子菌六胺银染色镜检和多聚酶链反应（PCR）监测结果而确诊PCP。予克林霉素（患者磺胺过敏）、卡泊芬静治疗6 d后痰液肺孢子菌DNA载量由104/ml减为102/ml,12 d后进一步降至101/ml,经卡泊芬静治疗21 d后痊愈出院。结论 痰液标本肺孢子菌六胺银染色镜检具有高度特异性,但敏感性较低。高敏感性的PCR方法不仅可以用于PCP筛查,还可用于疗效监测。     

Tubulointerstitial Nephritis Antigen (TIN-ag) is Expresses in Distinct Segments of the Developing Human Nephron

Tubulointerstitial nephritis antigen (TIN-ag) is a 58 kDa glycoprotein restricted within the kidney to basement membranes underlying the epithelium of Bowman's capsule and proximal and distal tubules. Autoantibody formation against this component has been described in association with primary immune-mediated tubulointerstital nephritis, membranous nephropathy and anti-glomerular basement membrane nephritis. In the present report, the ontogeny of this protein was studied in human fetal kidney tissue by immunohistochemical analysis of immature and developing nephrons using a panel of monoclonal and polyclonal antibodies. TIN-ag is first detected in basement membranes underlying the epithelium of Bowman's capsule of early capillary loop stage glomeruli and the primitive proximal tubule. No detectable expression is observed in the basement membranes of the branching ureteric bud, nephrogenic vesicle, or comma shape and s-shape stages of nephrogenic development. Increased staining of the proximal tubular basement membrane is associated with outgrowth of the primitive tubule from the urinary pole of the developing glomerulus. In more mature fetal tubules, TIN-ag expression closely resembles that of previously reported observations in mature tissue where it is present in high amounts in the basement membranes of proximal tubules, and to a lesser extent in Bowman's capsule and distal tubules. Our results suggest that TIN-ag expression is developmentally regulated in a precise spatial and temporal pattern throughout nephrogenesis.     

Heavily pigmented melanocytic neoplasms: comparison of two melanin-bleaching techniques and subsequent immunohistochemical staining

The effect of melanin bleaching using permanganate/oxalate and dilute hydrogen peroxide on subsequent immunohistochemical staining of heavily pigmented melanocytic neoplasms is investigated. Permanganate/oxalate precluded the use of some antibodies but allowed much faster bleaching times, whereas dilute hydrogen peroxide enabled a full range of antibodies to be used, yet bleaching times were far longer. Each technique has advantages; however, the choice of method should be determined by the nature of the information needed to make a diagnosis and the speed at which a report is required.     

Immunolocalization of entactin, a sulfated basement membrane component, in rodent tissues, and comparison with GP-2 (laminin).

Entactin is a sulfated glycoprotein in the extracellular basement membrane like matrix produced by M1536-B3 cells, a mouse endodermal line derived from an embryonal carcinoma. It has a molecular weight of 158,000 and is chemically and immunologically distinguishable from GP-2 (laminin) and fibronectin. Antibodies produced against entactin and GP-2 react with subepithelial and vascular basement membranes in rat lung, liver, spleen, and kidney and mouse placenta and kidney when examined by light microscopy. Both antibodies yield staining around the marginal sinus of the white pulp of the spleen. Antientactin reacts with basement membrane and mesangium in rat glomeruli, and anti-GP2 does not. Ultrastructurally, staining in kidneys is strongest at epithelial or endothelial cell membranes bordering basement membranes, with only moderate staining of the basement membrane proper. Intracellular staining is not present. The location of entactin suggests that it has a role in the interaction of cells with extracellular matrix, possibly in adhesion. Lack of intracellular staining suggests that the tissues studied are not actively producing entactin or GP-2 and that these substances may be fairly stable in adult organisms.     

鼻硬结病的临床特点及病理诊断研究

目的 探讨鼻硬结病的临床特点及病理诊断价值。方法 收集2014年4月~2018年12月复旦大学附属眼耳鼻喉科医院10例硬结病患者资料进行回顾性分析,对硬结病临床、病理学特征及六胺银染色结果进行分析研究。结果 9例患者病变累及鼻部（1例同时累及气管,1例同时累及软腭）,另1例侵犯会厌。最常见的症状为鼻塞（9例）,其次为流涕（2例）,鼻出血（2例）,头痛（1例）,嗅觉下降（1例）,呼吸不畅（1例）及咽部异物感（1例）。通过六胺银染色显示,5例形态不典型硬结病病例中有4例短杆状菌体。结论 硬结病常累及上呼吸道,鼻腔是常见发病部位,六胺银特殊染色在硬结病病理诊断有较大作用,是诊断硬结病的重要辅助手段。