Gram staining apparatus for space station applications.

A self-contained, portable Gram staining apparatus (GSA) has been developed for use in the microgravity environment on board the Space Station Freedom. Accuracy and reproducibility of this apparatus compared with the conventional Gram staining method were evaluated by using gram-negative and gram-positive controls and different species of bacteria grown in pure cultures. A subsequent study was designed to assess the performance of the GSA with actual specimens. A set of 60 human and environmental specimens was evaluated with the GSA and the conventional Gram staining procedure. Data obtained from these studies indicated that the GSA will provide the Gram staining capability needed for the microgravity environment of space.     

Enzyme immunoassay (EIA) in the rapid diagnosis of gonorrhoea

The diagnostic value of a new, modified enzyme immunoassay (EIA) (Gonozyme; Abbott Laboratories, North Chicago III) was evaluated for the rapid antigenic detection of Neisseria gonorrhoeae in endocervical and urethral specimens. EIA results were compared with those of Gram stain (GS) and conventional culture tests. EIA sensitivity and specificity for male patients attending dermatovenerological clinic were 100% and 96.8% respectively in comparison to 86.7% and 96.8% obtained by Gram staining. For female Obstetrics-Gynaecology patients EIA sensitivity of 100% was highly significant compared to 50% sensitivity by the Gram stain. In culture, 30 strains of N. gonorrhoeae were isolated from 125 male specimens and 2 from 105 specimens from females; this suggests a prevalence of N. gonorrhoeae of 24% in males and 1.9% in females. In vitro antibiotic sensitivity testing indicated 55% resistance to penicillin and 43% to ampicillin in these isolated strains; all were sensitive to erythromycin/tetracycline. 12% of the strains were beta-lactamase producers.     

A protocol for Papanicolaou staining of cytologic specimens following flow analysis

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman "Metricel" filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.     

痰标本涂片检查与培养结果分析

目的：探讨痰标本涂片检查在细菌培养以及临床治疗中的意义。方法：送检标本首先直接涂片革兰染色镜检,同时将合格痰标本划分为以阴性杆菌、阳性球菌、霉菌为主和普通标本四大类。再对合格痰标本行细菌学培养及药物敏感测定。结果：314份痰标本合格率为62．4％(196／314)。196份合格痰标本共检出145株病原菌,培养阳性率74．0％。涂片以阴性杆菌、阳性球菌、霉菌为主的标本与培养结果符合率分别为93．1％、68．4％和41．2％。结论：痰培养结果的准确性受痰标本是否合格、病原菌的生长速度以及是否使用过大剂量抗生素等因素直接影响。临床医生必须依据临床症状及涂片结果判断作出何种为真正的病原菌。     

Gram staining and lectin binding properties of Myxosporea and Sporozoea

The staining method developed by Christian Gram was introduced as a simple and highly selective tool for demonstrating myxosporean and coccidian sporogonic stages. When using standard blood staining procedures for those enigmatic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histological sections, but staining is variable in air dried fish organ imprints. To visualize the Gram-negative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gram protocol may be modified as follows: after application of 2% crystal violet (basic violet 3) and Lugol's solution, sections are stained with 0.1% nuclear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) dissolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ imprints containing myxospores or coccidia, but only in sections containing myxosporea. Staining for 1 min with an aqueous solution of 0.5% malachite green and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With regard to the role of acid mucopolysaccharides and other carbohydrates in the Gram reaction of spores, alcian blue 8GX staining was compared to the binding of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 μl/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the alcian blue staining, GS II resulted in a pattern similar to the Gram staining results. This binding was weak in untreated specimens, but was significantly enhanced when digested first within trypsin overnight in a humid chamber at 37 °C. The binding of GS II to both myxosporidian and coccidian spores suggests that they are both composed of polymers containing N-acetyl-D-glucosamine residues. Furthermore, the results suggest that this hexosamine plays a key role in the Gram reaction.     

革兰染色对患者阴道分泌物的检验价值

目的探讨革兰染色对患者阴道分泌物的检验价值。方法选择2015年4月至2017年4月于诸暨市中心医院进行阴道分泌物检验的阴道炎患者为研究对象,共1 600例。采用生理盐水镜检法和革兰染色法对分泌物进行检验,比较两种检验方法对分泌物病变的检出情况,同时比较两种检验方法的特异性和敏感性。结果革兰染色法对分泌物病变的检出率为32.50%,生理盐水镜检法的检出率为28.50%,二者差异有统计学意义(P0.05)。革兰染色法对阴道分泌物检验的准确性、敏感性及特异性分别为95.61%、96.34%、97.87%,均高于生理盐水镜检法,差异有统计学意义(P0.05)。结论阴道分泌物检验是诊断阴道炎最便捷的方法。与常规的生理盐水镜检法相比,革兰染色法具有更高的准确性、特异性和敏感性,对阴道炎的早发现、早治疗具有较高的应用价值。     

The Effect of the Microgravity Rotating Culture System on the Chondrogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

We investigated the influence of the microgravity rotating culture system on the chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs). During chondrogenic induction, MSCs combined with polyglycolic acid (PGA) were cultured by static culture or microgravity rotating culture and chondrocyte formation was confirmed by toluidine blue staining. Furthermore, the mRNA and protein expressions of a specific cartilage extracellular matrix protein (collagen type II and Aggrecan) were evaluated by real-time RT-PCR and western blot, respectively. Toluidine blue staining indicated the OD values of proteoglycans semi-determination were higher in the microgravity rotating culture group than the static culture group. Following chondrogenic induction, mRNA and proteins of collagen type II and Aggrecan were more significantly expressed in cells of the microgravity rotating culture group compared with the controls. Compared with routine three-dimensional static culture, the microgravity rotating culture system was more effective for the construction of tissue-engineered cartilage in vitro.     

Histochemical detection of glycogen using Griffonia simplicifolia agglutinin II

The utility of a lectin from Griffonia simplicifolia (GSA II) for demonstrating glycogen in situ was tested on fixed paraffin-embedded sections of a variety of tissues from rodents and man. The histochemical specificity of GSA II conjugated to horseradish peroxidase (GSA II-HRP) for glycogen was documented by the lability of such staining on adjacent sections treated with either malt diastase or alpha-amylase. In some tissue sites, the lectin-HRP conjugate imparted cytoplasmic staining that was diastase- and amylase-labile but resisted digestion with N-acetylglucosaminidase. Reactivity in the latter sites was attributed to glycogen and occurred in cell types having well-documented glycogen content, including liver hepatocytes, skeletal muscle fibres and polymorphonuclear leukocytes. In other tissue loci, GSA II binding was confined to cell surfaces or intracellular compartments, was not affected by prior diastase or amylase digestion, but was abolished with N-acetylglucosaminidase. Staining in these sites was attributed not to glycogen, but instead to glycoconjugate having sugar chains terminated with N-acetylglucosamine. These findings document the affinity of GSA II for glycogen in situ but do not conflict with the biochemically demonstrated affinity of GSA II for terminal N-acetylglucosamine. The results reported here show that the GSA II-HRP method may be useful for detecting physiological or pathological changes in the glycogen content of cells.     

Simulated Microgravity Using a Rotary Culture System Compromises the In Vitro Development of Mouse Preantral Follicles

Background

Growing cells in simulated weightlessness condition might be a highly promising new technique to maintain or generate tissue constructs in a scaffold-free manner. There is limited evidence that microgravity condition may affect development of ovarian follicles. The objective of the present study was to investigate the effects of simulated microgravity on the in vitro development of mouse preantral follicles.

Methods and Results

Ovarian tissue from 14-day-old mice, or preantral follicles mechanically isolated from 14-day-old mouse ovaries were cultured at a simulated microgravity condition generated using a rotating wall vessel apparatus. Follicle survival was assessed quantitatively using H&E staining. Follicle diameter and oocyte diameter were measured under an inverted microscope. Ultrastructure of oocytes was evaluated using transmission electron microscopy. We observed that simulated microgravity compromised follicle survival in vitro, downregulated PCNA and GDF-9 expressions, and caused ultrastructural abnormalities in oocytes.

Conclusion

This study showed for the first time that three-dimensional culture condition generated by simulated microgravity is detrimental to the initial stage development of mouse preantral follicles in vitro. The experimental setup provides a model to further investigate the mechanisms involved in the in vitro developmental processes of oocytes/granulosa cells under the microgravity condition.     

革兰氏染色三步法与质量控制

革兰氏染色(Gram stain),是细菌学中一个经常使用和十分重要的方法,自从1884年微生物学家Gram氏发明著名的革兰氏染色法以后,100多年来虽然经过后来学者的几次改进,但都仍然沿用着Gram氏原来的四步法,基本原理也没有改变。最近Allen氏对Ziehl-Neelsen抗酸菌染色法的改进,是一个良好的启示,使我们开始了革兰氏染色三步法的研究并取得了成功。现将我们建立的革兰氏染色三步法与质量控制报告如下。 1 材料和方法 1.1 结晶紫染色液 甲液:结晶紫2g;95％乙醇20ml。 乙液:草酸铵0.8g;蒸馏水80ml。 甲乙二液先分别溶解,然后混合在一起,过滤除去残渣后装入滴瓶中备用。     

Comparison of the quick Gram stain method to the B&M modified and favor methods

The Gram stain is an established method for bacterial identification, but the time needed to carry out this stain is 2-3 min. We attempted to shorten this time and stained a total of 70 clinical specimens isolated from using the Bartholomew & Mittwer (B&M) modified or Favor methods with a 3 s duration for washing and staining steps. Results were plotted and analyzed using a Hue Saturation Intensity (HSI) model. The range based on a plot of the two methods with the HSI model was presented as a reference interval. Our results indicated that 100% (35/35) of strains were Gram positive and 97.1% (34/35) were Gram negative for the quick B&M modified method. In the quick Favor method, 80.0% (28/35) were Gram positive and 68.6% (24/35) of strains were Gram negative. We propose that the quick B&M modified method is equivalent to the standard Gram staining method and is superior to the quick Favor method.     

Lectin-binding sites in isolated equine cumulus-oocyte complexes: Differential expression of glycosidic residues in complexes recovered with compact or expanded cumulus

Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA₁₂₀, and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of both types of COCs, (3) uniform staining with PNA only in Exp COCs, (4) uniform and trilaminar binding pattern with SNA in Cp and Exp COCs, respectively, and (5) major reactivity with GSA II in Exp COCs. Ooplasm showed similar staining intensity with Con A, HPA, GSA I-B₄, and WGA in both Cp and Exp COCs, with stronger reactivity to GSA II in Exp COCs, whereas SNA, UEA I, and LTA binding sites were present only in Cp COCs. Oocyte cortical granules of both Cp and Exp COCs reacted with Con A and WGA. These results suggest that, in the mare, viable (Cp) and atretic (Exp) COCs display different glycoconjugate staining pattern, which may account for the different maturation and developmental competence of COCs.     

Standardization of fine needle aspiration cytology of the breast – comparison of Auto Cyto Fix and conventional smears

In Japan, there are some problems with fine needle aspiration (FNA) cytology of the breast, such as insufficient smeared cells, air-drying artefact and excessive erythrocytes. Liquid-based cytology has been found to solve these problems. Equipment for such preparations has been developed, but can be expensive to purchase and operate. We developed Auto Cyto Fix 1000 (ACF), which is inexpensive and automatically smears and fixes cells. The purpose of this study was to compare the various cytological features of conventional and ACF specimens. We evaluated whether the ACF method would be able to replace the conventional method. Forty-eight FNA specimens of breast were studied. All specimens were prepared by the direct smeared (DS) and ACF methods and evaluated for unsatisfactory cell collection, air-drying artefacts, background findings and epithelial cell findings. Although ACF specimens were prepared using the cells remaining in the needle and syringe after preparing DS specimens, the cellularity of two of the ACF specimens was better than that of the corresponding DS specimens. ACF specimens never showed air-drying artefact. Unlike DS specimens, which have many erythrocytes in the background, erythrocytes were filtered out and the background of ACF specimens was clean. We believe that many problems attributable to conventional FNA specimen preparation have been solved in this study. Preparation using the ACF apparatus can reduce running costs and can be used to prepare FNA specimens of the breast for cytological examination as an alternative to the conventional method.     

Standardization of fine needle aspiration cytology of the breast -- comparison of Auto Cyto Fix and conventional smears.

In Japan, there are some problems with fine needle aspiration (FNA) cytology of the breast, such as insufficient smeared cells, air-drying artefact and excessive erythrocytes. Liquid-based cytology has been found to solve these problems. Equipment for such preparations has been developed, but can be expensive to purchase and operate. We developed Auto Cyto Fix 1000 (ACF), which is inexpensive and automatically smears and fixes cells. The purpose of this study was to compare the various cytological features of conventional and ACF specimens. We evaluated whether the ACF method would be able to replace the conventional method. Forty-eight FNA specimens of breast were studied. All specimens were prepared by the direct smeared (DS) and ACF methods and evaluated for unsatisfactory cell collection, air-drying artefacts, background findings and epithelial cell findings. Although ACF specimens were prepared using the cells remaining in the needle and syringe after preparing DS specimens, the cellularity of two of the ACF specimens was better than that of the corresponding DS specimens. ACF specimens never showed air-drying artefact. Unlike DS specimens, which have many erythrocytes in the background, erythrocytes were filtered out and the background of ACF specimens was clean. We believe that many problems attributable to conventional FNA specimen preparation have been solved in this study. Preparation using the ACF apparatus can reduce running costs and can be used to prepare FNA specimens of the breast for cytological examination as an alternative to the conventional method.     

State of Brassica rapa photosynthetic membranes in microgravity

The structural characteristics of the photosynthetic apparatus of Brassica rapa plants grown on board the space shuttle Columbia (STS-87) for 15 days were examined using the methods of transmission electron microscopy and statistic programme STAT. Maintaining of the same growth conditions for control plants was realized with great accuracy using the Orbiter Environmental simulator in Kennedy Space Center. A grana number per a medial section 1.8 times decreased in microgravity. Considerable changes were also revealed in the grana structure in microgravity in comparison with th ground control, namely: 1/a greater diversity in the thylakoid length with granae and 2/ lateral shifting of the thylakoids lateral shifting of the thylakoids relative one to another. The previous mentioned pheomenon was found for 64% of the invested granae. Shifting of the thylakoids in the granae in microgravity led to increasing of the grana thylakoid surface exposed to a stroma. In addition, the volume of stromal thylakoids increased. The peculiarities in the photosynthetic apparatus structure in microgravity are supposed to be an evidence of decreasing in the light harvesting complex amount of photosystem II (PSII).     

"Modeled microgravity" affects cell response to ionizing radiation and increases genomic damage

The aim of this work was to assess whether "modeled microgravity" affects cell response to ionizing radiation, increasing the risk associated with radiation exposure. Lymphoblastoid TK6 cells were irradiated with various doses of gamma rays and incubated for 24 h in a modeled microgravity environment obtained by the Rotating Wall Vessel bioreactor. Cell survival, induction of apoptosis and cell cycle alteration were compared in cells irradiated and then incubated in 1g or modeled microgravity conditions. Modulation of genomic damage induced by ionizing radiation was evaluated on the basis of HPRT mutant frequency and the micronucleus assay. A significant reduction in apoptotic cells was observed in cells incubated in modeled microgravity after gamma irradiation compared with cells maintained in 1g. Moreover, in irradiated cells, fewer G2-phase cells were found in modeled microgravity than in 1g, whereas more G1-phase cells were observed in modeled microgravity than in 1g. Genomic damage induced by ionizing radiation, i.e. frequency of HPRT mutants and micronucleated cells, increased more in cultures incubated in modeled microgravity than in 1g. Our results indicate that modeled microgravity incubation after irradiation affects cell response to ionizing radiation, reducing the level of radiation-induced apoptosis. As a consequence, modeled microgravity increases the frequency of damaged cells that survive after irradiation.     

改良革兰染色法在显示石蜡切片真菌中的应用

目的建立真菌的病理切片改良革兰染色法。方法选取确诊真菌感染的组织蜡块,切若干空白片,通过苏木素-伊红染色（HE）、高碘酸-无色品红染色（PAS）、六胺银染色（GMS）、改良革兰染色后,比较改良革兰染色法的染色效果。结果改良革兰染色法的染色效果好,该法具有易操作、染色效果稳定等优点。结论改良革兰染色法能够清晰地显示出组织切片中的真菌,并且染色效果稳定,在检测组织中的真菌时可发挥重要的辅助诊断价值,具有广泛的应用前景。     

Structural study on hen egg-white lysozyme crystals grown in gravity and microgravity

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Biofilm prevalence and microbial characterisation in chronic wounds in a Sri Lankan cohort

Biofilms have been associated with chronic wound infections in diabetic patients. The study assessed the occurrence of biofilms in chronic diabetic wounds (CDWs) in a Sri Lankan cohort. Tissue specimens collected during surgical debridement were analysed by quantitative differential viable counting, scanning electron microscopy (SEM), fluorescence insitu hybridization (FISH) and light microscopy with Gram and Haematoxylin-Eosin staining. All specimens harboured >5·0 log₁₀ CFU per g bacteria and 2–9 distinct species per specimen were recovered from twenty wounds by culture. The most frequently isolated bacterium was Pseudomonas spp. (12/20;60%). Strict anaerobes were isolated from 10/20 specimens. Gram and Haematoxylin-Eosin staining showed aggregated micro-colonies, embedded in the wound tissue bed (20/20) but the exopolymer matrix was not visible in all samples (13/20). Fluorescence microscopy using a eubacteria-specific FISH probe indicated the presence of bacterial aggregates within the deep layers of the wound tissues (20/20). SEM revealed the presumptive architecture of matrix-embedded microbial clusters (20/20). The approximate diameter of bacterial aggregates in tissues ranged between 12 and 400 µm. Bacterial infiltration into the internal portions of the tissues was apparent using FISH, Gram, and Haematoxylin-Eosin staining. All CDWs carried biofilm-specific morphological features. FISH was more specific than SEM and indicated the presence of microcolonies within deeper tissues.     

Improved Microscopic Detection of Bacteriuria

A simple method is described which permits the microscopic detection of bacteria in sediments of urine and other fluids, including bacteria that have eluded detection by conventional means. The method introduces increased centrifugal force and stepwise chemical fixation and then conventional staining. It is rapid, economical, and suitable for use in a physician's office. Use of this method immediately reveals those bacteria reported as “significant” by the conventional laboratory culture. More importantly, it immediately reveals the presence of bacteria, living or dead, which are missed by the conventional culture and by the conventional Gram staining procedure. These bacteria usually can be grown in special media and they appear to be related to systemic disease as evidenced by the clinical response to appropriate antibiotics.