A RAPID STAINING METHOD SHOWING AgNOR AND DNA IN PATHOLOGICAL DIAGNOSIS

In order to satisfy the need of rapid clinical diagnosis, a series of experimental studies for AgNOR (Argyrophilic NORassociated proteins) and DNA (Dereyribonucleic acid) staining was compared, from which a stable staining solution was selected to show mucin residence. Using the referred tables, with reference to the corresponding relation of the staining solution with temperature and time, reliable staining results for AgNOR and DNA could be obtained.These two modified staining methods are simple, reliable and easy to operate, able to provide good contrast for pathological diagnosis.

A Comparative Study on Four Staining Methods for Antitumor Active Fraction of Periplaneta americana

[ Objective ] This study aimed to compare four staining methods for proteins of SDS-polyacrylamide gel dectrophoresis （SDS-PAGE） and explore a suit- able staining method for the antitumor active fraction of P. americana after polyacrylamide gel electrophoresis. [ Method ] BSA was used as the standard for the comparison of Coomassie brilliant blue staining method, potassium staining method, calcium staining method and silver staining method, on the basis, antitumor ac- tive fraction samples of P. americana were used for SDS-PAGE electrophoresis and staining. [ Result] The results showed that silver staining method could be ac- curately, quickly and easily used for SDS-PAGE staining of the antitumor active fraction of P. amer/cana. [ Conclusion] This study laid the foundation for explo- ring the medicinal value of P. americana.

A NEW STAINING METHOD FOR THE MEASUREMENT OF PROTEIN USING TETRAPHENYLPORPHYRIN TETRASULFONATE(TPPS_4)

A new dye-staining method for protein assay is described.The reaction between TPPS_4 and protein molecule causes a shift in the absorption of TPPS_4 from 435 nm to 488nm.The absorbance at 488 nm is proportional to the concentration of protein.The range of Beer's law was 1-5 ug/ml and the Sandell's sensitivity was 0.0087 ug/cm^2.

COMPARISON OF DIFFERENT STAINING METHODS FOR ANALYSING ESTERASE (EST) ISOZYME OF FUNGI AND PLANTS

EST isozymes are one of the frequently used biochemical markers in genetic analysis of fungi. and the staining is an important process in electrophoresis analysis of studying Est.The effects were compared ainong different stain recipes for Est of 3 kinds of fungi-Lentinus edodes. Pleurotus sapidus. Phellinus igriarius and 2 kinds of plants-Populus sp and Brassica chinensis. Of the four kinds of Est staining recipes tested.the recipe α-acetic acid-naphther showed the best effect.and followed by β-aceticacid-naphther, semicontent α-aceticacid-naphther and α+β-aceticacidnathpher.

Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells

Objective To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.Methods Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient.Results One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50%-90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or AnnexinV-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type.Conclusions Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2.7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis.

Localization of Abscisic Acid Binding Proteins in Maize Root Tip Using Immunogold-Silver Staining Method

Antisera against abseisic-acid-binding proteins(Ab Ⅰ)and anti-idiotypic antibodies to ABAmonoclonal antibody(Ab Ⅱ)have been developed and tested to comparatively localize abacisic-acid-bindingproteins(ABBPs)in maize root tips using immunocytochemistry technique.Based on the staining pattern ob-served along the root tip,an apicobasal gradient of ABBP has been revealed with either Ab Ⅰ or Ab Ⅱ.Thelabels are mainly concentrated in,the root cap and apical meristem.In the elongation zone and root hair zone,only cells in pericycle and epidermis are obviously labeled.The silver labels are distributed evenly in thewhole cell of the apical meristem,but in the cells of the root cap and elongation zone,the positive stainingdegree in the cytoplasm is reduced even to completely negative staining;nevertheless,the nuclei and plasmamembranes are strongly labeled.As in the labeled cells of the root hair zone,only some immunoactive sitescan be seen in the nucleus compacted to the edge of the cells.The staining pattern in the whole root tip alsoshows obvious dependence of the ABBPs localization on the activity of the nucleus.The significance ofABBPs localization is discussed.

Introducing a Rapid and Safe Method for Myeloperoxidase Staining

Background: Myeloperoxidase staining is used to differentiate leukemias since several decades. Despite implementation of flow cytometric, cytogenetic and molecular techniques for identification of leukemic blasts, histochemical stains such as myeloperoxidase stain are persistently used for better classification of leukemias. The myeloperoxidase staining is a time consuming and hazardous procedure. The present report describes a sensitive, rapid and easy method for assessment of peroxidase activity. Materials and Methods: Bone marrow aspiration slides were stained with Dako product: Code number: K3467 containing DAB chromogen (3,3-diaminobenzidine in chromogen solution) and substrate buffer (Imidasole-HCL buffer, PH 7.5 containing hydrogen peroxide and an anti microbial agent) in a rapid procedure taking only ten minutes time. The staining needs no material preparation steps. Neutrophils in the slide are taken as positive control or another normal smear was costained to be used as control. All cases were followed up with flow cytometry and cytogenetic studies. Result: The reaction product of this stain is brown and granular. Promyelocytes and myelocytes are the most strongly staining cells with positive (primary) granules. Lymphoblasts are negative. The result of classification of leukemias with this technique was in concordance with flow cytometric immunophenotyping. Discussion: Many practical techniques have been described using benzidine as an indicator for myeloperoxidase staining. Benzidine is a carcinogenic material and its usage is severely restricted in laboratory. Formerly we prepared requisite materials for myeloperoxidase staining by hazardous ways (boiling), but we decided to apply ready to use 3,3-diaminobenzidine (DAB), which is used in final step of immunohistochemistry stains. Conclusion: Use of 3,3-diaminobenzidine (DAB) is highly recommended for myeloperoxidase staining, while the result is extraordinary and fully compatible with flow cytometry and the method is safe and rapid.

A revisit to staining reagents for neuronal tissues

In the early days of deciphering the injured neuronal tissues led to the realization that contrast is necessary to discern the parts of the recovering tissues from the damaged ones.Early attempts relied on available(and often naturally occurring)staining substances.Incidentally,the active ingredients of most of them were small molecules.With the advent of time,the knowledge of chemistry helped identify compounds and conditions for staining.The staining reagents were even found to enhance the visibility of the organelles.Silver impregnation identification of Golgi bodies was discovered in owl optic nerve.Staining reagents since the late 1800s were widely used across all disciplines and for nerve tissue and became a key contributor to advancement in nerve-related research.The use of these reagents provided insight into the organization of the neuronal tissues and helped distinguish nerve degeneration from regeneration.The neuronal staining reagents have played a fundamental role in the clinical research facilitating the identification of biological mechanisms underlying eye and neuropsychiatric diseases.We found a lack of systematic description of all staining reagents,whether they had been used historically or currently used.There is a lack of readily available information for optimal staining of different neuronal tissues for a given purpose.We present here a grouping of the reagents based on their target location:(I)the central nervous system(CNS),(II)the peripheral nervous system(PNS),or(III)both.The biochemical reactions of most of the staining reagents is based on acidic or basic pH and specific reaction partners such as organelle or biomolecules that exists within the given tissue type.We present here a summary of the chemical composition,optimal staining condition,use for given neuronal tissue and,where possible,historic usage.Several biomolecules such as lipids and metabolites lack specific antibodies.Despite being non-specific the reagents enhance contrast and provide corroboration about the microenvironment.In future,these reagents in combination with emerging techniques such as imaging mass spectrometry and kinetic histochemistry will validate or expand our understanding of localization of molecules within tissues or cells that are important for ophthalmology and vision science.

Comparative Study of Z N Staining vs. Flurochrome Staining and Impact of Sample Processing on Diagnosis of Tuberculosis from Various Clinical Samples

Background: Tuberculosis is a highly infectious disease and India has the highest burden with it. Diagnosis of tuberculosis in many countries is still dependent on microscopy. Although its sensitivity is low in comparison to culture and molecular methods, its sensitivity can still be improved by using fluorescence staining method and processing of samples by homogenization and concentration method. Material and methods: Samples were collected from all newly registered suspected cases of tuberculosis in tertiary care hospital from outward and indoor department during a period of one year. Smears were prepared for Ziehl Neelsen stain and fluorescence stain both before and after homogenization and concentration procedure by 4% NAOH-2.9% sodium citrate method and results of them were interpreted according to RNTCP criteria for grading of sputum samples. All the samples were cultured in liquid culture MGIT system (Mycobacterial Growth Indicator Tube) and results of microscopy were compared with liquid culture taken as gold standard. Data were analyzed by using SPSS software version 16. Result: 350 samples were collected during study period. Out of 350 samples, 48 samples were positive for M. tuberculosis by MGIT system. In comparison with MGIT system, sensitivity of Z N stain for detection of acid fast bacilli was 77% before decontamination procedure, which was increased up to 85.42% after decontamination and concentration process. Sensitivity of fluroscence stain was 85.42% before processing, which was increased up to 91.67% after processing of samples. Conclusion: Sensitivity of smear microscopy can be enhanced by use of fluroscence microscopy and concentration method.

Two New Identification Methods for Encephalitozoon cuniculi on Tissue Section

[ Objective] The paper aimed to search new identification methods of Encephalitozoon cuniculi on tissue sections. [ Method] Using improved Gram staining method and methyl green pyronin staining method, the pathological sections of sick rabbits were stained and identified. [ Result] The pathological changes in brain tissue could be clearly observed on sections, but parasites were not examined in pathological brain tissues stained by common staining method. When the pathological section was stained by improved Gram staining method, the pathological changes in brain tissue were not ouly stained very clearly, but blue parasites were also found in brain tissues. The parasites in epithelioid cells were stained into purple ones by methyl green pyronin staining method. [ Conclusion] The im- proved Gram staining method and methyl green pyronin staining method performed good staining effects of E. cuniculi in pathological sections, which were conducive to rapid diagnosis of encephalitozoonosis in rabbit.

不同类型多肉植物的花粉活力检测方法筛选及活力鉴定

以3个科6个属的13种多肉植物种质资源为材料,对比亚历山大染色法和碘-碘化钾(I2-KI)染色法染色后花粉活力的差异,筛选适宜不同类型多肉植物花粉活力的测定方法。结果表明,两种染色法对不同科属的多肉植物花粉染色结果差异极显著,I2-KI染色法检测的花粉活力平均值显著低于亚历山大染色法;种质资源的花粉活力普遍高于杂交组合,聚类分析结合差异显著性分析将26种目标种质资源及杂交组合的花粉活力划分为5个等级;显微镜下花粉形状呈现圆形、方形、椭圆形和不规则形,且同一品种存在一种到多种的花粉萌发孔形态。

72例头癣患儿真菌荧光染色与皮肤镜特征分析

目的研究拟通过真菌荧光染色镜检技术结合皮肤镜分析不同临床类型头癣的特点,为临床提供准确、快速诊断头癣的依据。方法收集72例头癣患儿,使用荧光染色法结合皮肤镜观察头癣病发的主要特征;观察随访患儿治疗后荧光染色法及皮肤镜特殊表征是否同时转阴。结果根据临床特征,72例头癣患儿以白癣为主,占58.33%(42/72);荧光染色法阳性率100%,发外孢子占比最高,为62.50%(45/72);皮肤镜诊断的阳性率100%,皮肤镜检查白癣以断发(90.48%)为主、脓癣以脓疱(100%)为主,以逗号发/螺旋发/条码样发为特殊的表征;随访率为59.72%(43/72)。结论真菌荧光染色法结合皮肤镜特征能对头癣疾病及其临床类型做出快速且准确的诊断。

样本直接涂片法与液基夹层杯涂片法查找抗酸杆菌的效果比较

目的探讨液基夹层杯涂片法查找抗酸杆菌检验方法的临床应用价值。方法从广州市胸科医院医学检验科LIS系统中收集、整理2021年1月至7月痰液与支气管冲洗液主要送检临床样本的涂片染色检查和分枝杆菌培养结果,对同一患者时间间隔不超过1周的直接涂片与液基夹层杯涂片查找抗酸杆菌检验的结果进行回顾性分析。结果在241份痰液样本、178份支气管冲洗液样本和全部419份样本的检测中,直接涂片法、液基夹层杯涂片法的阳性率分别为15.35%(37/241)和13.69%(33/241)、15.17%(27/178)和12.92%(23/178)、15.27%(64/419)和13.37%(56/419)。在同期分枝杆菌培养结果阳性的65份痰液样本、50份支气管冲洗液样本和115份全部阳性样本中,直接涂片法、液基夹层杯涂片法的阳性率分别为56.52%(65/115)和50.78%(65/128)、54.34%(50/92)和45.88%(50/109)、55.56%(115/207)和48.73%(115/236)。在241份痰液样本、178份支气管冲洗液样本检测中,直接涂片阳性且液基夹层杯涂片阳性、直接涂片阳性且液基夹层杯涂片阴性、直接涂片阴性且液基夹层杯涂片阳性、直接涂片阴性且液基夹层杯涂片阴性的样本占比分别为12.03%(29/241)、3.32%(8/241)、1.66%(4/241)、82.99%(200/241)和8.99%(16/178)、6.18%(11/178)、3.93%(7/178)、80.90%(144/178)。结论液基夹层杯涂片法查找抗酸杆菌检验方法总体阳性检出率略低于直接涂片检测法,但可以发现相当比例的直接涂片法阴性的病原学阳性结核病患者,尤其是在支气管冲洗液检测中,具有一定的临床应用价值。

碱性副品红染色法检测木质部导管栓塞技术改进

染色法是测量导管长度、直径、导管分布以及栓塞脆弱性的重要方法。在测量导管栓塞时,染色压力和时间条件将极大地影响实验结果的准确性。为准确利用染色法测定刺槐Robinia pseudoacacia导管栓塞状况,本研究通过使用充气组(人为栓塞100%)以及冲洗组(完全去除栓塞)对碱性副品红染色法进行了压力、时间条件的改良。结果表明,当压力在0.04kpa,染色时间60min时,实验结果最为准确可靠。在此压力下染色过程不会冲开栓塞导管,配合60 min的染色时间可对未栓塞导管进行充分染色。另外,本研究通过诱导组,利用离心机法对枝条诱导不同程度的栓塞与染色法进行了对比验证,结果表明在该染色条件下,可以很好地反映茎段的栓塞状况,保证了实验结果的准确性。在后续试验中,使用碱性副品红染色法研究木质部栓塞时可以沿用此压力及时间条件。

痰标本革兰染色联合肺炎链球菌抗原检测快速诊断肺炎链球菌性肺炎

目的:探讨肺炎链球菌抗原快速检测联合痰涂片革兰染色在肺炎链球菌肺炎感染中的临床应用价值。方法:收集临床痰液标本分离得到的草绿色链球菌群、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌、金黄色葡萄球菌、嗜麦芽窄食单胞菌、流感嗜血杆菌及卡他莫拉菌共8种细菌各5株,肺炎链球菌培养阳性的痰液标本30例及痰涂片镜下疑似肺炎链球菌且合格的痰液标本40例,利用BinaxNOW试剂盒进行胶体金法肺炎链球菌抗原检测。结果:痰标本中分离得到的8种细菌经抗原检测结果均为阴性;胶体金法检测30例肺炎链球菌培养阳性的痰液标本阳性率为100%;痰涂片联合抗原快速检测阳性率高于痰培养,差异具有统计学意义(P<0.05)。结论:痰液肺炎链球菌抗原检测法具有较好的特异性,与痰培养结果有较好的一致性,且痰涂片革兰染色联合抗原快速检测在早期鉴定肺炎链球菌肺部感染中具有较高的临床应用价值。

崇明岛河道纤毛虫原生动物群落结构及对水环境的指示

浮游纤毛虫原生动物是真核微型生物的重要类群,在微食物环中扮演着物质和能量转换的关键角色,可作为水环境质量的指示生物。但传统沉降计数法对纤毛虫的鉴定分辨率较低,常造成多样性被低估。为此本研究在2020年7月2021年5月期间利用可获得更高分类分辨率的定量蛋白银法(QPS),对崇明岛河道浮游纤毛虫的多样性和群落结构进行调查,同时用多元统计方法分析环境因子对其影响,并筛选潜在指示种。结果共检出纤毛虫131种,隶属于3纲11目,其中寡毛目相对丰度最高,是全年优势类群。Shannon-Wiener指数为1.53±0.06(范围为0.16~2.62),Pielou指数为0.75±0.02(范围为0.18~1.00),Margalef指数为1.07±0.06(范围为0.13~2.98),呈现明显的季节变化,与硝酸盐和叶绿素a浓度均呈显著相关。ANOSIM检验发现4个季节的纤毛虫群落结构差异极显著,存在明显演替过程。dbRDA分析显示,营养盐、叶绿素a、溶解氧和降水量是影响全年纤毛虫群落结构变化的主要环境因子,盐度对其无显著影响;而各季节纤毛虫群落结构的异质性也均与水质指标呈显著相关,盐度仅在秋季对纤毛虫群落结构有显著影响。12种纤毛虫对群落结构的累积贡献率达90%以上,以食藻性和杂食性种类为主,它们与富营养化指标显著相关。优势种中奇异海游虫(Pelagostrombidium mirabile)和透明裂隙虫(Rimostrombidium hyalinum)不易受盐度影响,可作为崇明岛水质状况的潜在指示生物。

大花黄牡丹花粉活力检测方法筛选

为筛选适合大花黄牡丹花粉活力检测的方法,以大花黄牡丹花粉为试验材料,采用离体培养法和染色法(TTC、碘-碘化钾、醋酸洋红)测定其花粉活力。结果表明,4种因素对大花黄牡丹花粉离体萌发率的影响依次为温度>蔗糖浓度>硼酸浓度>氯化钙浓度,其中温度和蔗糖浓度的主效应显著;现有因素水平下,1%琼脂+150 g/L蔗糖+20 mg/L氯化钙+50 mg/L硼酸+25℃恒温培养1.5 h是大花黄牡丹花粉离体培养的最佳条件。研究表明,花粉离体培养可快速、准确测定大花黄牡丹的花粉活力,碘-碘化钾染色法可用于大花黄牡丹花粉活力的快捷检测;TTC染色法和醋酸洋红染色法则不适用。

基于深度学习构建金黄色葡萄球菌和粪肠球菌的快速图像识别系统

目的基于GoogleNet、ResNet101和Vgg193种深度学习模型,对血流感染病原菌(金黄色葡萄球菌、粪肠球菌)进行高置信度的识别,比较模型间的性能与分类能力,探讨深度学习模型对血流感染病原菌快速识别的应用可行性。方法将革兰染色及摄像预处理后的细菌图像和空白对照图像输入模型,进行训练与验证,共采集1682张金黄色葡萄球菌、1723张粪肠球菌和688张空白对照显微图像,对其中1344张金黄色葡萄球菌、1376张粪肠球菌和544张空白对照图像进行训练,余下的图像用于验证。根据模型间的分类参数评估出性能最佳的模型。结果ResNet101模型识别三类验证集图像的交叉熵损失值(0.0087103)最低,Epoch值(93)最大且准确率(99%)最高;GoogleNet模型识别三类验证集图像的交叉熵损失值为0.06389,Epoch值为86,准确率为98.6%;Vgg19模型识别三类验证集图像的交叉熵损失值为0.035682,Epoch值为86,准确率为97.7%。结论ResNet101模型在对三类验证集图像的分类上性能最佳;深度学习模型可对金黄色葡萄球菌和粪肠球菌的革兰染色图像进行准确、可信的快速识别。

改良组织贴壁法分离培养新生乳小鼠原代肺成纤维细胞的研究

目的建立改良版简单高效的原代小鼠肺成纤维细胞(lung fibroblast,LFB)分离培养方案,研究其体外生长特性。方法无菌条件下,分别取3天龄小鼠和8周龄小鼠肺组织,剪成1mm3大小,分别采用含10g/dl胎牛血清(fetal bovine serum,FBS)和20g/dl胎牛血清的高糖DMEM培养液进行组织贴壁法培养。采用差时贴壁法对LFB进行纯化,倒置显微镜下动态观察细胞生长状态及贴壁状态,采用流式细胞特异性分子染色法对LFB进行鉴定,传代培养后的第3代细胞采用CCK-8(cell counting kit-8)法检测细胞活性。结果3天龄小鼠在20g/dl FBS浓度条件下采用改良组织贴壁法进行培养,培养后第2天开始向周边呈放射状长出细胞,第7天细胞生长密度达90%,细胞形态呈长梭形。经差时贴壁法纯化后,CD140a阳性且CD45阴性细胞占比可达90%以上,连续传代3次后保持较好细胞活性。结论改良的组织贴壁法可简单高效地获得大量纯化的、活性好的小鼠LFB,为肺部炎症、肿瘤及药物体外疗效等研究奠定基础。

包头地区1 249例浅部真菌病患者中荧光染色法和KOH湿片法应用对比研究

目的:分析比较荧光染色法和KOH湿片法检测浅部真菌病的效果及不同位置检测结果比较。方法:收集2021年10月至2023年3月在包头医学院第二附属医院皮肤科就诊拟诊断为浅部真菌病患者的标本,分别采用荧光染色法和KOH湿片法直接镜检,比较阳性率检出结果,并进一步针对不同检测部位检测阳性率比较分析。结果:KOH湿片法和荧光染色法分别检测558例和691例患者(阳性率分别为38.35%和58.47%),阳性率差异有统计学意义(P<0.001);且头面部、手部、足部、躯干、腹股沟及甲部位荧光染色法阳性检出率均高于KOH湿片法,差异有统计学意义(P<0.05),差异由大到小依次为足部、头面部、腹股沟、手部、甲部、躯干部(P<0.001)。结论:荧光染色法在浅部真菌病的实验室检验中阳性检出率明显优于KOH湿片法,可以降低临床漏诊误诊率,在甲部、足部、腹股沟、头面部的检测中可作为优选,手部、躯干部则根据临床医生的临床经验在KOH湿片法或荧光染色法中选择,以减轻患者经济负担。