痰涂片标本用于结核分枝杆菌耐药性检测的研究

目的 研究抗酸染色结核分枝杆菌(简称结核杆菌)阳性痰涂片标本直接用于耐药性检测的方法。方法 对18株临床分离培养的结核杆菌用利福平进行药敏试验。分别提取菌株DNA和与之对应的痰涂片标本的菌体DNA,用聚合酶链反应(PcR)扩增ropB基因后进行固相杂交和核酸测序检测结核杆菌的耐药性。结果 18株结核杆菌中有12株对利福平耐药。经PCR扩增的ropB片段与探针杂交后,敏感菌株未发现rpoB基因的突变,自耐药菌株提取的DNA中rpoB突变体的检出率为100％(12／12),痰涂片提取DNA的检出率为91．7％(11／12)。所有耐药菌株DNA与痰涂片DNA核酸测序结果相吻合,都有rpoB基因核心区域碱基突变。结论 抗酸染色痰涂片阳性标本可直接用于检测结核杆菌利福平耐药基因rpoB突变体,是一种值得临床实验室推广使用的耐药菌诊断方法。     

用于抗酸菌培养的痰标本室温保存方法的研究

在痰标本中加入适量的基础保存液,作为在室温下保存痰标本的方法。实验结果表明,基础保存液对抗酸菌H₃₇Rv株及草分枝杆菌株无抑制作用,但对变形杆菌等痰标本抗酸菌培养中常见的杂菌具杀灭作用。219例临床痰标本的实验结果显示:痰标本用此法处理在室温中保存28天,其阳性检出率不受影响,与未经任何处理的在同样室温中保存的痰标本相比两者具有极显著性差异(p<0.01).本方法可使痰标本在室温中保存21天,以保存14天以内为佳。     

乳腺肿瘤中抗酸菌L型感染的研究

用ＩＫ抗酸染色和卡介苗抗体免疫组化染色等方法对乳腺癌（９７例）,和乳腺纤维腺瘤（２１例）进行了研究。结果发现Ｌ型阳性例数在乳腺癌和乳腺纤维腺瘤间无显著差异（Ｐ〉０．０５）。乳腺癌中抗酸菌Ｌ型主要分布于癌细胞巢及其间质内,常集聚成堆,且该处癌细胞有不同程度变性或坏死。乳腺纤维腺瘤中的抗酸菌Ｌ型主要存在于间质巨噬细胞中。并讨论了抗酸菌Ｌ型感染与乳腺癌发生的可能关系。     

结核菌荧光抗体的制备及其临床应用

本文介绍以无毒人型结核菌菌悬液大剂量(80mg/ml)经皮下多点、静脉注射免疫家兔。获得凝集效价1:160—1:320免疫血清。提取免疫球蛋白制备免疫荧光抗体。用该抗体检查结核菌,其敏感性与荧光素染色和抗酸染色基本相似。与22种抗酸菌和3种非抗酸菌进行交叉试验,除与牛型结核菌、堪萨斯杆菌有交叉反应外,其它被试细菌均呈阴性反应。用该抗体检查肺结核病人痰标本155例,阳性率为43.87%,培养法阳性率27.74%,两法差异显著(P<0.005)。结果表明,结核菌免疫荧光抗体可用做肺结核病细菌学的快速诊断。     

恶性肿瘤中抗酸菌L型检出意义的研究

本文用改良抗酸染色法和卡介苗抗体免疫组化染色法对４２０例恶性肿瘤（乳腺癌１１２例肠腺癌１００例、恶性淋巴病５４例、子宫颈癌７０例、绒毛膜癌８４例）进行了研究。结果发现４２０例恶性肿瘤中４７例（１１．２％）,１Ｋ抗酸染色阳性和４９例（１１．６％）免疫组化染色阳性,其中以恶性淋巴瘤抗酸菌Ｌ型检出率最高（４４．４％）绒毛膜癌最低（２．４％）,抗酸菌Ｌ型主要（８５％）分布于癌巢或肿瘤实质内该区５１．１％癌细胞呈增生活跃状态,３４％癌细胞呈空泡样变性,颇似病毒感染的凹空细胞,２．１％的瘤细胞发生坏死。结合文献复习,提示抗酸菌Ｌ型感染可能与肿瘤发生有关。     

1例L型支气管戈登菌肺部感染的报道

放线菌目戈登菌属支气管戈登菌存在于环境中,在机体免疫力下降时可感染致病。从1例肺部感染4年的78岁患者痰标本中分离出1株菌株,经聚合酶链反应(PCR)扩增16SrRNADNA,序列比对分析证实为支气管戈登菌。该菌株在含蔗糖的Middlebrook7H9(7H9-L)半固体培养基上呈煎蛋样嵌入性生长,在含氯化钠的92-3TB(92-3TB-L)液体培养基也生长,而在罗氏培养基、BACTECMGIT-960、不含氯化钠的92-3TB(92-3TB-B)液体培养基及不含蔗糖的Middlebrook7H9(7H9-B)半固体培养基不生长。涂片抗酸染色显微镜下可见大小不等的抗酸染色阴性球状或短杆状菌。传代培养2代后可见橘红色菌落,涂片抗酸染色可见少量抗酸染色弱阳性杆状菌,革兰染色及扫描电子显微镜观察菌体为杆状。结果表明,从患者痰液中分离的菌株为L型支气管戈登菌,提示肺部感染迁延不愈的免疫力低下患者需考虑感染L型支气管戈登菌的可能。     

抗酸染色后不同复染方法的比较

目的比较抗酸染色后3种复染剂的染色效果,选择更适于抗酸菌感染临床病理诊断的复染剂种类。方法用确诊结核菌或麻风菌的病例进行抗酸染色后用Mayer氏苏木精、亚甲蓝、甲基绿分别复染。结果用Mayer氏苏木精进行衬染的阳性位置定位清楚,对比清晰,背景干净。结论用Mayer氏苏木精复染有利于病理医生的观察,对提高抗酸菌阳性检出率具有一定的实用价值。     

聚合酶链式反应检测结核杆菌的研究

以人型结核杆菌基因组DNA为模板,合成二段引物各20个碱基进行聚合酶链式反应(PCR)。经琼脂糖凝胶电泳证实,获得一条245bp扩增带。PCR检测的敏感性染色体基因组DNA为1pg,菌悬液为13个活菌／ml。在特异性试验中,人型结核杆趋,牛型结核杆菌、BCG可见此扩增带。被试的其它14种扰酸菌以及变铅青链霉菌、大肠杆菌质粒Puc19、星状诺卡氏菌、红球菌均未见该扩增带。54例肺结核痰标本3种方法检查的阳性率分别为：萋尼氏抗酸染色16．7％,培养法14．8％,PCR 37．0％。前2种检查方法分别与PCR比较,经统计学处理均有显著性差异(P<0．01)。12例非结核性肺部疾患痰标本抗酸染色和PCR均为阴性。结果表明,PCR技术是快速、敏感、特异诊断结核病的方法。     

抗酸性颗粒的结核流行病学意义

抗酸性颗粒的结核流行病学意义江苏省武进市人民医院武进２１３０００孙建中国内外结核病发病呈上升趋势,探讨抗酸性颗粒（即Ｌ型）对控制结核流行病学意义及临床诊断治疗都是有价值的。１材料与方法１．１标本采自肺结核患者２６２例痰标本,常规涂片,抗酸染色。１．２...     

微波在抗酸染色中的应用改进

抗酸染色以Ziehe—Neelsen抗酸杆菌法的应用最为广泛,但操作过程中温度及复染背景难以控制,在总结了我科10例麻风活检标本抗酸染色基础上,我们对抗酸染色法进行改良,效果显著,现介绍如下。     

Differential Stain for acid fast Bacteria and Spores

A modified method of staining acid-fast organisms is described. After staining with carbol-fuchsin as usual in the Ziehl-Neelson method, wash with water and while the slide is still wet cover with a saturated acetone solution of malachite green for three to five minutes. Wash and examine. The acid-fast organisms and spores are red in a green background. If the smear is thick and appears too dense, dry for three minutes and hold over the mouth of a bottle of ammonia until decolorized to suit. Upon exposure to the air the green returns. This can be prevented by keeping the smear alkaline, by the addition of sodium bicarbonate.

A second method is described for use with sputum in which acid-fast organisms are scarce. It permits the examination of thick smears and therefore increases the chances of finding tubercle organisms when few in number. Stain with carbol fuchsin as in the Ziehl-Neelson method. Decolorize with 30% phenol-disulfonic acid in water for a few seconds or until decolorized. Wash and examine at once. If color returns upon washing decolorize again. The tubercle organisms appear red in a colorless background.     

结核菌L型感染与无反应性结核病关系的研究

本文应用改良抗酸染色及免疫组织化学染色等方法对４２例非特异性炎、肉芽肿性炎及肿瘤进行抗酸及免疫组化染色,观察抗酸菌Ｌ型感染与无反应性结核病的关系进行了初步研究。抗酸染色结果阳性率为８０．９５％;免疫组化染色为７８．５７％。     

Demonstrating Weakly Acid-Fast Tubercle Bacteria in Sputum

Acid-fastness of the tubercle bacteria has long been used as the common method of diagnosis in sputum. It has been suggested sometimes that tuberculosis could occur without demonstrable bacteria, as well as with acid-fast bacteria, non-acid-fast bacteria or granules. It is shown in this paper that some of the sputa which are negative to the standard staining technic will show rods, rods with round polar bodies, or similar bodies without the rod portion. It is also pointed out that the decolorization of the smears by acid alcohol be shortened to approximately 3 to 5 seconds and picric acid be used as a counterstain. These forms are apparently the varying stages of the loss of acid-fastness. It is essential that a counterstain be used which will not interfere, and yellow is indicated because it does not absorb the red rays. Sputa which are negative to the standard acid-fast staining technic but which come from persons with a variable intermittent fever should be stained by this modified technic before they are pronounced germ-free.     

植物标本制作工艺的改良

用于教学的植物标本主要有浸液标本和腊叶标本。蜡叶标本在使用过程中破损严重,浸液标本囡其笨重的外形不适用于大范围的教学研究。现通过叶片含水量检测、植物种子超干实验等一系列对比实验,针对教学标本工艺进行改良,以册的形式,对标本进行整理归纳,形成适应普通教学的简易植物标本数据库。     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.     

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.     

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.     

Survival of tubercle bacilli in heat-fixed and stained sputum smears

We used a slide culture technique to detect tubercle bacilli surviving in sputum smears (n=46) after conventional heat fixation and Ziehl-Neelsen staining. In all heat-fixed sputum smears, tubercle bacilli survived after time 0 (n=22), 24 h (n=7), 48 h (n=7), 72 h (n=4), and seven days (n=6). None of the stained sputum smears showed growth on slide cultures. Viable tubercle bacilli remaining in heat-fixed sputum smears for at least seven days may present an infection risk to laboratory staff. Thus, sputum smears should be stained immediately by the Ziehl-Neelsen method or stored in a safe container to avoid transmission of tuberculosis.     

评价空肠弯曲菌外膜蛋白PEB1介导的重组BCG与上皮细胞的结合能力

目的：评价PEBl介导的屋尘螨抗原（Derp2）重纽BCG疫苗（PEBI-Derp2．rBCG）与人上皮细胞的结合能力。方法：采用体外细胞培养的方法,分别将普通BCG、胞壁型Derp2-rBCG和胞壁型融合蛋白PEBl-Derp2-rBCG与HeLa细胞及人类肠粘膜上皮细胞（HIEC）进行共孵育,利用HE和抗酸染色法对各组细胞与疫苗的黏附结果进行染色,光学显微镜下计数各组的黏附率,并进行比较;对以上各组分别加入PEBl蛋白,进行黏附阻断,观察对结合能力的影响。结果：孵育24小时后,无论HeLa细胞还是H匝CPEBl-Derp2．rBCG组较普通BCG组和Derp2-rBCG组的黏附率明显提高,差异有显著性（P〈0．05）;PEBl蛋白的加入对PEBl．Derp2-rBCG的黏附功能有明显抑制作用（P〈O．05）;但是,Derp2-rBCG组与普通BCG组比较没有明显差异（P〉o．05）,PEBl蛋白的加入对二者的黏附亦无影响（P〉0．05）。结论：PEBl具有介导增强PEBl-Derp2-rBCG与上皮细胞黏附的能力。