克氏原螯虾(Procambarus clarkii)血细胞染色方法的研究

甲壳类血细胞的形态和分类是甲壳类免疫学研究的基础.本文选择三种甲壳类血细胞的常用染料:瑞氏染液、姬姆萨染液和瑞氏-姬姆萨混合染液,研究它们对于克氏原螯虾血细胞的染色效果.通过改变染色时间、染色温度、分色方式以及分色时间等染色条件,观察、比较不同染色条件下血细胞内的颗粒、细胞核和细胞质的着色情况,以及细胞整体轮廓清晰程度,确定适用于克氏原螯虾血细胞染色的理想染色方法,并建立相应的操作程序.     

东亚飞蝗血细胞形态学研究

通过Wright's染色和光学显微镜对不同地区东亚飞蝗的血细胞进行了观察,发现东亚飞蝗Locusta migratoria manilensis(Meyen)的血细胞包括原血胞、浆血胞、粒血胞和类绛血胞4种类型.原血胞的细胞核为红色,而其它3种血细胞的细胞核均被Wright's染液染成红色和蓝色2种类型.故认为浆血胞是...     

黑水虻幼虫血细胞类型的研究

黑水虻Hermetia illucens(L.)是一种重要的资源昆虫。本文旨在筛选出适合黑水虻血细胞观察的染色方法,明确黑水虻血细胞类型、数量及组成,为黑水虻血细胞免疫研究奠定基础。采用Giemsa和Giemsa-Wright's染色方法和血球计数板法,对黑水虻血细胞染色方法和血细胞数量及形态进行研究。结果表明,甲醇固定4 min,Giemsa-Wright's染液染色9 min、pH 7.2磷酸盐缓冲液分色10 min是黑水虻幼虫血细胞最佳染色方法;黑水虻幼虫血细胞包括原血细胞、浆血细胞、粒血细胞、类绛血细胞、珠血细胞5类;4龄黑水虻幼虫血细胞数量大约为2917个/μL,其中浆血细胞占53.20%±2.78%,粒血细胞占37.49%±3.96%,原血细胞占7.97%±1.51%,类绛血细胞占1.02%±0.24%,珠血细胞占0.62%±0.08%。Giemsa-Wright's染色法为黑水虻幼虫血细胞最佳染色方法,黑水虻幼虫血细胞可分为5类10种。     

鲫鱼(Carassius auratus)外周血细胞显微和亚显微结构的观察

本文记载了用光镜和电镜观察鲫鱼外周血细胞所得的结果。 用光镜观察以Wright氏染液染色的血涂片,可区分出下列各种类型的血细胞:红血细胞,淋巴细胞,血栓细胞,单核细胞,中性、嗜酸和嗜碱粒细胞。此外,在外周血液中还可以看到少量未成熟的红血细胞。 在外周血液中,嗜酸和嗜碱粒细胞的数量极少。但在头肾和脾等造血组织中这类细胞的数量较多。 鲫鱼外周血液中各种血细胞的超微结构,基本上和人类以及其他鱼类相应血细胞的超微结构相似。     

对DNA与RNA区分染色实验的改进

核酸是主要的遗传物质 ,核酸的主要成分在细胞核中是 DNA,在细胞质与核仁中的是 RNA。由于 DNA与RNA在化学组成与分子结构上存在一定差别 ,因而对不同染料有不同的染色反应 ,可以根据这一原理来定性鉴定细胞中 DNA与 RNA的存在与分布。实验所采用染料为甲基绿 -焦宁 (Netyl- Green-Pyronin)染液 ,其中染液中的甲基绿能使细胞核中的DNA呈现绿色 ,而焦宁则能把细胞质与核仁中的 RNA染成红色。因此可以根据细胞中不同部位呈现颜色的不同来进行定性鉴定。1　材料与方法为了使实验方法快速简易而效果清晰准确 ,我们从染液的配方、材料…     

血涂片制作的几点体会

制作方法：血涂片Wright’s染色法。①采血、制血膜、水平放置血膜片晾干。②血膜片固定于甲醇2-3min,取出晾干。③用蜡笔在血膜片两端划一染色区,避免染色时染液外溢。④往血膜片滴加瑞氏染液（瑞氏染料0．1g＋甲醇60ml）,数十秒钟后,再加一倍于染液的蒸馏水,染色5-8min。     

改良Pereira髓过氧化物酶快速染色法及应用

目的　为了使髓过氧化物酶 (MPO)染色快速准确、安全可靠 ,对Pereira碘化钾MPO染色方法做了进一步改进。方法　采用将碘化钾溶于Wright Giemsa染色液中的新配方 ,使试剂更稳定、保存时间长 ,并简化了操作、缩短了染色时间。结果　此法与Washburn联苯胺法比较 ,两者阳性率十分相近 ,差异无显著性意义 (P >0 0 5 )。结论　改良Pereira髓过氧化物酶法阳性反应标本存放多年不褪色 ,是目前众多碘化钾法中较理想的MPO染色方法之一。     

介绍一种陈旧苏木精染液复苏的方法

苏木精是组织切片制作常用细胞核染料 ,以Harris苏木精染液最为常用。Harris苏木精染液在使用一段时间后 ,染色能力逐渐减弱 ,造成细胞核着色困难 ,染色时间延长 ,虽能着色 ,但依然着色浅淡 (图 1,2 )。为此 ,我们采用在陈旧Harris苏木精染液中加入苏木精酒精液 ,经煮沸冷却后加冰醋酸的方法 ,使Harris苏木精染液恢复染色能力 ,收到了较好的效果 ,现介绍如下。1　材料与方法1 1材料 (1) 5 %苏木精酒精液苏木精(E·Merck进口分装 ) 5 g溶于无水乙醇 10 0ml,放置 4～ 5周后使用。 (2 )陈旧Harris苏木精染液80 0ml。1 2方法在 80 0ml陈旧Ha…     

滇金丝猴的血细胞学检查

本文首次报道了滇金丝猴的血细胞学检查结果,内容包括骨髓细胞分类计数和外周血象。前者检查了一例成年 雌性猴,骨髓取自肋骨,涂片用Wright氏染液染色和用Giemsa氏染液复染,两次取样检查,每次计数500个骨髓有核细胞;后者检查了一例成年雌性猴和一例幼年雌性猴,每例检查二次取其平均值。结果如表1、2、3。 检查结果中,值得注意的是:1.骨髓细胞中退化细胞较多,出现率达16.4％;2.骨髓细胞中出现少量环形核粒细胞。据张耀平等(1986),树鼩骨髓细胞中出现少量环形核粒细胞,这在啮齿动物大、小鼠中是常见的,但在灵     

硫堇显示细胞核DNA组织化学染色技术

细胞DNA含量反映细胞生长及分化状态,测定其含量的变化对判断肿瘤的性质及预后具有重要意义。在进行各种肿瘤细胞核DNA含量图像分析时,发现细胞核DNA染色质量的好坏直接影响DNA含量测定结果的准确性。应用中发现传统的Feulgen染色技术存在染色特异性不强,背景易共染,染液配制较复杂,对反应条件要求严格,染色时间较长,切片易褪色等缺点。     

4种碘化钾-过氧化氢法髓过氧化物酶染色结果比较

目的为了探讨较理想的髓过氧化物酶（MPO）染色方法。方法采用我室新建立的碘化钾-吡啰红B法（KI-PyB法）及碘化钾-吡啰红G法（KI-PyG法）,以及碘化钾-煌焦油蓝法（KI-BCB法）、碘化钾-wright-Giemsa法（KI-WG法）四种MPO染色法,同时对80例骨髓涂片标本进行了MPO染色。结果KI-PyB法MPO阳性产物呈鲜红色颗粒状,细胞核为蓝绿色;KI—PyG法MPO阳性产物呈棕黑色颗粒状,细胞核为浅红色;KI-BCB法阳性产物为蓝黑色颗粒状,细胞核为紫红色;KI-WG法阳性产物为深红色至棕黑色颗粒状,细胞核为浅蓝色或浅紫红色。4种碘化钾法MPO染色平均阳性率分别为60．9％、62．1％、56．3％及61．3％,差异无统计学意义（P〉0．05）。结论4种碘化钾法MPO染色中KI-PyG法的阳性率相对较高、且阳性反应易于判断,是较理想的MPO染色方法。     

Mechanism of Intranuclear Crystal Formation of Herpes Simplex Virus as Revealed by the Negative Staining of Thin Sections

Structural alterations induced in HeLa cells by herpes simplex virus and the mechanism whereby the virus is formed in the nucleus in crystal arrays were studied by electron microscopy with both the usual and negatively stained sections. Aggregates of granular and filamentous material were observed in the cytoplasm of infected cells with both sections. On the other hand, no remarkable alterations in appearance of the cytoplasmic ground substance were observed with the usual sections of infected cells. However, the cytoplasmic ground substance of infected cells when negatively stained consisted of granular material which was different in appearance from the spongy material constituting the cytoplasmic matrix of uninfected cells. In the nucleus of infected cells, complexes consisting of round bodies, amorphous material, aggregates of uniform granules in rows, and viral crystals were often observed near the nuclear membrane in both types of sections. Examinations of the granular aggregates with negatively stained sections suggested that each granule represents a subunit and that the several adjoining subunits (approximately eight) constitute the requirement for formation of a single viral capsid with a core. Thus, rapid and simultaneous formation of the core and capsid within the aggregate would replace the rows of the granules with the viral crystal. The advantages of negative staining of thin sections for visualization of fine structural alterations are discussed.     

A method for the differentiation of T and B lymphocytes and monocytes migrating under agarose.

This report describes alterations in the agarose lymphocyte migration technique which resulted in satisfactory differentiation of T and B lymphocytes and monocytes which have migrated as a monolayer for 1-3 days. The Wright-Giemsa staining used in the original method did not permit identification of individual migrating cell types. The most important modifications were changing from a plastic to a glass migration surface, and significantly reducing the overlying thickness of agarose which permitted a short fixation time and easy preparation of permanent slides stained for nonspecific esterase. The esterase staining of monocyte cytoplasm was intense and diffuse. One or two small, discrete areas of cytoplasmic esterase activity were identified in the majority of T lymphocytes. B lymphocytes showed either a trace or no evidence of esterase activity. The modified method should prove useful for the histochemical differentiation of migrating subpopulations of mononuclear cells.     

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach

Summary A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substrances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.     

嗜酸粒细胞过氧化物酶快速染色的研究

目的建立嗜酸粒细胞过氧化物酶快速染色方法,完善包括嗜酸粒细胞脱颗粒或中性粒细胞粗颗粒等各种情况下嗜酸粒细胞准确并快速计数的质量控制。方法随机选取75例血液病患者的骨髓涂片标本,要求常规瑞-姬染色骨髓分类嗜酸粒细胞≥3.5%,取材3d内。每份标本中选取两张,分别划入实验组和对照组。实验组标本进行嗜酸粒细胞过氧化物酶快速染色,对照组标本进行常规瑞-姬染色。分别计数嗜酸粒细胞百分数。结果实验组嗜酸粒细胞颗粒染成黑色,对包括中性粒细胞在内的其他细胞染色效果同瑞-姬染色,显微镜下嗜酸粒细胞显示醒目,可快速准确计数。结果与对照组比较,经t检验,P0.05,无统计学差异。结论嗜酸粒细胞过氧化物酶快速染色方法比较常规瑞-姬染色具有快速染色,对嗜酸粒细胞的显示更加醒目的优点,且对包括中性粒细胞在内的其他细胞染色兼有瑞-姬染色的效果。该方法值得推广用于快速骨髓细胞染色,且适用于包括嗜酸粒细胞形态不典型及中性粒细胞颗粒粗大等各种情况下的嗜酸细胞计数的质量控制,从而准确有效地服务于临床诊治。     

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.     

Neutrophil granulation stained with May Grunwald Giemsa or pure Azure B-eosin Y quantified by image analysis

Neutrophil granulation was quantified after staining with May Grunwald Giemsa or with the pure dyes Azure B and eosin Y. Spinner slides of the buffy coat of 3 normal subjects and 14 persons with different grades of toxic granulation were studied. Morphometric parameters were measured using an image analysis computer (Texture Analysis System, Leitz Wetzlar, FRG). The parameters for granulation varied over a wider range in Azure B (AzB) than in May Grunwald Giemsa (MGG) stained granulocytes. This is in accordance with the visual microscopy observation, that granulation is more pronounced after staining with AzB than MGG. The predominant shade of the nucleus was similar in both stains, whereas considerable and variable differences in the shade of the cytoplasm were found.     

Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.     

Preferential C-banding of wheat or rye chromosomes

Summary Using different stains, wheat chromosomes could be distinguished from rye chromosomes by preferential staining. C-bands of rye chromosomes were preferentially stained with Giemsa while those of wheat chromosomes were preferentially stained with either Leishman or Wright stain. Preferential staining aids the identification of wheat and rye chromosomes and chromosome segments and in particular the recognition of wheat/rye chromosome substitutions and translocations.     

Fluorescence of yeast vitally stained with ethidium bromide and propidium iodide

Both ethidium bromide and propidium iodide stain growing yeast. As visualized in the fluorescence microscope, ethidium stains the nucleus and cytoplasm in wild type yeast and in those grown in 10% dextrose, with brightly fluorescent cytoplasmic granules being present in both. Under the latter conditions, the mitochondria are repressed but not absent. In rho 0 cells, in which the mitochondrial DNA is absent, ethidium appears to bind to the cell wall or membrane preferentially with no cytoplasmic granules being visible. In all cell types, propidium appears to bind the cell wall or membrane with no cytoplasmic granules being visible in any cell. The staining patterns thus suggest greater differences in the binding of these two types to mitochondrial DNA in situ than is suggested by their in vitro behavior. These differences in binding could explain their different mutagenic capacities..