戊二醛浓度对免疫电镜胶体金标记密度影响的定量分析

经４％多聚甲醛＋０或０．５％戊二醛固定（１小时）大豆子叶细胞,大部分细胞器和蛋白体发生较大程度的变形,甚至解体,随着固定剂中戊二醛浓度升高对１．５、２．５％。细胞超微结构保存效果提高,蛋白体内反映７Ｓ蛋白质存在的胶体金颗粒密度分别为１９．７％和１１．４个／μｍ＾２。     

易褐变材料荔枝果皮透射电镜制样固定方法的改进

荔枝果皮组织在切块固定过程中 ,快速褐变引起细胞超微结构变化。用戊二醛固定剂固定也无法终止褐变反应。如在戊二醛液中添加 0 .5 %抗坏血酸 ,即可有效地克服褐变反应 ,因而固定的样品能更好地保持原有的结构。     

低浓度戊二醛对红细胞膜力学特性的影响

采用显微动态图像分析技术无扰、实时、在位地研究了低于0．005％浓度的戊二醛对年轻、老龄红细胞膜变形能力的作用．实验结果表明随着戊二醛浓度的增加,年轻、老龄红细胞膜的弯曲弹性模量明显变大,而老龄红细胞增加幅度更大;且随着戊二醛固定时间的延长,年轻、老龄红细胞膜变形能力的差距越来越大．说明作为免疫分析中常用固定剂的戊二醛对年轻、老龄红细胞细胞骨架的影响差异较大。研究结果可为适当选取在免疫分析测定中,以及以年轻红细胞作为药物载体时需采用的戊二醛浓度提供依据。     

不同固定条件下细胞与活细胞的原子力显微镜实时观察

用原子力显微镜（atom force microscope,AFM）观察固定细胞的最佳条件并在生理溶液中对活细胞实时观察.用不同固定剂和同一固定剂的不同浓度处理细胞;不加任何固定剂而直接在生理溶液中对细胞进行AFM成像.以戊二醛为固定剂并使用0.5%～1%的浓度固定细胞,后用缓冲溶液漂洗,再对细胞进行成像时可获得质量良好的图像.直接在生理溶液中进行观察,成像质量低于使用固定剂的细胞,但保持了细胞的生活原貌.在用原子力显微镜高分辨率观察生理条件下细胞的特点时,需要在制样与观测系统两方面进行改进.     

大黄蒽醌类化合物的组织化学定位研究

利用新鲜材料的秆手切片直接观察,５％ＮａＯＨ水溶液显色、荧光显微镜观察和戊二醛及饿酸固定薄切片法对大黄根茎蒽醌类化合物进行了组织化学定位研究。结果发现大黄根茎内蒽醌类化合物主要积累在次生木质部的木射线和次生韧皮部的韧皮射线细胞中。根据显色程度的不同,可以判断早期形成的维管射线细胞蒽醌类化合物的含量较晚期形成的射线细胞的含量高,二年生根茎的含量高于一所生根茎的含量。     

酶的微波孵育

我们在酶组织细胞化学技术中,应用微波辐射对生物组织进行快速孵育处理,获得满意的效果。具体操作;将小鼠断椎处死后立即打开腹腔,取出心、肾等组织,用刀片切成1mm~3左右的小块,放入2％多聚甲醛和2.5％戊二醛混合固定液中固定2小时。再用0.1mol/L二甲胂酸钠缓冲液浸洗3次,每次10分钟。     

白腐真菌漆酶的固定化及其应用研究*

以尼龙网为载体,戊二醛为交联剂,进行固定化真菌漆酶的条件优化和性质研究,优化条件为：尼龙网在5％的戊二醛溶液中交联6h后,加入30U漆酶溶液固定8h,酶活回收率为50．3％。与游离酶相比,固定化漆酶的热稳定性明显提高,最适pH值略有下降。用该固定化漆酶处理低浓度造纸废水,经过8批次连续试验,酶活保留52％。     

用过氧化氢酶电镜细胞化学技术显示人肝细胞的过氧化物酶小体

本文采用新鲜的人肝组织标本,进行过氧化氢酶细胞化学反应,以显示肝细胞中的过氧化物酶小体。结果表明,此细胞器圆形或椭圆形直径0.4—0.6μm。同时,要获得满意的反应效果,须使用低浓度的戊二醛短时间固定,孵育液由0.15％H_2O_2和4mg/ml的DAB配制(pH10.0),孵育时间为60分钟(25℃)。     

用共聚焦扫描显微镜检术观察水稻胚囊发育

介绍了一种快速简便观察分析水稻胚囊发育程序的方法。水稻子房经固定和脱水,用冬青油整体透明及丁香油封片,在共聚焦扫描显微镜检术下,发育中的胚囊产生自发荧光、可分辨出胚囊内部结构。ＦＡＡ和４％戊二醛两种固定液效果不同,后者效果较佳。     

不同固定液对Vero E6细胞形态和染色的影响及在病毒噬斑分析中的应用

病毒噬斑形成实验是确定病毒滴度的重要方法,其中噬斑染色是非常关键的步骤,而良好的固定液不仅能够维持细胞形态,也能起到助染作用,是有效识别病毒噬斑的关键因素.为了研究不同固定液对Vero E6细胞形态和结晶紫染色效果的影响,本研究采用五种固定液对Vero E6细胞进行固定(100％甲醇、4％甲醛、10％甲醛、75％乙醇和95％乙醇).以只加PBS缓冲液的细胞作为对照组.细胞固定30 min后利用1％结晶紫进行染色.利用光学显微镜和肉眼观察细胞形态,从而评价固定和染色效果.结果 显示,10％甲醛的细胞固定效果最佳,细胞形态良好,细胞染色均匀且着色深,利用该固定剂能在病毒噬斑实验中很好的识别噬斑,是Vero E6较为理想的固定剂.     

Restrictions on rotational and translational diffusion of pigment in the membranes of a rhabdomeric photoreceptor

Individual, isolated rhabdoms from dark-adapted crayfish (Orconectes, Procambarus) were studied with a laterally incident microbeam that could be placed in single stacks of microvilli. Concentration gradients of metarhodopsin along the lengths of microvilli were produced by local bleaches, accomplished by irradiation with small spots of orange light at pH 9 in the presence of glutaraldehyde or formaldehyde. No subsequent redistribution of pigment was observed in the dark, indicating an absence of translational diffusion. On the basis of comparison with other systems, glutaraldehyde, but not formaldehyde (0.75%), would be expected to prevent diffusion of protein in the membrane. Under the same conditions photodichroism is observed, indicating an absence of free Brownian rotation. Photodichroism is larger in glutaraldehyde than in formaldehyde, suggesting that the bifunctional reagent quiets some molecular motion that is present after treatment with formaldehyde. Quantitative comparison of photodichroism with mathematical models indicates that the pigment absorption vectors are aligned within +/- 50 degrees of the microvillar axes and are tilted into the surface of the membrane at an average value of about 20 degrees. The photoconversion of rhodopsin to metarhodopsin is accompanied by an increase in molar extinction of about 20% at the lambda maxand a reorientation of the absorption vector by several degrees. The transition moment either tilts further into the membrane or loses some of its axial orientation, or both. The change in orientation is 3.5 time larger in formaldehyde than in glutaraldehyde.     

Effect of Fixation on Demonstration of Phosphatases of Eimeria tenella Grown in Chick Kidney Cell Cultures

SYNOPSIS. The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase > thiamine pyrophosphatase > 5'-nucleotidase > adenosine triphosphatase > acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase. TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase.
Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.     

Immunocytochemistry of glutamate at the synaptic level

High concentrations of glutaraldehyde (2-5%) were found optimal for fixation of glutamate. In the absence of glutaraldehyde, (para)formaldehyde does not permanently retain L-[3H]-glutamate or D-[3H]-aspartate previously taken up into brain slices. Rats were fixed by rapid transcardial perfusion with 2.5% glutaraldehyde/1% (para)formaldehyde, and brain samples osmicated, embedded in epoxy resin, sectioned, and exposed to specific antisera to glutamate (conjugated to carrier protein by glutaraldehyde), followed by colloidal gold-labeled second antibody. The gold particle density was higher over putative glutamatergic nerve terminals than over any other tissue elements (two to three times tissue average in cerebellum and hippocampus). Calibration by test conjugates containing known concentrations of fixed glutamate processed in the same fluid drops as the tissue sections indicated that the concentration of fixed glutamate in putative glutamatergic terminals in hippocampus CA1 was c. 20 mmol/liter. The grain density over the parent cell bodies was only slightly higher than the tissue average. (Grain densities over test conjugates of other amino acids, aldehyde-fixed to brain macromolecules, were similar to that over empty resin. Labeling was blocked by glutamate-glutaraldehyde but not by other glutaraldehyde-treated amino acids.) In other experiments, brain slices were incubated in oxygenated artificial cerebrospinal fluid (CSF) and then immersion-fixed and processed as above. Here, the ration of grain densities in putative glutamatergic terminals vs other tissue elements was greater than in perfusion-fixed material. Comparison of intra-terminal areas poor and rich in synaptic vesicles suggested that in this preparation vesicles contained at least three times the glutamate concentration of cytosol. In the glutamatergic synapses of the giant reticulospinal axons in lamprey the ratio was over 30. Prolonged K+ depolarization of hippocampal and cerebellar slices reduced the nerve terminal glutamate immunoreactivity in a Ca2(+)-dependent manner. The results suggest that glutamate is released by exocytosis at excitatory synapses and show that immunocytochemistry can be used to study the cellular processing of small molecules.     

Indole reactions of mast cells and enterochromaffin cells related to fixations with and without aldehydes

Summary Survey of a considerable number of rat, mouse and hog tissues which presented large numbers of mast cells in preparations stained with toluidine blue and other metachromatic or basic dyes at low pH levels, revealed numbers of oval bodies of about the same size as mast cells which reacted weakly or even moderately to the postcoupled benzylidene indole reaction. The numbers of these were always less than that of mast cells in toluidine blue sections of the same blocks. They never occurred in clusters of perhaps 15–20 in a single high power field, as mast cells often do. Smooth and especially striated muscle which often formed the background tissue where most mast cells are found with metachromatic stains, regularly present indole reactions due to protein tryptophan. This is usually equal to or stronger than that in the supposed mast cells.Indole reactive bodies whose morphology suggests mast cells are also present in similar numbers in formaldehyde and glutaraldehyde fixed tissue as well as with aldehyde free fixations. Glutaraldehyde and formaldehyde are known to inhibit the benzylidene reaction of 5-HT in vitro (30 min for glutaraldehyde, 3 h for formaldehyde) (Lillie, 1977). This action was avoided in mercury and lead heavy metal fixations and in acetone, Carnoy, chloroform methanol and similar fixations.The mast cell-like bodies are best explained as tangential or oblique sections of individual muscle fibers. We have described the same phenomenon with the ferric ferricyanide (Golodetz-Unna, 1909) reaction (Lillie et al., 1978a), and the PCB reaction is that of tryptophan in these muscle cell sections.In contrast to the DMAB type reaction failure acid diazosafranin successfully demonstrated mast cells with both aldehyde and aldehyde free fixations. This reaction has been shown to occur with 5-HT and 5-HTP (Lillie et al., 1973).     

Improved utilization of tissue blocks for electron microscopy. Effect of various concentrations of formaldehyde and glutaraldehyde.

Liver tissue from miniature pig fetuses was immersion-fixed in fixative mixtures with various concentrations of formaldehyde and glutaraldehyde. The preservation quality of hepatocytes was evaluated ultrastructurally in a peripheral zone (30--130 micron below the surface) and a central zone (500 micron below the surface). In the peripheral zone the best preservation was obtained with a fixative mixture containing 2% formaldehyde and 2% glutaraldehyde and in the central zone with a fixative mixture containing 8% formaldehyde and 8% glutaraldehyde. It is concluded that a better utilization of fairly large tissue blocks for ultrastructural investigation can be obtained by division of the block and subsequent fixation in fixatives containing various concentrations of formaldehyde and glutaraldehyde.     

Effect of fixation on demonstration of phosphatases of Eimeria tenella grown in chick kidney cell cultures.

The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase greater than thiamine pyrophosphatase greater than 5'-nucleotidase greater than adenosine triphosphatase greater than acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase, TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase. Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.     

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue.

Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5--01%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.     

UV Micrographs and Photomicrographs from the Palynological Laboratory,Stockholm-Solna

In Equisetum both the spore and the rhizoidal and first-formed prothallial cells contain sac-like cytoplasmic particles limited by a unit membrane. After KMnO₄ fixation these bodies resemble the spherosomes described from earlier studies (e.g. Frey-Wyssling et al., 1964). They are bounded by a unit membrane, have a diameter of 0.8–1.7 μ and a fine granular content. After double fixation with buffered glutaraldehyde combined with an osmium post-fixative, or triple fixation with buffered formaldehyde added, the bodies resemble microbodies in fine structure. They have a thin unit membrane, a more coarsely granular matrix than after KMnO₄ fixation and very often a core like a 0.5 μ cluster of tubules or discs of around 200 Å in diameter. When spores are fixed in glutaraldehyde the bodies often show cytoplasmic invaginations or inclusions, which is never the case when they are fixed with KMnO₄     

Potential biological indicators for glutaraldehyde and formaldehyde sterilization processes

The present study aimed to isolate, select, and evaluate bacterial isolates with potential for use as biological indicators for sterilization with glutaraldehyde and/or formaldehyde. A total of 340 local Bacillus isolates were screened for glutaraldehyde and/or formaldehyde resistance by determination of minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and extinction time and were compared with B. subtilis (var. niger) ATCC 9372, the biological indicator for ethylene oxide sterilization, as reference. Of these, 85 isolates had glutaraldehyde MICs of 0.5% or higher, while 29 had formaldehyde MICs of 0.04% or higher. Of the 29 resistant isolates, 15 had MBCs of 0.05% or more. Extinction times were used to evaluate the bactericidal/sporicidal activity of glutaraldehyde. Eight had inactivation times of more than 5 h in 2% glutaraldehyde (pH 8), whereas 12 had inactivation times of more than 3 h in l% formaldehyde, with one isolate in common. These 19 isolates were selected and evaluated as potential biological indicators for aldehydes by determination of the decimal reduction times (D values), compared with the reference strain. Eight glutaraldehyde-resistant isolates exhibited D values 2.0- to 3.5-fold higher than the reference strain (30 min.). Only five of 12 formaldehyde resistant isolates had D values higher than that of the reference strain. Using six resistant isolates, temperature coefficient values between 2.11 and 3.02 were obtained for 2% formaldehyde. Finally, 14 isolates were tested for potential pathogenicity and were identified to species level. All of the eight glutaraldehyde-resistant isolates, including the isolate with dual resistance, and three formaldehyde-resistant isolates were B. licheniformis, while two other formaldehyde-resistant isolates were B. cereus. Six of the selected B. licheniformis isolates are potential biological indicators for sterilization processes using aldehydes. Three can be suggested for glutaraldehyde only and three for both aldehydes. Electronic Publication     

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue

Summary Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5–1%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.