Electron Microscopic Observations on Mitochondria of Rat Testes Fixed in Potassium Permanganate

A morphological investigation of mitochondria within the seminal epithelial cells of rat testes fixed in potassium permanganate reveals differences in electron opacity between the internal mitochondrial membranes of spermatogenic cells and those of Sertoli cells. Some interpretations of the apparent density differences are briefly discussed. It is concluded that the different effects of permanganate fixation upon the mitochondria of these cell types may reflect a significant structural difference between them.     

Alkaline Bismuth Solution as an En Bloc Stain for Formaldehyde-Glutaraldehyde Potassium Permanganate Fixed Fungal Spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

多种农药与次氯酸钠、高锰酸钾和高铁酸钾的反应活性

为探究4类,10种广泛使用的农药(苯氧羧酸类,芳香酸类,取代脲类和烟碱类)与3种氧化剂(次氯酸钠,高锰酸钾和高铁酸钾)的反应活性,本研究在温度(25±2)℃、pH值为8的条件下,分析10种农药分别与3种氧化剂NaClO、KMnO4、K2Fe O4在不同浓度下的反应活性,采用HPLC检测法,对比降解效能,探究氧化剂性质与有机物结构导致的反应活性的差异。实验结果表明,不同种类农药的结构性质对反应活性有重要影响,3种氧化剂的氧化降解能力有明显差异。苯氧羧酸类和芳香酸类农药结构较简单,并含有稳定的苯环或吡啶环结构,氧化降解较困难。取代脲类和烟碱类农药结构较复杂,氧化剂可攻击其不饱和官能团,反应活性较高。NaClO对取代脲类农药的降解率明显优于其他2种氧化剂,3种氧化剂对烟碱类农药的氧化降解效果依次为NaClO>KMn O4>K2Fe O4。研究多种农药与次氯酸钠、高锰酸钾和高铁酸钾的反应活性对降解去除水体中的农药残留对水环境的治理具有重要意义。     

The Action of Ribonuclease on Fixed Tissues

To study the optimal conditions for histochemical use of ribonuclease on fixed tissues, the factors of (1) type of fixation, (2) temperature, pH, type of buffer and length of incubation, (3) concentration of enzyme, and (4) staining and dehydration of sections were observed on rabbit pancreas.

The fixing fluids studied were sublimate-alcohol, Bouin's, Zenker-acetic, Zenker-formol, Petrunkevich's cupric-paranitrophenol, 10% neutral formalin, SUSA, Carnoy, Bensley's chrom-sublimate, absolute ethyl alcohol and acetone. Formaldehyde was a satisfactory fixative, although others might be preferred for special purposes. Of the five buffers tested, McIlvaine's citric-acid-disodium-phosphate mixture was the most satisfactory, whereas veronal-acetate extracted considerable stainable cytoplasmic material. The optimum concentration of ribonuclease and length of incubation varied greatly after the 11 different types of fixation. For example, with ribonuclease buffered by Mcllvaine's fluid, the intense cytoplasmic staining of formaldehyde-fixed tissues was removed by concentrations as low as 0.001 mg./ml., whereas, with sections fixed in Zenkers fluid some cytoplasmic staining persisted even after 3 hours in 0.2 mg./ml. Under the conditions employed the temperature and hydrogen-ion concentration during incubation were less important. Examples of nonspecific action of ribonuclease were noted. Until the degree and optimum conditions of specific action have been more precisely established by further experiments, it is suggested that this histo-chemical reaction only be interpreted as a confirmatory test which is, under the best conditions, only relatively specific for ribonucleic acid and not highly quantitative.     

感染大丽菊花叶病毒的大丽菊细胞超微结构观察

本文描述了大丽菊花叶病毒(DaMV)感染大丽菊叶片细胞的超微结构变化。受侵细胞内形成切面直径约1.6—3.1μm的球状或卵球状缅胞贡内含体。病株细胞内过氧物酶体的数量显著增加。内含体周围较细胞其它部位有较多的核糖体。高尔基体及微泡。叶绿体,线粒体正常。     

Ultrastructural and Biochemical Changes in Cotyledon Reserve Tissues3

Changes in major protein, lipid and carbohydrate reserves duringthe germination of Citrus limon (L.) Burm. f. seeds have beenstudied. The rate of release of amino acids and soluble sugarshas been evaluated. Mobilization of protein reserves began 4 d after the onset ofimbibition. The main period of hydrolysis occurred between 8d and 24 d after the start of germination. Ultrastructural studies showed the presence of protein bodiesin quiescent cotyledon cells. These bodies virtually disappeared14 d after the start of germination. The nitrogenous compoundsthat were liberated and subsequently translocated were predominantlyin the form of asparagine, arginine, and proline. The cotyledonshad a lipid content representing 51.7% of their dry weight.Lipid reserves in quiescent cotyledons were laid down in theform of oil-bodies. These organelles rapidly disappeared asgermination progressed, and were replaced by vacuoles. The starch content of quiescent cotyledons is very low, butit increased considerably up to 20 d after germination started. Key words: Proteins, lipids, carbohydrates     

Induction of Defense-Related Ultrastructural Modifications in Pea Root Tissues Inoculated with Endophytic Bacteria

The stimulation exerted by the endophytic bacterium Bacillus pumilus strain SE34 in plant defense reactions was investigated at the ultrastructural level using an in vitro system in which root-inducing T-DNA pea (Pisum sativum L.) roots were infected with the pea root-rotting fungus Fusarium oxysporum f. sp. pisi. In nonbacterized roots, the pathogen multiplied abundantly through much of the tissue including the vascular stele, whereas in prebacterized roots, pathogen growth was restricted to the epidermis and the outer cortex In these prebacterized roots, typical host reactions included strengthening the epidermal and cortical cell walls and deposition of newly formed barriers beyond the infection sites. Wall appositions were found to contain large amounts of callose in addition to being infiltrated with phenolic compounds. The labeling pattern obtained with the gold-complexed laccase showed that phenolics were widely distributed in Fusarium-challenged, bacterized roots. Such compounds accumulated in the host cell walls and the intercellular spaces as well as at the surface or even inside of the invading hyphae of the pathogen. The wall-bound chitin component in Fusarium hyphae colonizing bacterized roots was preserved even when hyphae had undergone substantial degradation. These observations confirm that endophytic bacteria may function as potential inducers of plant disease resistance.     

Combined Carbowax-Paraffin Technic for Microsectioning of Fixed Tissues

A combined Carbowax-paraffin technic for microsectioning fixed tissues gave ribbon sections as do paraffin infiltrated and embedded tissues. Blocks of formalin or alcohol fixed tissues 2 mm. thick were infiltrated with H.E.M. (Polyethlene Glycol: Carbowax, Hartman-Leddon Co.) or with one of the following polyethylene glycol ester waxes (Glycol Products Co., Inc.) for 4 hours at (61°C.): Polyethylene Glycol 600(Di) Stearate; Carbowax 1000-(Mono) Stearate; Carbowax 4000 (Mono) Stearate; Carbowax 4000 (Mono) Laurate; Carbowax 6000 (Mono) Oleate. The Carbowax infiltrated tissues were placed for 10 minutes in xylene (61° C.), into paraffin (61° C.) for 30 minutes, then into molten paraffin contained in separate molds. (The xylene passage can be excluded for preparations which preclude its use). The blocks were hardened rapidly by submerging in ice water and were fastened to carriers as in the usual paraffin technic. Tissues were cut 6 µ thick. Segments of ribbon were spread on a water bath and mounted on slides. After drying, tissues were stained directly with hematoxylin-eosin or were carried through xylene and alcohols as in routine paraffin preparations prior to staining. The Sudan III fat stain and Best's carmine stain for glycogen were applied as in usual technics. Cellular detail was well preserved and structures did not show the extent of distortion and shrinkage encountered in ordinary paraffin technic preparations.     

非酒精性脂肪肝病肝组织超微结构特点

目的:阐明非酒精性脂肪肝病(NAFLD)的超微结构特点。方法:收集我校和其他单位送检的3例单纯性非酒精性脂肪肝,16例NASH患者和4例NAFLD肝硬化患者的肝穿刺组织。用2.5%戊二醛、1%锇酸双固定、Epon 812包埋,超薄切片70nm,醋酸铀和柠檬酸铅染色后,JEM-2000EX透射电镜观察。结果:单纯性脂肪肝患者主要表现为大小不等的脂滴沉积、以小脂滴为主,可互相融合。NASH患者的肝细胞都可出现大量脂滴积聚,为大小脂滴混合型、内容物主要为中等电子密度、比较均一的甘油三酯,部分脂滴周围可见磷脂成分,NASH患者肝细胞内脂滴也互相融合。肝细胞线粒体的超微结构改变包括多形性线粒体、基质颗粒增多、线粒体增大和嵴的丧失是主要的电镜异常发现,线粒体内还可见副晶格样包涵体。部分NASH患者肝细胞内可见Mallory小体。NASH患者肝细胞周围可见淋巴细胞浸润。肝血窦Kupffer细胞增生不明显,NAFLD肝硬化患者Disse间隙和肝细胞间可见胶原纤维增生。结论:NAFLD具有较为明确的超微结构改变,电镜检查有助于诊断。     

A Simple Polychrome Stain for Conventionally Fixed Epon-Embedded Tissues

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO₄ fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H₂O₂, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic' fibers. Intra cytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes¹, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.     

Improved Paraffin Schedules for Plant Tissues

The two paraffin schedules here presented have produced less distortion in plant tissues than those commonly used. Both are modifications of the schedule described by Hemenway.¹ Schedule A is very similar to that described by Hewitt.² Schedule B usually causes less distortion than A but staining is not so bright as after A.     

Kinetic Modelling for the Assay of Nortriptyline Hydrochloride Using Potassium Permanganate as Oxidant

Kinetic methods for accurate determination of nortriptyline hydrochloride have been described. The methods are based on the oxidation of nortriptyline hydrochloride with KMnO₄ in acidic and basic media. In acidic medium, the decrease in absorbance at 525.5 nm and in basic medium, the increase in absorbance at 608.5 nm were measured as a function of time. The variables affecting the reactions were carefully investigated and optimised. Kinetic models such as initial rate, rate constant, variable time and fixed time were employed to construct the calibration curves. The initial rate and fixed time methods were selected for quantification of nortriptyline hydrochloride. In acidic medium, the calibration curves showed a linear response over the concentration range 10–50 μg mL⁻¹ for initial rate and 10–60 μg mL⁻¹ for fixed time method (2 min). In basic medium, the calibration graphs were linear over the concentration range 10–100 μg mL⁻¹ for initial rate and fixed time methods (4 min). In acidic medium, the limits of detection for initial rate and fixed time methods (2 min) were 1.02 and 3.26 μg mL⁻¹, respectively. In basic medium, the limits of detection were found to be 1.67 and 1.55 μg mL⁻¹ for initial rate and fixed time methods (4 min), respectively. The initial rate and fixed time methods have been successfully applied to the determination of nortriptyline hydrochloride in commercial dosage form. Statistical comparison of the results of the proposed methods with those of reference method exhibited excellent agreement and there is no significant difference between the compared methods in terms of accuracy and precision.KEY WORDS: dosage forms, fixed time method, initial rate method, nortriptyline, potassium permanganate     

Optimization of PAHs Oxidation from a Concentrate of Soil Attrition Using Potassium Permanganate

The treatment of soils contaminated with organic compounds, such as polycyclic aromatic hydrocarbons (PAHs), by attrition produced large amounts of highly concentrated attrition sludge (PAH – attrition concentrate – PAC). This paper studied the performance of an oxidation process using potassium permanganate (KMnO₄) to degrade PAHs that were initially present in attrition concentrates. The influence of operating conditions (temperature, concentration of KMnO₄ and reaction time) was studied, and these parameters were optimized using a response surface methodology (RSM). The results showed that the temperature and the reaction time had a significant and positive effect on the degradation of PAHs for the experimental domain studied (temperature between 20 and 60°C and reaction time between 1 and 7 h). The interaction between the temperature and the concentration of KMnO₄ significantly influenced the degradation of the PAHs. The temperature and the concentration of KMnO₄ were the main parameters that influenced the degradation of both phenanthrene (Phe) and benzo [a] pyrene (BaP). For benzo [a] anthracene (BaA), the temperature was the most influential factor. According to our results, the optimal conditions were defined as [KMnO₄] = 0.4 M for 5.5 h at 60°C. These optimal conditions led to degradations of 42.9%, 40.8%, 41.0% and 46.0% of the total PAHs, Phe, BaA and BaP, respectively.     

In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate–fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies. (J Histochem Cytochem 57:615–622, 2009)