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.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

En Bloc Staining of Articular Cartilage and Bone

Blocks of canine and porcine articular cartilage were stained en bloc with Weigert's iron hematoxylin or Harris' hematoxylin with or without eosin Y counterstaining and cleared in methyl salicylate. The morphology and three-dimensional relationships of chondrocytes were best demonstrated with Weigert's iron hematoxylin. The morphology of the cartilage and chondrocytes was superior to that in sections of routine hematoxylin and eosin stained, paraffin processed samples. The three-dimensional localization of intracellular lipids in individual and clones of chondrocytes was observed when cartilage samples were stained with oil red O and mounted directly in a water-based medium. Blocks of decalcified bone were stained en bloc with Weigert's iron hematoxylin and cleared with methyl salicylate. The three-dimensional orientation of osteocytes around osteonal canals, in circumferential lamellae, and in interstitial lamellae was demonstrated. The morphology of “cutting cones” in cortical bone also was observed.     

Application of the Periodic Acid-Schiff Procedure to Tissue Blocks

The periodic acid-Schiff procedure can be used for staining en bloc by incorporating the periodic acid with the fixing fluid. After simultaneous fixation and oxidation for 48 hr at room temperature and subsequent staining in Schiff reagent the tissues are dehydrated, embedded in paraffin and sectioned. Of two fixatives used, 95% alcohol proved superior to 10% formalin. Various concentrations of periodic acid (0.1-2.0%) yielded equally good results, thus the use of the lower concentrations is feasible and preferable. Fixation and oxidation simultaneously or separately yielded equally satisfactory results and in view of the time saved in the simultaneous method the authors recommend it. Using similar time of fixation and oxidation, satisfactory results were obtained with the intestine of rat after 3 hr of exposure to Schiff reagent. A longer period of exposure (up to 48 hr) was needed for comparable results with the kidney and liver.     

Preservation and Long-Term Storage of Feulgen-Stained Mouse Tissues for Subsequent Autoradiography

Tissue of the jejunal crypts of mouse intestine was fixed for 24-48 hr in acetic-ethanol, 1:3, and stained en bloc by the Feulgen reaction. The stained preparations were then stored 4 mo at -25 C in either 45% acetic acid alone or in dimethyl sulfoxide (DMSO) or glycerol to which 45% acetic acid had been added to make 15% of the total volume. Such storage preserved not only the stain but allowed autoradiographs to be made. No loss of silver grains or a decrease of labeling index was observed. The procedures are equally successful when used with double-labeling experiments. Solid, transplantable, experimental carcinomas can be preserved in a manner identical to that suggested for the epithelial cells of the crypts.     

Identification of Cells by Backscattered Electron Imaging of Silver Stained Bulk Tissues in Scanning Electron Microscopy

Anuran tadpole tail muscle was stained en bloc by a modified light microscope silver stain for light microscopy and freeze-fractured in liquid nitrogen after partial dehydration with ethanol. The fractured specimens were observed in both secondary electron and backscattered electron modes in a scanning electron microscope. Since the cell nuclei specifically stained with silver provided high contrast against the unstained background due to atomic number contrast of backscattered electron image, various cells were easily identified by a comparison of secondary electron images and compositional images of backscattered electron signals.     

A Hot Aldehyde-Peroxide Fixation Method for Electron Microscopy of the Free-Living Nematode Caenorhabditis Elegans

An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis clegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of micro filaments and micro tubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.     

Electron Dense Artefactual Deposits in Tissue Sections: The Role of Ethanol, Uranyl Acetate and Phosphate Buffer

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

篮状菌属岛篮状菌组的三个中国新记录种

根据形态学和CaM、Rpb2及rDNA ITS1-5.8S-ITS2序列的分子系统学分析,界定篮状菌属岛篮状菌组Talaromycessect.Islandici的3个中国新记录种,即螨生篮状菌T.acaricola、根篮状菌T.radicus和哒叻篮状菌T.tratensis。螨生篮状菌生长局限,形成绒状菌落,在MEA上产生稀疏的灰绿色分生孢子,菌丝淡绿黄色,分生孢子梗双轮生、三轮生和不规则生,帚状枝排列较松散,瓶梗安瓿形,孢子梭形至椭球形,壁光滑。根篮状菌生长较慢,但在37℃可生长,形成致密短絮状菌落,菌丝体绿黄色,孢子稀疏,灰绿色,分生孢子梗双轮生,帚状枝排列紧密,瓶梗圆柱形至披针形,孢子椭球形,壁光滑。哒叻篮状菌生长缓慢,在25℃培养7 d后未产分生孢子,菌丝体呈浅橙黄色,产生适量橙黄色裸囊壳,子囊孢子椭球形,壁光滑至稍粗糙。     

篮状菌属的三个中国新记录种

报道依据形态学和基于BenA、Rpb2和rDNA ITS1-5.8S-ITS2序列的分子系统学分析确定的篮状菌属篮状菌组Talaromyces sect. Talaromyces的3个中国新记录种,即蛇床篮状菌T. cnidii,苹果篮状菌T. malicola和丘陵篮状菌T. tumuli。T. cnidii生长较快,形成典型的绒状菌落,产生大量橄榄绿色分生孢子,在CYA和MEA上菌落背面呈深红色;其帚状枝为双轮生兼不规则生,分生孢子椭球形至卵形,壁光滑,大小不一。T. malicola生长适中,在CYA上形成絮状兼绳状菌落且只产生少量分生孢子,但在MEA上产生大量灰绿色分生孢子;其帚状枝主要为紧密双轮生,偶尔单轮生,分生孢子球形至近球形,壁光滑至稍粗糙。T. tumuli生长适中,形成絮状兼绳状菌落并产生大量灰绿色分生孢子;其帚状枝为双轮生兼不规则生,排列不紧密,瓶梗安瓿形,分生孢子椭球形至柠檬形,壁光滑至稍粗糙。     

Silver Staining, Featuring Rapid Reduction, For Whole Mounts of Retina and Optic Pathways in Chick Embryos

Developing and established nerve fibers in the retina and in superficial tracts of the brain can be stained and viewed en bloc. The method was developed on chick embryos of 2 days of incubation to several months post-hatching but could be used on other material provided that the objects of interest were within 35 μ of the surface. Procedure: (1) Place the entire eye or head in 50% pyridine for at least 16 hr. (2) Wash well for 5-7 hr with hourly changes of distilled water or with running tap water for 4-6 hr followed by several changes of distilled water. (3) Transfer to 95% ethanol for 16-48 hr. (4) Impregnate with 1.5% AgNO₃ for 2 days at 37 C. (5) Submerge in water and, when staining the retina, remove the vitreous body and apply an aqueous solution of 0.25% pyrogallic acid in 1.25% formalin by directing a narrow stream of this reducer against the retina for 2-5 sec. Wash the eye with distilled water 30-60 sec after applying the reducer. When staining the brain, remove the meninges under water, direct the stream of reducer against the brain for 20-30 sec, and rinse the brain immediately after the nerves have stained. (6) Dissect the specimen and make temporary mounts in glycerol; or, dehydrate and clear for resin mounting. The technique stains both mature and growing axons with their growth cones and sometimes their cell bodies. The fiber patterns show best on the surfaces of the retina and brain. The stain works consistently and is suited to the study of both normal and abnormal development.     

Epidemiological studies on horses infected with nematodes of the family Trichonematidae (Witenberg, 1925)

Observations are reported on seasonal changes in the age structure of populations of nematodes of the family Trichonematidae recovered at regular intervals from the lumens of the large intestines of horses slaughtered in S.W. England. The results show that changes in the size of parasitic populations of Trichonema nassatum follow seasonal variations in the rate of infection, more individuals maturing during summer/autumn than during winter/spring because of proportional differences in the numbers of infective larvae ingested from the pasture. In contrast, larvae of T. longibursatum, T. catinatum and T. goldi ingested by grazing horses during summer accumulate in the gut wall rather than promptly returning to the lumen and developing to maturity. Their development is apparently inhibited until the following spring when 4th-stage larvae emerge en masse and quickly reach the adult stage. The relationship between fluctuations in the size of the adult populations of the 4 species studied and the characteristic seasonal variations in horse faecal egg counts is discussed.     

Technical Considerations in Evaluating the Adhesion of Leukocytes to Aortic Endothelium of the Rat

Comments on techniques for characterizing leukocytes adhered to the aortic endothelium of the rat are given. Alpha-naphthyl acetate esterase positive leukocytes were studied by optical microscopy of en face intima-media preparations. Results indicate 1) 1% paraformaldehyde-2% glutaraldehyde is a better fixative than formalin-calcium or 4% paraformaldehyde with or without 1.5 mM CaCl₂; the latter produces distortion of leukocytes, endothelial desquamation and enzymate inhibition, 2) washing the aorta with phosphate-buffered saline for 90 sec prior to fixation-perfusion produces a notable decrease in the number of leukocytes adhered, 3) diazotized pararosaniline is better than fast blue RR salt as coupling agent in the esterase reaction, and 4) counterstaining with 1% methyl green for 1 min, before or after the esterase reaction, is not adequate because of limited contrast and the heavy staining of smooth muscle. Counterstaining with Gill's hematoxylin No. 3 for 90 sec is adequate only when done before the esterase reaction. Inhibition of endothelial esterase activity by hematoxylin decreases background, favors contrast of adhered leukocytes and makes it possible to observe nucleus-cytoplasm relations.     

On the identity and reactivity patterns of the "second oxidant" of the T252A mutant of cytochrome P450cam in the oxidation of 5-methylenenylcamphor

Density functional calculations show that in the absence of Compound I, the primary oxidant species of P450, the precursor species, Compound 0 (FeOOH), can effect double bond activation of 5-methylenylcamphor (1). The mechanism is initiated by homolytic cleavage of the O–O bond and formation of an OH radical bound to the Compound II species by hydrogen bonding interactions. Subsequently, the so-formed OH radical can either activate the double bond of 1 or attack the meso position of the heme en route to heme degradation. The calculations show that double bond activation is preferred over attack on the heme. Past the double bond activation, the intermediate can either lead to epoxidation or to a glycol formation. The glycol formation is predicted to be preferred, although in the P450_cam pocket the competition may be closer. Therefore, in the absence of Compound I, Compound 0 will be capable of epoxidizing double bonds. Previous studies [E. Derat, D. Kumar, H. Hirao, S. Shaik, J. Am. Chem. Soc. 128 (2006) 473–484] showed that in the case of a substrate that can undergo only C–H activation, the bound OH prefers heme degradation over hydrogen abstraction. Since the epoxidation barrier for Compound I is much smaller than that of Compound 0 (12.8 vs. 18.9 kcal/mol), when Compound I is present in the cycle, Compound 0 will be silent. As such, our mechanism explains lucidly why T252A P450_cam can epoxidize olefins like 5-methylenylcamphor but is ineffective in camphor hydroxylation [S. Jin, T.M. Makris, T. A. Bryson, S.G. Sligar, J.H. Dawson, J. Am. Chem. Soc. 125 (2003) 3406–3407]. Our calculations show that the glycol formation is a marker reaction of Compound 0 with 5-methylenylcamphor. If this product can be found in T252A P450_cam or in similar mutants of other P450 isozymes, this will constitute a more definitive proof for the action of Cpd 0 in P450 enzymes.     

基于MTT染色法对葡萄生单轴霉孢子囊存活力的检测

本文利用MTT染色法和点接生物测定法对不同温度干燥处理36h后的葡萄生单轴霉孢子囊存活力和致病力进行了检测,并应用单因素试验和正交试验对其孢子囊进行了MTT染色条件的优化。结果表明：葡萄生单轴霉孢子囊MTT染色的最佳条件为温度36℃、MTT浓度 0.05%、染色48h,孢子囊染色率可达83.0%。20℃恒温干燥处理36h显著提高了孢子囊存活力,葡萄生单轴霉孢子囊的蓝色染色率为79.3%,叶片点接发病率为98.9%,显著高于对照的蓝色染色率52.0%和发病率62.7%。MTT染色得到的孢子囊蓝色染色率与点接生物测定法得出的发病率存在很好的线性关系y=1.276 1x-1.939 1,R²=0.996 1,孢子囊的蓝色染色率与叶片点接发病率呈正相关。本研究表明MTT染色法可以用于葡萄生单轴霉孢子囊存活力的快速和准确检测。     

Staining of plant Materials Cleared in NaOH

Stains are listed which have proved suitable for staining the epidermis, mesophyll, and sclerenchyma and tracheary elements, respectively, of cleared leaf material of Mouriri and Linociera. Too rapid leaching is avoided by overstaining high in the dehydration series, destaining briefly in the same solvent, and moving through to xylene. Twenty to thirty minutes staining time is generally sufficient. Concentrations and solvents can be varied widely. If destained too much, the material can usually be replaced in the dye with no ill effects. A double stain schedule (Bonnett) of five to ten minutes in 1% Bismarck brown Y in 95% alcohol followed by one to two minutes in 1% fast green FCF in 100% alcohol may be advantageous for thin-walled cells in thick material. It may be preferable to treat thinner material with tannic-acid-iron-chloride followed by safranin (Foster). The effects of bleaches and clearing compounds other than NaOH on staining have not been investigated; however, Dr. Bonnett finds that lactic acid used after NaOH improves clearing and also improves the staining of his combination (above). Mordants can doubtless be used to advantage.     

Staining Streptomyces Scabies in Lesions of Common Scab of Potato

Toluidine blue can be used to stain Streptotnyces scabies distinctively in slide cultures or in the lesions of common potato scab. This staining method is based on the metachromaticism of volutin, a constant constituent of the spores and mycelium of S. scabies. Either sections or smears are fixed in FPA (formalin, 5 parts; propionic acid, 7.5; 50% alcohol, 87.5), stained in a 1:100 dilution of saturated aqueous toluidine blue from 20 minutes to 24 hours, dehydrated in an acetone-xylene series and mounted. Cellular constituents of the potato tuber stain blue or are colorless whereas the mycelium of Streptomyces appears as a series of red volutin spheres in the blue stained cytoplasm. The criteria of volutin and cytoplasmic staining along with the 1 µ diameter of the mycelium make it possible to distinguish Streptomyces from the other microorganisms and cells in the lesion region.     

The Value of the Smear Method for Plant Cytology

The smear method of plant cytology as described by Taylor, presented difficulties in obtaining an adequate stain with the iron-alum haematoxylin combination. The writer has largely avoided those difficulties by curtailing the times of mordanting and of staining. The smear method has been applied by the writer in the study of microsporogenesis in the monocotyledonous genera Tradescantia and Rhoeo and in the dicotyledonous genus Podophyllum. Due to the rapidity with which permanent preparations can be completed by the smear method, there is presented a valuable means for securing a more critical evaluation of the available preservatives.     

Triton X-100 in Giemsa Staining of Blood Parasites

The effects of a surface active agent, Triton X-100, were studied in the routine Giemsa staining of seven blood parasites: Plasmodium vivax, Trypanosoma cruzi, T. lewisi, Leishmania donovani, Toxoplasma gondii, and microfilariae of Dirofilaria immitis and of Wuchereria bancrofti. Concentrations of Triton X-100 ranging from 0.01% to 0.5% were used in staining (a) both thick and thin blood films of all organisms except L. donovani, (b) tissue smears of L. donovani, and (c) tissue and peritoneal fluid smears of T. gondii. In general, the addition of the detergent to the Giemsa solution resulted in cleaner preparations and better stained organisms. Morphological details were more distinct, thus facilitating microscopical detection and identification of species. The most beneficial concentration of Triton X-100 was found to be 0.1%. Since it has a hemolytic effect on erythrocytes, concentrations above 0.01% cannot be used in staining thin blood films. It is suggested also that the use of Triton-Giemsa may help prevent transfer of some of these organisms from one slide to another during mass staining procedures as it has been demonstrated to do with malaria parasites (Brooke and Donaldson, 1950).