A Rapid Method for Demonstrating Skeletal Muscle Motor Innervation in Frozen Sections

Longitudinal 50-100 μm-thick frozen sections of muscle are picked up on slides coated with 3% EDTA and after drying are incubated to demonstrate acetylcholinesterase. Subsequent incubation in 0.5% K₃Fe(CN)₆ is followed by fixation for 30 minutes in formol-calcium or formol-saline. After washing, the slides are incubated in 20% aqueous AgNO₃ containing 0.1% CuSO₄ for 2-30 minutes at 37 C. Following development in a 1% solution of quinol (w/v) 5% with respect to Na₂SO₃ (w/v), axons and subneural apparatus stain dark brown to black in contrast to the less well stained muscle fibers and nuclei. This procedure permits study of the pattern of neuromuscular innervation in skeletal musk 31/2-4 hours after receipt of a sample, and makes possible determination of the terminal innervation ratio.     

Localization of Myoglobin in Striated Muscle of the Domestic Pig; Benzidine and NADH2-TR Reactions

Tissue blocks 1 cm³ from longissimus (white) and trapezius (red) muscles of adult pigs were fixed in phosphate-buffered 2.5% glutaraldehyde, pH 7.4, for 4 hr at about 25 C; washed 4 hr in running tap water, and immersed in 30% w/v sucrose solution for 16 hr or more. After freezing in liquid N₂, cryostat sections were cut and floated into saturated aqueous benzidine containing 0.15% H₂O₂ at 25 C for 30 min. Stained sections were washed in distilled water and mounted on slides with glycerol jelly. Three distinguishable gradiations of color intensity were found: strong, intermediate, and negative. The trapezius had a greater number of myoglobin-positive fibers than the longissimus muscle. Myoglobin-positive and myoglobin-negative staining occurred in red and white fibers, respectively; intermediates were apparently more closely related to the red than to the white fibers. The NADH₂TR reaction showed the same sites as did the benzidine reaction.     

Improvement of silver impregnation technique (protargol) to obtain morphological features of protists ciliates, flagellates and opalinates

The research on ciliates, flagelates and opalinates have been widespread by the utilization of techniques employing silver impregnation (protargol), modified by several authors. However, these are time consuming and its results are variable. The present work is a variant of the technique described by Tuffrau (1964, 1967) showing some adaptations made in our laboratory. The organisms can be preserved by different fixatives (alcoholic Bouin, Stieve's fluid, 2.5% glutaraldehyde and others) and then rinsed in destilled water followed by a fast clarification by 3% sodium hypochloride. If the organism is very sensitive to hypochloride, 4% sodium lauryl sulfate may be used and then washed 3 times in distilled water. The protista can be adhered to the glass slides with Mayer's glycerinated-albumin (1 glycerin vol. to 1 or 2 albumin vol.), diluted in water at a proportion of 1:10 Cv/v., or with 1% polylysine followed by fast washes with distilled water. After the slide preparation, they were covered with a layer of 0,8% Silver proteinate. Right after that, the slide has to be placed in a glass tray lined with moist tissue and covered to prevent the proteinate to dry. The tray was placed in a incubator at 40 degrees - 50 degrees C for 30 minutes. The slides are rinsed for 1 minute. with warm (35 degrees C) distilled water. The development of the material should be done with 0.4% hydroquinone with a maximum incubation time of 1 minute. It should be developed gradually, controlling the silver impregnation intensity by observation under optical microscope. Next, rinse in distilled water for 1 minute, and then, fix in 2,5% Sodium thiosulfate. Rinse the slide for two minutes before dehydrating it in an alcoholic serial 50-100 degrees. Finally rinse the slides in xylene. Mount the slides with Entellan MerckTM or Canada balsam.     

Immunofluorescence Microscopy of Cyanurated Tissues

Test tissues consisted of: (1) popliteal lymph nodes of rabbits, removed 6 hr after injection of hind footpads with 0.2 ml of 125 mg/ml solution of 5× crystallized chicken ovalbumin, and (2) lungs from guinea pigs, passively sensitized with rabbit antiovalbumin serum, then anaphylactically shocked by intracardial injection of a 1% chicken ovalbumin solution. Similar control tissues from normal rabbits, and lungs of passively sensitized guinea pigs, but shocked with histamine instead of ovalbumin, were included. Pieces of fresh tissue not exceeding 2 mm³ were fixed as follows: (1) Cyanuration—lymph nodes, 1 hr; lung, 0.5 hr; both at 23-27 C—in anhydrous methanol containing 0.5% w/v cyanuric chloride and 1% v/v N, N-diethylaminoethanol. (2) Control fixatives—all specimens 18-24 hr at 4—6 C—absolute methanol; 95% ethanol; neutral buffered 10% formalin; and an FAA mixture (formalin, conc., 6; glacial acetic acid, 2; 30% ethanol, 92). Freeze-dried material was either left unfixed (a control) or fixed in xylene or toluene containing 0.5% w/'v cyanuric chloride and 1% v/v N, N-diisopropylaminoethanol; time and temperature as for fresh tissues. All tissues were routinely dehydrated, cleared, and vacuum embedded in an ester wax, diethylene glycol distearate, or in paraffin at 52 C. Sections 2-4 μ thick were attached to gelatin-coated slides, the wax removed in petroleum ether, and stained 20 min at 23-27 C in a 0.10% solution of fluorescein isothiocyanate-conjugated rabbit antiovalbumin globulin, washed in phosphate buffered saline 10 min, dehydrated, cleared and covered in a nonfluorescent medium. With ultraviolet illumination, brightly immunofluorescent, anti-genically specific staining was obtained in cyanurated fresh and freeze-dried lymph node and lung tissues. In contrast, specific staining was diminished or absent in comparable tissues reacted in the control fixatives.     

Techniques for Studying Adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are frozen rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl₂ (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl₂ (2.5%). When procedures described above for immature adipose depots are combined with esterase fining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

石牌广藿香原生质体的分离及培养研究

以石牌广藿香悬浮细胞为材料,对影响其原生质体分离和培养的酶浓度、作用时间、溶液渗透压和材料的生理状态等因素进行了研究。结果表明:以0.5%的果胶酶、0.2%离析酶和0.8%的纤维素酶组合处理继代培养3～11d的悬浮细胞8h,渗透压调节剂为9%甘露醇,原生质体产量达1.65×106 protoplasts·mL-1 PCV,活力超过86%。在原生质体的液体浅层培养中,细胞分裂频率为13.5%。     

A Simple Procedure for in Situ Immunolabeling, Embedding and Sectioning of Layers of Cultured Endothelial and Smooth Muscle Cells for both Light and Electron Microscopy

We present a simple procedure for in situ immunolabeling, embedding and sectioning of layers of cultured endothelial and smooth muscle cells for both light and electron microscopy. Endothelial and smooth muscle cells were seeded in tissue culture chambers /slides precoated with 30% (w/v) gelatin drops fixed with 0.5% glutaraldehyde. Live endothelial cell layers were labeled with an antibody against the surface membrane protein, anti-CD 13. After labeling, the cell layers were fixed and separated from the chambers/slides by lifting all of the samples with a spatula. Sections (1-2 mm) were cut, embedded and processed further for light or electron microscopy. Because of the delicate cell layers and the importance of preserving maximum integrity, labeling was performed under standard culture conditions and treated in situ during the entire procedure. Moreover, the small chamber size of the tissue culture dishes generated the additional advantages of requiring only a limited number of cells, small volumes of media, and little antibody.     

Method for Staining Bacterial Flagella

The following method of staining bacterial flagella is ecommended for use on smears made from suspensions of 10 to 16-tour agar slant cultures, incubated 30 minutes at 37°C before spreadng on thoroly cleaned and named slides:

1. Cover with fixative (100 cc. of 1/4 sat. aqu. solution picric acid, with 5 g. tannic acid and 7.5 g. ferrous sulfate).
2. Wash with tap water, dry and cover with Fontana spirochaete stain; heat to steaming and allow to act for 1 to 2 minutes. Wash in ap water. The stain is prepared as follows: To 25 cc. 2% AgNO₃ add dilute ammonia till the precipitate which forms redissolves; then add more AgNO₃ till a faint turbidity results. A clear solution is useess.

     

Techniques for studying adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl2 (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl2 (2.5%). When procedures described above for immature adipose depots are combined with esterase staining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

Determination of carbon monoxide (CO) in rodent tissue: effect of heme administration and environmental CO exposure

Carbon monoxide (CO), produced endogenously during heme degradation, is considered a messenger molecule in vascular and neurologic tissues. To study this role, it is important to determine CO concentration in target tissues pre- and post-perturbations. Here, we describe a sensitive and reproducible method, which is linear and accurate, and provide some examples of its application for quantitation of CO concentrations in tissues pre- and post-perturbations. Tissues from adult rats and mice were sonicated (20% w/w), and volumes representing 0.04-8 mg fresh weight (FW) were incubated at 0 degrees C for 30 min with sulfosalicylic acid. CO liberated into the headspace was quantitated by gas chromatography. Tissue CO concentrations (mean+/-SD, pmol CO/mg FW) were as follows: blood (47+/-10, 45+/-5), muscle (4+/-4, 10+/-1), kidney (5+/-2, 7+/-2), heart (6+/-3, 6+/-1), spleen (11+/-3, 6+/-1), liver (4+/-1, 5+/-1), intestine (2+/-1, 4+/-2), lung (2+/-1, 3+/-1), testes (1+/-1, 2+/-1), and brain (2+/-1, 2+/-0) in untreated rat (n=3) and mouse (n=5), respectively. Between the rat and the mouse, only CO concentrations in the muscle and spleen were significantly different (p0.05). Endogenous CO generation, after administration of heme arginate to mice (n=3), increased CO concentrations by 0-43 pmol/mg FW. Exposure of mice (n=3) to 500 ppm CO for 30 min yielded significantly elevated CO concentrations by 4-2603 pmol/mg FW in all tissues over the native state. While blood had the highest CO concentration for all conditions, muscle, kidney, heart, spleen, and liver, all rich in hemoglobin and/or other CO-binding hemoproteins, also contained substantial CO concentrations. Intestine, lung, testes, and brain contained the lowest CO concentrations.     

Controlled Formaldehyde-Catecholamine Condensation in Cryostat Sections to Show Adrenergic Nerves by Fluorescence

Three sets of sections of freshly removed tissue are cut at 18 μ in a cryostat and dried on slides for 1.5 hr over P₂O₅. Each set of sections is incubated with a differently hydrated paraformaldehyde (prepared by storing paraformaldehyde powder over 21%, 25% or 28% aqueous H₂SO₄ for 1 wk) at 80 C for 1 hr before being mounted in glycerol and viewed with a fluorescence microscope. At least one set of specimens shows optimal fluorescence. The entire procedure from removing the tissue to observing fluorescence microscopically is accomplished readily within 4-8 hr. Adrenergic axons in the medial muscle of the cat nictitating membrane, the myometrium of the cat uterus and the adventitia of arterial vessels in rat pancreas are demonstrated.     

Augmentation of Soil with Sporangia of Actinoplanes spp. for Biological Control of Pythium Damping-off

A method was developed for applying strains of Actinoplanes spp. that are hyper-parasites of oospores of Pythium ultimum to soil for reducing Pythium damping-off of plants. The method is based on the augmentation of soil with sporangia of a strain of Actinoplanes spp. borne on clay granules. In vitro sporulation of strains K30, W57, W257 and 25844 was: (1) greater for most strains on dilute Czapek-Dox agar than on four other agar media; (2) inhibited by continuous exposure to fluorescent light of intensity 4-150 μEm^-2s^-1, but not by exposure to 1 μEm^-2s^-1 or darkness; (3) greater at 20-307deg;C than at 10°C;and (4) greater at pH 6-7 than at pH 5 or 8. On solid carriers treated with dilute Czapek-Dox broth (pH 7) and incubated in the dark at 30°C for 3 weeks, strains sporulated poorly or not at all on vermiculite, perlite and rice hulls, but sporulated abundantly (10⁷-10⁹ colony-forming units (CFU) g^-1 of granules) on montmorillonite clay granules. When strains 25844, W57 and W257 were applied as granules (4 10⁷ - 4 × 10⁸ CFU g^-1) at 5% (w/w) to field plots infested with 750-1000 oospores of P. ultimum g^-1 of soil, only strain 25844 consistently increased emergence and reduced root rot of table beets 8- 1 at 24-28 days after planting compared with controls. Strain 25844 (10⁸ CFU g^-1 of granules) at 1% (w/w) also increased the emergence of bush beans at 28 days after planting in P. ultimum-infested plots, but lower rates were ineffective. The inoculum viability of strain 25844 on clay granules declined 100-fold during 2 months of storage at 5-35°C, but thereafter remained stable for another 4 months. Strain 25844 on 6-month-old granules retained a high degree of hyper-parasitic activity toward oospores of P. ultimum. Augmentation of field soil with sporangia of Actinoplanes spp. is a valid approach to the biological control of pythium damping-off.     

A simplified method for micronucleus preparation from hepatic cells

A simplified method for micronucleus preparation from regenerating hepatocytes has been developed. Small pieces of the regenerating portion of the liver are incubated in 1% sodium citrate solution containing collagenase Type 1A (final concentration 0.005% w/v) at 37 C for 10-15 min with occasional gentle agitation. The larger particles are discarded. Drops of the thick homogeneous citrate suspension of liver cells are put on the slides and drawn back immediately into the pipette, leaving only the drop marks. This simplified method, which gives good preparations with many intact hepatocytes, was validated in a model experiment using mitomycin C. The data revealed a distinct dose-response effect.     

A Simplified Method for Micronucleus Preparation from Hepatic Cells

A simplified method for micromicletu preparation from regenerating hepato-cytes has been developed. Small pieces of the regenerating portion of the liver are incubated in 1% sodium citrate solution containing collagenase Type 1A (final concentration 0.005% w/v) at 37 C for 10-15 min with occasional gentle agitation. The larger particles are discarded. Drops of the thick homogeneous citrate suspension of liver cells are put on the slides and drawn back immediately into the pipette, leaving only the drop marks. This simplified method, which gives good preparations with many intact hepatocytes, was validated in a model experiment using mitomycin C The data revealed a distinct dose-response effect.     

Hematoxylin Staining of Tissues Embedded in Epoxy Resins

Sections of 0.5-2 μ thickness are affixed to slides with albumen adhesive, thoroughly dried, and placed in xylene or toluene for 1 hr, then brought through ethanol to water. Sections of tissue fixed in O_sO₄ are treated first in 0.1% KMnO₄, then with 1.0% oxalic acid, and after rinsing, incubated at 60 C for 12-24 hr in hematoxylin (Harris's or Ehrlich's) and counterstained 10-15 min with 0.5% phloxine B. Permanent preparations are made by clearing and mounting in a synthetic resin. The method requires only easily available reagents and is suitable for routine processing of epoxy sections.     

Silver Staining of Nerve Fibers in Cardiac Tissue

Fresh hearts of dog were perfused through the coronary vessels with 1000 ml. of fixative (chloral hydrate, 5 g. per 100 ml. of 70% ethyl alcohol) and blocks of tissue 2 × 5 mm. from epicardium to endocardium fixed 48 hours in the same fixative. The blocks were placed in 95% alcohol containing 0.3% addition of strong ammonia for 4 hours, followed by 2 changes of plain 95% alcohol of 1 hour each, then cleared and infiltrated with paraffin. Mounted sections 12-15 µ thick were incubated in 1% silver proteinate (obtained from Serumvertrieb, Marburg, Germany)² at 38° C. for 48 hours in the presence of 10 g. of 15 gauge copper wire per 200 ml. of solution. The slides were rinsed gently in 3 changes of distilled water for 2 minutes, 1 minute and 1 minute, respectively, and reduced in 1% hydroquinone and 5% sodium sulfite for 5 minutes. They were washed 5 minutes in tap water and 5 minutes in 2 changes of distilled water and toned 3-5 minutes in 0.25% gold chloride, rinsed in distilled water 10 seconds, reduced 10 seconds in 1 % oxalic acid, rinsed 1 minute, fixed in 5% sodium thiosulfate 5 minutes, washed in tap water through 3 changes, dehydrated, cleared and covered. All solutions were made with distilled water except where otherwise specified. The results gave good impregnation of fine nerve fibers without the usual confusing staining of reticular tissue.     

Application of Methods for Mammalian Chromosome Spreads to the Cells of Cestodes

Tapeworm cells obtained by physical maceration between ground-glass surfaces are incubated for 3 hr in Hanks' balanced salt solution (BSS) supplemented with colchicine to a concentration of 10-⁴ M. After washing in BSS, the cells are incubated for 10 min in 1/4 strength BSS then centrifuged 10 min. Fixation of the intact button of cells (or alternatively, by squirting the cells directly into the fixative) in Carnoy's alcohol-chloroform-acetic acid (6;3:1) for 30 min follows, and cells, dispersed and washed in the fixative, are flattened by dropping the suspension on clean, water-wet slides which are then air-dried and stained with Giemsa diluted 1 ml;47 ml with distilled water to which 2 ml of buffer—M/15 KH₂PO₄, 32 ml, mixed with M/15 Na₂HPO₄, 68 ml—is added. After staining 15 min and washing in distilled water, slides are air-dried and mounted with resin. Well separated and well stained chromosomes have resulted.     

Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027

A low-cost medium, MGRS, has been developed for growth and lipase production from Bacillus THL027 at 65 degrees C and pH 7.0. MGRS was composed of 2% (v/v) buffer solution (7.3% (w/v) Na(2)HPO(4), 3.2% (w/v) KH(2)PO(4), pH 7.2), 40 microg ml(-1) FeSO(4) and 40 microg ml(-1) MgSO(4), 0.1% (w/v) (NH(4))(2)SO(4) supplemented with 3% NaCl, 0.1% glucose, 1.0% rice bran oil and 0.5% (w/v) rice bran. The lipase was purified 2.6-fold to apparent homogeneity by ultrafiltration and gel filtration chromatography. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties.     

Solubilization of insoluble inorganic phosphates by a novel salt- and pH-tolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere

To develop environment-friendly biofertilizer solubilizing insoluble phosphates, salt- and pH-tolerant, insoluble inorganic phosphate-solubilizing bacterium was isolated from soybean rhizosphere. On the basis of its physiological characteristics and Vitek analysis, this bacterium was identified as Pantoea agglomerans. The optimal medium composition and cultural conditions for the solubilization of insoluble phosphate by P. agglomerans R-42 were 3% (w/v) of glucose, 0.1% (w/v) of NH4NO3, 0.02% (w/v) of MgSO4 x 7H2O, and 0.06% (w/v) of CaCl2 x 2H2O along with initial pH 7.5 at 30 degree C. The soluble phosphate production under optimal condition was around 900 mg/l, which was approximately 4.6-fold higher than the yield in the MPVK medium. The solubilization of insoluble phosphate was associated with a drop in the pH of the culture medium. P. agglomerans R-42 showed resistance against different environmental stresses like 5-45 degrees C temperature, 1-5% salt concentration and 3-11 pH range. Insoluble phosphate solubilization was highest from CaHPO4 (1367 mg/l), hydroxyapatite (1357 mg/l) and Ca3(PO4)2 (1312 mg/l). However, the strain produced soluble phosphate to the culture broth with the concentrations of 28 mg/l against FePO4, and 19 mg/l against AlPO4, respectively.     

Halothane anaesthesia of normal and dystrophic hamsters.

Induction, carried out in a small clear-plastic box with 3-5% (v/v) halothane in 30:70 (v/v) oxygen: nitrous oxide, was quiet and rapid. Recovery was almost instantaneous. 2% halothane in the oxygen-nitrous oxide mixture was sufficient for maintenance anaesthesia. The anaesthetic mixture was given by face mask in an open circuit specially designed to function at low gas-flow rates. The halothane content of the muscle and blood after 25 min anaesthesia was estimated by gas chromatography of n-heptane extracts. The mean level (+/- s.e.m.) in blood was 22-8 +/- 2-7 mg/100 ml (n=4), and in dystrophic muscle 226 +/- 36-8 mg/100 g wet weight of tissue (n=4): there was a positive correlation (r=0-94) between them (p less than 0-02).