Preservation of fine Structures in Yeast by Fixation in a Dimethyl Sulfoxide-Acrolein-Glutaraldehyde Solution

The primary fixative containing 2% acrolein, 2% glutaraldehyde in 50% aqueous dimethyl sulfoxide (DMSO) buffered at pH 7.4, was applied for 7 hr in the cold. After a short wash in 0.02 M s-collidine buffer, pH 7.4, containing 0.2 M sucrose and 0.001 M CaCl₂, the yeast cells were postfixed in 3% OsO₄ at pH 4.0 (veronal acetate buffer). This method preserves many cytoplasmic features such as lipid deposits and ribosomes which are usually destroyed by permanganate fixation. DMSO apparently acts as a permeating agent allowing maximum penetration of the cell wall by the fixative without disrupting cellular fine structure     

Brazilin-Toluidine Blue O and Hematoxylin-Darrow Red Methods for Brain and Spinal Cord

Tissue fixed in 10% formalin, formalin-95% ethanol 1:s CaCO₂ or phosphate buffer neutralized formalin, or methanol-chloroform 2:1, was dehydrated and embedded in paraffin or double-embedded by infiltration in 1% celloidin followed by a chloroform-paraffin sequence. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 24-26 C. For either method, mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. For brazilin-toluidne blue O, myelin was stained for 20-60 min, depending upon section thickness, in a self-differentiating solution consisting of: 0.15% Li₂CO₃ 75 ml; 6% brazilin in 95% ethanol, 25 ml; and NaIO₃ 75 mg. After a thorough washing, Nissl material was stained for 3-8 min in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; and 1% toluidine blue 0, 2.5 ml. For hematoxylin-Darrow red, myelin was stained for 2-6 hr in a self-differentiating solution consisting of: 0.15% Li₂,CO₃ 95 ml; 10% hematoxylin in 95% ethanol, 5 ml; and NaIO₃ 25 mg. After a thorough washing, Nissl material was stained for 20 min or less in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; Darrow red, 25 mg. This mixture was first boiled, cooled to room temperature and filtered. In both methods, washing, dehydration, clearing, and mounting completed the process. In the brazilin-toluidine blue technic, myelin sheaths were stained reddish purple; neuronal nuclei light blue with dark granules of chromatin; nucleoli dark blue; and cytoplasm blue with dark blue Nissl granules. In the hematoxylin-Darrow red procedure, myelin sheaths were blue-black; nuclei light red with dark granules of chromatin; nucleoli almost black; and cytoplasm red with bright red Nissl granules.     

Effects of dimethylsulfoxide on the ultrastructure of fixed cells

It has been reported that the use of dimethylsulfoxide (DMSO) as a solvent for fixatives enhances preservation of cellular ultrastructure. By contrast, we have shown that DMSO alters the ultrastructural integrity of glutaraldehyde fixed cells. The cell membrane, nuclear envelope, endoplasmic reticulum, ribosomes, microtubules and intracytoplasmic organelles are most susceptible to the action of DMSO. We hypothesize that DMSO exerts intracellular alterations via its interaction with remnant interfacial water in fixed cells. DMSO-induced alterations of these and related cellular components may result in the formation of artefactual structures and networks. Thus, it appears that DMSO containing glutaraldehyde neither accelerates fixation nor enhances stabilization of cellular ultrastructure. For these reasons, addition of DMSO to fixatives is not recommended.     

Effects of dimethylsulfoxide on the ultrastructure of fixed cells

It has been reported that the use of dimethylsulfoxide (DMSO) as a solvent for fixatives enhances preservation of cellular ultrastructure. By contrast, we have shown that DMSO alters the ultrastructural integrity of glutaraldehyde fixed cells. The cell membrane, nuclear envelope, endoplasmic reticulum, ribosomes, microtubules and intracytoplasmic organelles are most susceptible to the action of DMSO. We hypothesize that DMSO exerts intracellular alterations via its interaction with remnant interfacial water in fixed cells. DMSO-induced alterations of these and related cellular components may result in the formation of artefactual structures and networks. Thus, it appears that DMSO containing glutaraldehyde neither accelerates fixation nor enhances stabilization of cellular ultrastructure. For these reasons, addition of DMSO to fixatives is not recommended.     

A Simple Histochemical Reaction for Aldehydes

A study has been made on the possibility of replacing leucofuchsin by colored basic fuchsin for the histochemical demonstration of aldehydes. Several tissues from mammals and various pertinent fixatives were used. Aldehydes were freed from carbohydrates by oxidation and from thymonucleic acid by hydrolysis.

It was found that the colored form and not necessarily the leucoform of basic fuchsin can be used histochemically in demonstrating aldehydes. The technic used is as follows: (1) Treat with 1.0-0.5% H₅IO₆ (or in 1% KIO₄ in M/1 H₂SO₄) for 5 to 10 min. and wash thoroughly. For thymonucleic acid hydrolize with N HCl 5 min. at room temperature, 10 min. at 60°C. and 5 min. at room temperature. (2) Stain for 2-3 min. with 0.05% basic fuchsin in 5% ethanol, 3% phenol. (3). Transfer immediately to 1 or 2 changes of 1% sodium bisulphite or potassium metabisulphite in 0.1-0.2 N H₂SO₄ for a total of 5 min. (4) Rinse with water and treat with M H₂SO₄ in 95% ethanol for 3-5 min. 6. Wash thoroughly in water and dehydrate, clear, and mount. For glycogen and mucin the following counterstaining solution is recommended: orange G, 0.25 g.; light green SFY, 0.10 g.; phosphotungstic acid 0.50 g.; 50% ethanol, 100 ml.; glacial acetic acid, 0.25 ml.     

Straight Oso4 versus Glutaraldehyde-Oso4 In Sequence as Fixatives for the Granular Vesicles in Sympathetic Axons of the Rat Pineal Body

Pineal bodies were removed immediately after death from 6 rats: representing both sexes, and adult and 21-day postnatal ages; cut into 2 or 3 pieces, and subjected to experimental fixations at pH 7.3, 0-4 C as follows: 1-2 hr in 1% OsO₄, with veronal-acetate buffer of phosphate buffer; 3-4 hr in 3% or 6% glutaraldehyde in 0.1 M or 0.2 M phosphate buffer, with or without 1% sucrose. Specimens from OsO₄ were dehydrated, and embedded in epoxy resin; those from glutaraldehyde were allowed to soak in buffer for 12-16 hr, then transferred to 1% OsO₄ at 0-4 C for 2 hr, and embedded in the same manner as the ones fixed directly in OsO₄. Representative electron micrographs of postganglionic sympathetic endings were studied for the morphology and frequency of granular vesicles. No consistent difference was shown between vesicles fixed in OsO₄ buffered by phosphate or by veronal-acetate, nor was there any effect caused by the different concentrations used for the glutaraldehyde solution; however, vesicles fixed by the glutaraldehyde-OsO₄ sequence showed an enhancement in the graininess of their membranes, were slightly larger, and had a much larger dense core than those fixed by OsO₄ alone. After glutaraldehyde-OsO₄, granular vesicles showed a frequency of 81%, whereas after direct fixation in OsO₄, only 40% without significant change their number per unit area. Therefore, glutaraldehyde-OsO₄ seems to be more effective than straight OsO₄ for the demonstration of granular vesicles in the autonomic nervous system.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

A Silver Impregnation Method for Nervous Tissue Suitable for Routine use with Mounted Sections

A simple, reliable silver impregnation method for nervous tissue is described for tissues fixed in various fixatives including formalin, Bouin, and Sum. Sections are impregnated in a solution containing 1 g Protargol, 2 ml of a 1% Cu(NO₃)₂ solution, 2 ml of a 1% AgNO₃ solution, and 2-4 drops 30% H₂O₂ in 100 ml distilled water. Sections are impregnated 4-5 days at 37 C and thereafter reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction, dehydration and mounting. This method has been found to be very reliable and selective.     

On the lability of dimethylsulfoxide (DMSO) coordinated to the [Ru(II)-NO(+)] species: X-ray structures of mer-[RuCl(3)(DMSO)(2)(NO)] and mer-[RuCl(3)(CD(3)CN)(DMSO)(NO)

The syntheses of nitrosyl–dimethylsulfoxide–ruthenium(II) complexes with general formula mer-[RuCl₃(L)(DMSO)(NO)] (L=DMSO or CD₃CN) is reported. The mer-[RuCl₃(DMSO)₂(NO)] (1) complex was obtained from the reaction of [RuCl₃(NO)] with the sulfoxide ligand in acetone. The mer-[RuCl₃(CD₃CN)(DMSO)(NO)] (2) compound was obtained from mer-[RuCl₃(DMSO)₂(NO)] maintained in deuterated acetonitrile. These data suggest a slow kinetic reaction due the low lability of the DMSO ligand coordinated to the {Ru^II–NO⁺} species. The crystal and molecular structures of (1) and (2) have been determined from X-ray studies. Crystal data: for (1), monoclinic, P2₁/c, a=8.8340(2) Å, b=12.0230(3) Å, c=13.7064(4) Å, β=114.546(2)°, Z=4, R1=0.0429; for (2), monoclinic, P21/n, a=10.0180(7) Å, b=9.5070(7) Å, c=13.3340(9) Å, β=102.264(4)°, Z=4, R1=0.0472. The spectroscopic characterization of (1), in solid state (infrared spectrum) and in solution (nuclear magnetic resonance and cyclic voltammetry) is also described.     

Geometrical configuration of monomethyl–platinum(II) complexes driven by the size of entering nitrogen ligands

The reaction of the monoalkyl complex trans-[Pt(DMSO)₂Cl(CH₃)] with a large variety of heterocyclic nitrogen bases L, in chloroform solution, leads to the formation of uncharged complexes of the type [Pt(DMSO)(L)Cl(CH₃)], containing four different groups coordinated to the metal center. Only two out of the three different possible isomers were detected in solution. These two trans(C,N) and cis(C,N) species can be unambiguously identified through ¹H NMR spectroscopy. For the trans(C,N) isomers, average values of ²J_PtH=75±4 Hz and ³J_PtH=36±4 Hz have been observed for the coordinated methyl and DMSO ligands, respectively. In the case of the cis(C,N) isomers, these values increase to ²J_PtH=83±2 Hz, and decrease to ³J_PtH=26±3 Hz due to the mutual exchange of ligands in trans position to CH₃ and DMSO. In the case of bulky asymmetric ligands, such as quinoline, 2-quinolinecarboxaldehyde, 2-methylquinoline, 5-aminoquinoline, 2-phenylpyridine and 2-chloropyridine, slow rotation of the hindered group around the Pt---N bond makes the coordinated DMSO ligand prochiral. NMR experiments have shown that the first reaction product is the trans(C,N) isomer as a consequence of the very fast removal of one DMSO ligand by the nitrogen bases from the starting complex trans-[Pt(DMSO)₂Cl(CH₃)]. This trans kinetic product undergoes a geometrical conversion into the more stable cis(C,N) isomer through the intermediacy of fast exchanging aqua-species. The rate of isomerization and the relative stability of the two isomers depends essentially on the rate of aquation and on the steric congestion imposed by the new L ligand on the metal.     

A Rhodocyan Technic for Staining the Anterior Pituitary

A reproducible, one-step, differential staining technic which uses routine formalin-fixed tissue and gives brilliantly contrasting results is produced by incubating sections for 1 hr in a 60° C oven in the following dye mixture: 1% eosin B (CI#771), 8 ml; 1% anilin blue (CI#707), 2 ml; and buffer solution (0.1M citric acid, 1.1 ml; 0.2M Na₂HPO₄, 0.9 ml; distilled water, 28.0 ml) at pH 4.5. No differentiation is necessary. The method can be modified for duodenal enterochromaffin cells and alpha cells of pancreatic islets by adjusting the buffer to pH 3.6 and staining for only 3 min at 60° C.     

响应面法优化荷叶离褶伞菌丝体产漆酶条件

为了对荷叶离褶伞产漆酶条件进行优化,在单因素实验基础上,通过最陡爬坡实验(PB)对培养基8因素进行筛选,获得影响产漆酶的3个显著性因素:葡萄糖,pH和KH₂PO₄;通过中心组合(CCD)设计及响应面分析确定了最优发酵条件:葡萄糖20.09g/L,酪蛋白1.5g/L,酵母提取物1.5g/L,MgSO₄ 3g/L,CuSO₄ 3.75mg/L,KH₂PO₄ 3.97g/L,pH 4.98,VB₁ 0.1g/L,愈创木酚12mg/L,该条件下,漆酶酶活为829.83U/mL,较未优化对照提高46.6%.     

Possibility of the long-term fixation of the cells of a HeLa culture without disturbing their ultrastructure

It is shown that the composition of fixatives (2.5% glutaraldehyde in 0.1 M phosphate buffer with pH 7.2-7.4, and the mixture of 2.5% glutaraldehyde with 2% paraformaldehyde in 0.1 M phosphate buffer with pH 7.2-74) and duration of fixation (30 minutes, 24 hours, 7 days, and 30 days) under the room temperature exerts no influence on preservation of HeLa cultured cells. The ultrastructure of all the organelles of these cells is similar in any cases examined. All the membrane structures are well preserved; no condensation of chromatin is observed; the widths of the canals of endoplasmic reticulum, and of the intracristal and lateral spaces of mitochondria are invariable. Polysomes are present in the cytoplasm throughout the period of fixation.     

Formalin-Chloride Fixation to Improve Silver Impregnation of the Golgi Apparatus

The addition of a 1% concentration of alkaline earth or heavy metal chloride to a 15% neutral formalin solution was found to be much superior to a similar addition of nitrate. Of the 19 salts tried, BaCl₂ and CoCl₂ generally gave the best results with the tissues of Pheretima posthuma, Pila globosa, Rana tigrina, Cauia porcellus and Mus rattus. The CoCl₂ fixative was found to work more successfully with shorter time for fixation and subsequent impregnation by silver, than da Fano's cobalt nitrate technique. The results obtained with BaCl₂ fixative, were equally good and in some cases even excelled those obtained with Aoyama's CdCl₂. Fixatives containing MgCl₂ and ZnCl₂ were satisfactory but the results were not so good as were those obtained with barium and cobalt chloride fixatives. Fixatives containing nitrates generally required longer periods for impregnation and reduction, than those containing chlorides.     

The Fixing and Staining of Liriodendron Tulepifera Root Tips and their Mycorrehizal Fungus

Root tips of Liriodendron Tulipifera forming with a fungus of the Mycelium Radicis group an endotrophic mycorrhiza, were subjected to several different fixations in which the action of cationic chromium and anionic chromium were compared. Anionic chromium in the form of chromic acid was combined with several substituted benzene compounds while cationic chromium in the form of chromic sulfate (Cr₂(SO₄)₃·15 H₂O) in 4% formaldehyde (HCHO) was used with the same ring compounds. In addition, several fixatives not containing chromium were tested.

Five percent chromic sulfate (Cr₂(SO₄)₂·15 H₂O) in 4% formaldehyde (HCHO) or in 1% osmic acid with saturated aqueous salicylic and/or picric acid preserved the histological and cytological details of the mycorrhiza, as was clearly demonstrated when followed by staining in 3% acetic acid saturated with orseillin BB and counterstained with 1% crystal violet in clove oil.

Two percent ferric chloride (Fe Cl₃) in 4% formaldehyde showed the relationship of the tannin contents of the cells to the invading hyphae when followed by suitable staining.

Cationic chromium appeared to be superior to anionic chromium in preserving cell walls as well as the general histologic features of the material investigated.     

椭圆背角无齿蚌胃的组织学及铜对其结构的影响

硫酸铜常用于水产养殖的疾病防治,但当水体中的铜离子浓度超过一定范围时便对软体动物产生一定的毒害作用.研究选取椭圆背角无齿蚌(Anodonta woodiana elliptica)作为研究对象,采用苏木精-伊红染色方法进行组织石蜡切片制作,从组织学角度研究了浓度为0.01 mg·L^-mg·L^-1、0.1 mg·L^-1、1 mg·L^-1和10 mg·L^-1的硫酸铜作用不同时间后椭圆背角无齿蚌的胃结构的变化.结果表明,0.01 mg·L^-1硫酸铜处理7d和14d后,仅胃绒毛变短,随着硫酸铜浓度的增大和处理时间的延长,胃绒毛逐渐变短,肌层变薄,腺细胞数量减少直至空化;低于10mg·L^-1的硫酸铜中毒7d和14d后,胃晶杆头直径和胃楯长度出现增大的现象.     

六妹羊肚菌工厂化制备液体菌种繁育条件的优化

为适合大规模工厂化制备羊肚菌液体菌种,以提高菌丝体生物量为目的,控制菌丝球直径,使用六妹羊肚菌Morchella sextelata,采用单因素、Plackett-Burman、响应面Box-Behnken实验摇瓶培养优化液体菌种制备条件,发酵罐扩大培养,具体参数为玉米淀粉2.5%、麸皮6%、酵母粉0.4%、葡萄糖2.5%、蛋白胨0.25%、磷酸二氢钾0.1%、硫酸镁0.05%、接种量10%、pH 5.5、转速200-500r/min、温度25℃、培养1-3d,在该条件下羊肚菌菌丝体干重可达16mg/mL,菌丝球直径降至1.5mm以下,密度达到2 967个/mL,从母种到栽培种仅需13-19d。本研究结果为羊肚菌工厂化液体菌种制备和栽培提供了理论基础。     

介质pH缓冲系统选择及其对龙须菜生长的影响

该文选择红藻门大型植物龙须菜(Gracilaria lemaneiformis)作为研究对象,通过黑白瓶法筛选出有机试剂POPSO(哌嗪-N,N-双(2-羟基乙烷磺酸)),能较好地缓冲室内龙须菜培养中介质pH的波动。在此基础上,探讨了POPSO对龙须菜生长的影响,结果表明:15 mmol·L ^-1及以上浓度的POPSO能较好地稳定介质pH值,减少系统中DIC浓度的急剧变化,但POPSO对CO₂浓度波动不起作用;试验结果还表明,介质中的CO₂浓度与龙须菜的生长速率间存在着类似酶动力学方程的关系,当介质中的CO₂浓度下降到5.25 μmol·L ^-1以下时,龙须菜生长出现抑制现象。相对稳定pH的培养环境,更有利于介质DIC中的HCO^-₃、CO^2-₃向CO₂的转换,缓解龙须菜生长的C抑制。     

Lysozyme for capture of microorganisms on protein biochips

Lysozyme placed on the SiO₂ surfaces that have previously been derivatized with C₁₈ coating will capture both Escherichia coli and Listeria monocytogenes cells from PBS buffer at pH 7.2. This phenomenon is of significance for the design and fabrication of protein biochips that are designed to capture bacteria from buffer or water so that these can be further interrogated with respect to possible pathogenicity. Fluorescent microscopy shows that two types of bacteria (gram-negative E. coli and gram-positive Listeria spp.) will be adsorbed by lysozyme placed on the surface of the biochip but that strong adsorption of the bacteria is reduced but not eliminated when Tween 20 is present (at 0.5%) in the PBS buffer in which the cells are suspended. In comparison, Tween 20 and Bovine Serum Albumin (BSA) almost completely block adsorption of these bacteria on C₁₈ coated surfaces. The combination of a lysozyme surface with Tween 20 gives a greater degree of adsorption of L. monocytogenes than E. coli, and hence suggests selectivity for the more hydrophobic E. coli may be reduced by the Tween 20. This paper presents protocols for preparing protein-coated, SiO₂ surfaces and the effect of buffer containing Tween 20 on adsorption of bacteria by SiO₂ surfaces coated with C₁₈ to which BSA, lysozyme or C11E9 antibody is immobilized at pH 7.2 and ambient temperature.     

Enhanced production of free trans-astaxanthin by oxidative stress in the cultures of the green microalga Chlorococcum sp

Free trans-astaxanthin accumulated in the alga Chlorococcum sp. was markedly enhanced from 3.664 mg g⁻¹ cell dry weight to 5.724 mg g⁻¹ cell dry weight when the culture was supplemented with hydrogen peroxide (0.1 mM) under mixotrophic conditions of growth. After saponification, a total of 7.086 mg astaxanthin per g cell dry weight was achieved. Similarly, in heterotrophic cultures, the total astaxanthin content was increased from 1.034 mg g⁻¹ cell dry weight without H₂O₂ to 1.782 mg g⁻¹ cell dry weight with 0.1mM H₂O₂. Results indicate that hydrogen peroxide effectively induces the formation of free trans-astaxanthin in Chlorococcum sp.