A Trichrome Stain for Insect Material

Deparaffinized insect sections are brought down to water and overstained in a 0.1% solution of azocarmine G in 1% acetic acid. They are then destained in a saturated solution of orange G until the azocarmine G is removed from the endocuticle and the latter is colored pale yellow. After washing, the sections are transferred to a 5.0 % solution of phosphotungstic acid in water for 3 min. They are then rinsed in distilled water and stained in a 0.1% solution of methyl green in 1% acetic acid until the endocuticle is green. Differentiation is done in 2 changes of 95% alcohol. The sections are then dehydrated either in absolute alcohol or dioxane, cleared in a mixture of “camsal”, eucalyptol, dioxane, and paraldehyde (1:2:2:1), and mounted in Mohr and Wehrle's medium, a mountant of the Euparal type.     

A Trichrome Stain for Insect Material

Deparaffinized insect sections are brought down to water and overstained in a 0.1% solution of azocarmine G in 1% acetic acid. They are then destained in a saturated solution of orange G until the azocarmine G is removed from the endocuticle and the latter is colored pale yellow. After washing, the sections are transferred to a 5.0 % solution of phosphotungstic acid in water for 3 min. They are then rinsed in distilled water and stained in a 0.1% solution of methyl green in 1% acetic acid until the endocuticle is green. Differentiation is done in 2 changes of 95% alcohol. The sections are then dehydrated either in absolute alcohol or dioxane, cleared in a mixture of “camsal”, eucalyptol, dioxane, and paraldehyde (1:2:2:1), and mounted in Mohr and Wehrle's medium, a mountant of the Euparal type.     

淋洗法修复化工厂遗留地重金属污染土壤的可行性

通过室内模拟试验,采用振荡淋洗的方法研究了蒸馏水、HCl、H₃PO₄、草酸、CaCl₂等淋洗剂对化工厂遗留地污染土壤中重金属的淋洗效果,探讨了淋洗剂浓度、淋洗时间、淋洗次数等对淋洗效果的影响,并研究了HCl处理前后土壤中重金属形态的变化.结果表明：蒸馏水、H₃PO₄、CaCl₂等对Cr、Pb、Zn、Cu、Cd的去除率较低,大多数条件下重金属去除率均在1%以下,最大去除率仅为3.58%.2 mol·L^-1 HCl在土水比为1∶3、反应时间为1 h、2次淋洗的条件下可以达到最佳淋洗效果,Cr、Pb、Zn、Cu、Cd的去除率分别达到80.75％、88.69％、98.00％、79.33％和95.52％.形态分析结果表明,HCl能有效去除土壤中各种结合形态的重金属.     

Demonstration of Degenerating Nerve Fibers by a Modified Cajal Technic

Whole brains of cat were fixed in two changes of cold acetone (24 hours each) and embedded directly in paraffin. The degeneration time recommended is 5 days. Mounted sections 15-20 μ thick were deparaffined, washed in absolute alcohol and given successive treatments of 6 hours each with 1% ammoniated absolute alcohol and pure pyridine, washing well with distilled water between them and after the pyridine. Impregnation in 2% silver nitrate 12 hours at 30°C., rinsing in absolute alcohol and reducing in a 95% alcoholic solution of pyrogallol and formalin (3% and 5%) was followed by 50% alcohol, thorough washing in distilled water, toning in 1% gold chloride and intensification in 1% oxalic acid. Treatment in 10% sodium thiosulfate solution, washing, dehydrating and covering completed the procedure. Normal fibers, degenerating fibers and terminals were stained specifically.     

Hatching of Meloidogyne incognita Eggs in the Neutral Carbohydrate Fraction of Root Exudates of Gnotobiotically Grown Alfalfa

Meloidogyne incognita eggs were hatched in soil sterilized by gamma kradiation and wetted with root exudates from alfalfa plants in different stages of development and subjected to various levels of clipping. Carbohydrate components of the exudates were identified by gas chromatography-mass spectrometry. Although significant stimulation of hatch was detected in exudates of seedling and flowering plants, the practical importance of the increase is doubtful as hatch in distilled water was always greater than 50%. Hatch did not differ among exudate samples from clipped plants. Incubation of eggs in soil moistened with 10⁻⁷ to 10⁻³ M solutions of glucose did not result in increased hatching over that in distilled water.     

Quantitative sampling method for fungus gnat (Diptera: Sciaridae) eggs in soilless growing media

A simple method is described for separating fungus gnat eggs from soilless growing media. The flotation/extraction method primarily involves the use of a MgSO4 solution (density 1.065 g cm(-3)) and a series of sieving procedures. Bradysia sp. nr. coprophila Lintner eggs were collected in an isolation chamber containing adult fungus gnats. Three soilless growing media were used: Metro-Mix 560 with Scott's Coir, Sunshine LC1 Mix, and Universal SB 300 Mix. Each growing medium was inoculated with 100 fungus gnat eggs. Growing medium samples were processed using separatory funnels and a MgSO4 solution. Eggs were washed through a 250-microm sieve and collected on a 53-microm sieve, and then the eggs were rinsed into glass petri dishes. The sieving process separated fungus gnat eggs from the larger growing medium particulates (perlite, bark, and peat), providing a sample consisting of water, eggs, and fine growing medium particulates (peat). Using a vortex for 60 s increased the number of fungus gnat eggs recovered (37.4 +/- 3.7-67.1 +/- 1.9 [mean +/- SEM]) for all three growing media. The mean number of fungus gnat eggs recovered from the three soilless growing media was significantly different, with the growing medium containing coir (Metro-Mix 560) having the highest recovery rate of 61.0 +/- 2.1. For all three growing media, there was > 50% recovery of fungus gnat eggs. In addition to fungus gnat eggs, this method also may prove useful for quantitative recovery of shore fly (Scatella sp.) eggs and the eggs of other arthropods from soilless growing media.     

Factors affecting the lytic susceptibility of some marine and terrestrial bacteria

Eighteen gram-negative marine bacteria and two terrestrial species, Escherichia coli and Pseudomonas aeruginosa, were examined for their sensitivity to lysis in distilled water after exposure to a salt solution containing a sea water concentration of Mg2+ (0.05 M) or to 0.5 M NaCl. A spectrum of lytic susceptibility was observed among the marine bacteria ranging from those organisms which lysed in distilled water after exposure to the Mg2+-containing solution, through organisms which could be sensitized to lysis by washing with the NaCl solution, to organisms which failed to lyse in distilled water even after having been washed with a solution of 0.5 M NaCl. Pseudomonas aeruginosa and E. coli fell within this spectrum, the former being capable of being induced to lyse in distilled water by washing with 0.5 M NaCl, while the latter failed to lyse in distilled water after this treatment. It was thus concluded that no overall distinction could be made between marine and terrestrial bacteria on the basis of the sensitivity of the two groups of organisms to lysis in freshwater. Quite large decreases in optical density and increases in the release of ultraviolet-absorbing material took place when cells preexposed to the Mg2+-containing solution or to 0.5 M NaCl were subsequently suspended in distilled water even though in some cases no loss of cell numbers could be detected. In most cases two to three times as much K+ as Na+ and 1/10 to 1/100 as much Mg2+ was required to prevent these changes. For three of the marine bacteria and P. aeruginosa grown in a terrestrial type medium little difference in the requirements for Na+ and K+ to prevent the optical density changes was noted. For P. aeruginosa grown in a marine type medium, cells required more K+ than Na+ to prevent these changes.     

A Quantitative Assessment Method for Ascaris Eggs on Hands

The importance of hands in the transmission of soil transmitted helminths, especially Ascaris and Trichuris infections, is under-researched. This is partly because of the absence of a reliable method to quantify the number of eggs on hands. Therefore, the aim of this study was to develop a method to assess the number of Ascaris eggs on hands and determine the egg recovery rate of the method. Under laboratory conditions, hands were seeded with a known number of Ascaris eggs, air dried and washed in a plastic bag retaining the washing water, in order to determine recovery rates of eggs for four different detergents (cationic [benzethonium chloride 0.1% and cetylpyridinium chloride CPC 0.1%], anionic [7X 1% - quadrafos, glycol ether, and dioctyl sulfoccinate sodium salt] and non-ionic [Tween80 0.1% -polyethylene glycol sorbitan monooleate]) and two egg detection methods (McMaster technique and FLOTAC). A modified concentration McMaster technique showed the highest egg recovery rate from bags. Two of the four diluted detergents (benzethonium chloride 0.1% and 7X 1%) also showed a higher egg recovery rate and were then compared with de-ionized water for recovery of helminth eggs from hands. The highest recovery rate (95.6%) was achieved with a hand rinse performed with 7X 1%. Washing hands with de-ionized water resulted in an egg recovery rate of 82.7%. This washing method performed with a low concentration of detergent offers potential for quantitative investigation of contamination of hands with Ascaris eggs and of their role in human infection. Follow-up studies are needed that validate the hand washing method under field conditions, e.g. including people of different age, lower levels of contamination and various levels of hand cleanliness.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

Selective Silver Impregnation of Degenerating Axons In The Central Nervous System

Rat and rabbit brains containing surgical lesions of 5-10 days' duration were fixed in 10% formalin (neutralized with calcium carbonate) for 1 week to 6 months. Frozen sections (15-20 n) were rinsed and then soaked 7 minutes in a 1.7% solution of strong ammonia in distilled water. Subsequent treatment was as follows: rinse; 0.05% aqueous potassium permanganate 5-15 minutes; 0.5% aqueous potassium metabisulfite, 2 changes of 2.5 minutes each; wash thoroughly in 3 changes distilled water; 1.5% aqueous silver nitrate, 0.5-1.0 hr.; 1% citric acid, 5-10 sec.; 2 changes distilled water; 1% sodium thiosulfate, 30 see.; 3 changes distilled water. Each section is then processed separately. Ammoniacal silver solution (450 mg. silver nitrate in 10 ml. distilled water; add 5 ml. ethanol; let cool to room temperature; add 1 ml. strong ammonia water and 0.9 ml. of 2.5% aqueous sodium hydroxide), 0.5-1.0 min. with gentle agitation. Reduction of about 1 minute is accomplished in: distilled water, 45 ml.; ethanol, 5 ml.; 10% formalin, 1.5 ml.; 1% citric acid, 1.5 ml. Rinsing; 1% sodium thiosulfate, 10 sec.; thorough washing followed by dehydration through graded alcohol and 3 changes of xylene or toluene complete the staining process. Normal nerve fibers are slightly stained to unstained, degenerating fibers, black. The treatment in potassium permanganate is critical since too little favors overstaining of normal fibers and too much abolishes staining of degenerating fibers.     

Field Assessment of Adhesion and Hatch ofChrysoperlaEggs Mechanically Applied in Liquid Carriers

A mechanical technique was evaluated for releasing green lacewing eggs in liquid suspensions. Deposited eggs were enclosed within a circle of nonpoisonous adhesive to protect them from predation and to prevent escape of hatched larvae. Released eggs were monitored daily for 5 days after release by measuring three response variables: adhesion rate of eggs to foliage, hatch rate of eggs, and “yield” of larvae from discharged eggs; “yield” was the product of egg adhesion and egg hatch. Factors tested were: egg conditioning prior to release (incubated or refrigerated), carrier (distilled water or commercial carrier solution), application technique (mechanical or hand application), and row facing (North or South). Release technique did not significantly effect egg hatch on any day. Conditioning eggs prior to release had the greatest effect on hatch of eggs and resulting yield of larvae during the 5-day monitoring period. Carrier had a significant effect on adhesion of eggs to leaves and hatch of eggs. Commercial carrier solution increased egg adhesion but decreased egg hatch compared to water. Overall mean yield of larvae from incubated eggs distributed mechanically was not significantly different for eggs suspended in water (36.4% on day 5 post-release) and for eggs suspended in commercial carrier solution (36.1% on day 5 post-release). Hand-applied eggs had a higher hatch and subsequent yield of larvae than mechanically released eggs; however, the hand technique was labor intensive.     

Silver Protein Staining of Nerve Fibers in Celloidin Sections

Celloidin sections from formalin-fixed brain and spinal cord of primates are stored in 70% alcohol after cutting, soaked in 2% pyridine in 50% alcohol for 6-8 hr at 37 C, and transferred to 1% concentrated NH₄OH in 50% alcohol 15-18 hr at 20-25 C. After washing and flattening, the sections are transferred to 1% silver protein solution containing 30 ml of 0.2 M H₃BO₃/100 ml. Impregnation is accomplished in 50 ml screw-top jars, 50 mm in diameter, which are filled to a depth of 35 mm, and have 1 gm of copper foil, 0.002 inch thick added. The foil is folded in loose accordion-fashion, pierced and threaded, cleaned in 5% HNO₃, rinsed in distilled water, and suspended in the solution just above the sections by fastening the thread to the jar lid. The sections are impregnated for 24 hr at 37 C, rinsed in distilled water, reduced in a solution of 5% Na₂SO₃ and 1% hydroquinone for 10 min, washed in distilled water and toned in 0.2% gold chloride for 5 min. After rinsing in distilled water, the sections are transferred to 1% oxalic acid for 45-60 sec, washed in distilled water and placed in 5% Na₂S₂O₃ for 5 min. Sections are then washed, dehydrated to 95% alcohol, cleared in terpineol, followed by 3 changes in xylene, and mounted.     

Staining of Nervous Tissue by Protein-Silver Mixtures

A staining method for nerves in paraffin sections is described in which an egg albumen-silver nitrate mixture is the impregnating solution. Blocks of tissue are fixed in Bouin's fixative, formol, Huber's fixative or formol-acetic-alcohol, and decalcified if necessary in Bensley's decalcifier. Sections are impregnated overnight, in the dark, at 37-56°C in a solution containing 50 ml of filtered, aqueous 0.5% dried egg albumen with 1.8-2.5 ml of 2% silver nitrate and adjusted to pH 8.2-8.3 by the addition of ammonia. The sections are then rinsed in distilled water and the silver reduced in a mixture of hydroquinone, 1 gm; anhydrous sodium sulfite, 10 gm and distilled water, 100 ml. The remainder of the process consists of washing, gold toning, fixing in 5% sodium thiosulfate, washing, dehydrating, clearing and mounting. Casein may be used as an alternative to egg albumen in the impregnating solution (0.5% casein, 50 ml; 2% silver nitrate, 1 ml). The pH value of the solution may be adjusted by a boric acid-borax buffer or ammonium hydrogen tetraborate in the place of ammonia.     

Fluorescence Microscopy for Measuring Fibril Angles in Pine Tracheids

Observation and measurement of fibril angles in increment cores or similar small samples from living pine trees was facilitated by the use of fluorescence microscopy. Although some autofluorescence was present, brighter images could be obtained by staining the specimens with a 0.1% aqueous solution of a fluoro-chrome (Calcozine Ha vine TG extra concentrated, Calcozine red 6G extra, rhodamine 6G, rhodamine 6GD extra, or a succession of flavine and rhodamine). Staining for 2—5 min followed by a 5-10 sec washing in distilled water and drying 15 min at 190-195°C prepared radially split surfaces of specimens for microscopic observation with ultraviolet light. Measurement of fibril angles, important for the determination of wood strength and the properties of its pulp, was made with a protractor eyepiece. Photomicrography was feasible also, and the need of preparing microtome sections was obviated.     

云锦杜鹃种子萌发及对干旱胁迫的响应

以不同渗透势的PEG-6000溶液模拟干旱胁迫条件, 研究云锦杜鹃种子的萌发、幼苗生长和累积吸水率对干旱胁迫的响应。结果显示：种子于第14 d开始萌发,萌发期为8 d,总萌发率为45.5%±1.7%。在渗透势为-0.3～-0.6 MPa的溶液中, 种子的累计吸水率在第5 d就超过对照(蒸馏水中), 种子的萌发和幼苗的生长也优于对照, 说明云锦杜鹃种子不适合过于湿润的土壤环境中萌发。但溶液的渗透势≥-0.9 MPa时, 种子的吸水减缓、萌发率下降, 同时幼苗的生长也慢, 而当渗透势≥-1.8 MPa时, 种子不能充分吸涨、萌发, 说明云锦杜鹃种子对干旱胁迫比较敏感。另外, 经PEG溶液浸种2天后移入蒸馏水中,萌发率均有一定程度的提高, 其中经-0.3～-1.5 MPa的PEG溶液浸种后, 萌发率显著高于对照。提示用一定浓度范围的PEG溶液浸种可使休眠种子活化, 提高种子繁育的效率。     

Aerosol ot Solution—An Effective Softener of Herbarium Specimens for Anatomical Study

Dried plant parts are cut into convenient sizes and soaked in a solution containing 2.5-3.3% Aerosol OT in distilled water for 5 or more hours until well penetrated by the Aerosol. After brief washing in distilled water the material can be embedded, can be sectioned freehand or, if the nature of the material permits, with a sliding microtome, without embedding. Although it is a good wetting agent, Aerosol is chemically neutral; therefore, microchemical tests can be performed successfully on material treated with it. Refractory plant tissues embedded in paraffin can be successfully softened if one face of the block is trimmed to expose the tissue, then soaked in an Aerosol solution before sectioning.     

A Technic for Differential Staining of Nucleoli and Chromosomes

A procedure is described which enables a stain to be definitely located in the substance of the nucleolus. Material is fixed in either Navashin or Levitsky; the chromatin is stained by means of the improved Feulgen technic introduced by de Tomasi, and preparations brought thru the washing solutions down to distilled water. From distilled water the material is transferred to a mordant solution, 5% sodium carbonate in water, in which it is left for at least one hour. After mordanting wash well with water then stain for ten minutes in light green solution (90% alcohol, 100 cc, light green SFY, 0.5 g, aniline oil, 2 drops, well shaken); differentiate in alcoholic sodium carbonate solution, (70% alcohol saturated with carbonate); treat with 95% alcohol, absolute alcohol, equal parts xylene and absolute alcohol, clear in pure dry xylene and mount in neutral balsam. Cytoplasm and karyolymph should be quite clear, with magenta chromatin and well defined green nucleoli. The light green does not behave like a simple counterstain as in previous technics but as a definite stain for nucleolar material.     

Viability of the R, S and M variants of Mycobacterium lacticolum under various preservation conditions

The survival rate of Mycobacterium lacticolum and the proportion between its R, S and M variants were studied in the course of 12 months under different conditions of storage (freeze-drying, under vaseline oil, in 0.85% NaCl solution and in distilled water). A high survival rate of the variants was found in cells freeze-dried in a protective medium containing 10% of sucrose +1% of gelatin as well as in a 0.85% solution of NaCl.The survival rate of te variants differed by 2--3 orders of magnitude in cells freeze-dried with sodium glutamate or suspended in distilled water. The proportion between the R, S and M variants in the population noticeable changed after storage under these conditions.     

Controlled Differentiation of Cells and Connective Tissue Fibers in Silver Staining

Controlled silver staining of connective tissue fibers and sometimes of these fibers and cells simultaneously can be obtained. 1. Fix in 10% formalin. Embed in paraffin and cut sections as usual, but do not mount them on slides. Deparaffinize and hydrate through xylene, alcohols and distilled water and henceforth treat them the same as frozen sections. Real frozen sections can also be used. 2. Treat with a freshly prepared 1% solution of KMnO₄, usually 15-60 sec, sometimes up to 10 min. 3. Wash in distilled water, 5-10 sec. 4. Decolorize in 2% potassium metabisulfite, 10-20 sec. 5. Place in distilled water, 1 min. 6. Sensitize with 2% iron alum, 1 min. 7. Place in distilled water, 1 min. 8. Impregnate in Gomori's silver oxide solution, 2 min. 9. Wash in a 1.5% aqueous solution of pyridine, about 15 sec. 10. Reduce in a mixture containing 0.25% gelatin and 2% formalin 1 min. 11. Repeat steps 7 to 10 once or several times until the connective tissue fibers are completely stained. For cell staining (which may fail) proceed as follows: After the first insufficient staining of the connective tissue fibers, rinse in distilled water, dip for 1 sec in Gomori's solution and reduce immediately in gelatin-formalin without previous washing in pyridined water. This step can be repeated. 12. If the staining is too strong, decolorize as needed in 2% iron alum. 13. Toning in 0.2% gold chloride, 5 min or more, followed by fixation in 5% sodium thiosulfate, 1 min, is optional. Counterstain as desired. 14. Wash in tap water, dehydrate, clear in xylene and mount in balsam. The same technique applied to sections attached to slides gives good results but inferior to that obtained in paraffin sections processed in the loose, unmounted condition.     

Impregnation of Oligodendroglia in Nervous Tissue Kept in Formalin for Many Years

A method for impregnating oligodendroglia in nervous tissue (monkey) fixed and preserved in formalin for many years is described. This tissue is reconditioned by placing 12 to 30μ frozen sections of it in concentrated ammonia (sp. gr. 0.90) and by washing them slowly for 24 hours with a 1 mm. stream of water. The fluid is then poured off the sections; the jar is refilled with concentrated ammonia; and washing is repeated for another 24 hours. The sections are then plunged into concentrated ammonia for 7 minutes.

After treatment in ammonia, the sections are incubated for one hour at 38^oC. in Globus' 5% hydrobromic acid solution. They are washed again, in distilled water, and then impregnated in a “medium” strength ammoniacal silver carbonate solution (5 ml. of 10% AgNO₃ added to 15 ml. of 5% Na₂CO₃. The precipitate is dissolved in concentrated ammonia and diluted to SO ml. with distilled water). Impregnation is followed by reduction in 1% formalin without agitation; fixation in 5% Na₂S₂O₃; dehydration, and mounting in clarite.

Typical oligodendroglia (Fig. 1) were made visible by use of the method outlined in this paper.