Orseillin Bb for Staining Fungal Elements in Sartory's Fluid

A method is described for the squashing and permanent mounting of preparations, similar to the nucleal squash technic, to be used after hematoxylin, coal tar dyes and other stains. The colored pieces are immersed in glycerin, squashed or smeared, the glycerin is slowly removed with water by capillarity and the water is afterwards similarly substituted by alcohol. The cover glass is then removed and after a rapid rinsing in alcohol the materials are permanently mounted in alcohol-soluble resin.     

Permanent Stained Preparations of Thick Blood Films

Fixing thick films in alcoholic solution of dye after the usual staining-and-laking procedure preserves the appearance of parasites and blood elements very similar to that of the usual thick films (not fixed) for the diagnosis of malaria and relapsing fever.

Procedure recommended: Films are stained and laked for 15 minutes in diluted Giemsa—1 to 3 drops of stock solution (0.4 g. in 60 ml. equal parts absolute methyl alcohol and glycerin) per ml. distilled water; rinsed in water and allowed to dry. They are then immersed in, or flooded with, May-Griinwald's stain (0.5% in absolute methyl alcohol) for 30 seconds, rinsed in water and allowed to dry. Solutions of MacNeal's tetrachrome stain in methyl alcohol and glycerin may be substituted for Giemsa and a solution in methyl alcohol may be substituted for May-Griinwald. With slight modification of the procedure, both thick and thin films on the same slide may be stained together.

Films stained and fixed as described, and mounted in Diaphane, have shown no evidence of fading in 3 years.     

The use of basic Fuchsin in Plant Anatomy

A method useful in tracing the vascular bundles of herbaceous plants and the water conducting system of woody plants is presented. A stock solution of 2.5% basic fuchsin in 95% alcohol is prepared. A mixture of 1 to 2 parts of the stock solution to 100 parts of tap water (slightly alkaline) is made, standing 12-48 hours and being filtered before use. Cut ends of the living plants to be examined are immersed in the working solution until the dye appears in the parts desired.

The material may be examined by serial, hand sections mounted in 50% to 75% glycerin, or it may be split, laid open, and examined with a low power lens. Clearer views may be procured by dehydrating in an alcohol series and clearing in methyl salicylate. Permanent mounts may be made by removing the methyl salicylate with xylol and mounting in balsam. Structures such as leaves, which contain considerable chlorophyll, should be first boiled in slightly alkaline 85% alcohol until the green color is removed.     

Biodiesel Production from Waste Cooking Oil Using Anion-Exchange Resin as Both Catalyst and Adsorbent

A novel production process of biodiesel fuel was developed using an expanded-bed reactor packed with an anion-exchange resin having catalytic and adsorption abilities. Waste cooking oil was used as a cheaper feedstock, and methanol was added at the stoichiometric molar ratio of 3:1. The main constituent of the feedstock, triglyceride was completely converted to the biodiesel by the resin??s catalytic ability. The impurities of the feedstock, free fatty acid, water, and dark brown pigment were removed from the product by the adsorption on the resin. In addition, the by-product glycerin was also removed from the product by the adsorption on the resin. The product eluted from the reactor was directly used for the standard tests of the biodiesel properties. The eluted product almost met the biodiesel quality standards (EN14214 in Europe and ASTM D6751 in North America). Thus, the proposed system permitted the simple production of biodiesel from the waste cooking oil without the upstream processing to remove the impurities (free fatty acid and water) and the downstream processing to remove the catalyst and by-products (glycerin and soap).     

Optimization of biogas production from cattle manure by pre-treatment with ultrasound and co-digestion with crude glycerin

Biogas production by co-digestion of cattle manure with crude glycerin obtained from biodiesel production was studied after pre-treatment of the cattle manure or mixtures of cattle manure with different amounts of added glycerin with ultrasound. Batch experiments with 1750 mL of medium containing 1760 g of screened cattle manure or mixtures of cattle manure (screened or ground) and 70-140 mL or crude glycerin were incubated under mesophilic and thermophilic condition in stirred tank reactors. Under mesophilic conditions, the addition of 4% glycerin to screened manure increased biogas production by up to 400%. Application of sonication (20 kHz, 0.1 kW, and 4 min) to a mixture of manure + 4% glycerin increased production of biogas by up to 800% compared to untreated manure. The best results were obtained under thermophilic conditions using sonicated mixtures of ground cattle manure with 6% added glycerin (348 L methane/kg COD removed were obtained).     

Methods for the Study of Leaf Anatomy in Palms

Large size, hardness, combinations of thick-walled fibers and sclereids with thin-walled parenchyma cells, and the occurrence of silica, calcium oxalate, and tannins make anatomical preparations of palm leaves difficult. Samples for anatomical study should encompass one-half a pinna or a comparable portion from palmate and entire leaves including the midrib, all large ribs, and the margin. Similar pieces from herbarium specimens are reconstituted in glycerin alcohol, aerosol OT and distilled water (10:3:90). All samples are fixed in formol-acetic-alcohol (FAA) but stored in glycerin alcohol to minimize hardening. Transverse and longitudinal sections IS microns thick, epidermal macerations, and pieces for clearing and for scanning electron microscopy are prepared from the FAA fixed material. Samples for electroscanning are gradually changed to 100% acetone, critical point dried with CO₂, and coated with 100-300 angstroms of gold. Leaf material for microtomy is treated with hydrofluoric acid, embedded in Paraplast, and sectioned at 15 microns at a temperature of 7.2 C. Paraplast sections are floated directly on a modification of Sass' Adhesive III, mounted unstained or stained in safranin and fast green, and observed in polarized light. Epidermal peels are prepared by soaking pieces 5 mm square for 12-24 hours in undiluted bleach. Pieces for observation of transverse veins are cleared by treatment in 5% sodium hydroxide in a 60 C oven, washed rapidly in three changes of distilled water, and placed in one-third strength commercial bleach until clear. The same procedures can be used to prepare reproductive material for anatomical observations, but time schedules must be increased for larger specimens.     

Demonstration of the Ground Substance of Cartilage, Bone, and Teeth

A slab of bone about 5 mm. thick is decalcified in 5% HCl, washed, and placed for several days to 2-3 weeks in 3% KOH in 20% glycerin (if the bone is medium sized); for small bones the KOH should be decreased to 1%, and for large bones it may be increased to 5%. The solution is changed frequently. When the bone begins to dissociate, it should be removed and washed in water till all traces of alkali are removed. The specimen is passed through 3 changes of dioxane into paraffin, and then through a second paraffin bath into the final paraffin. Sections are cut at 10-12 μ and stained with VanGieson's picro-fuchsin or with orcein.     

Improvements In The Paraffin Method

Dilute hydrofluoric acid alone and in conjunction with glycerin and ethyl alcohol was employed successfully to soften various types of refractory plant materials embedded in paraffin. Serial sections were cut at 6-8 μ and no appreciable deleterious effects on cell walls, cell contents, or staining procedures occurred. “Tannins” and “phlobaphene compounds” can be removed from tissues softened by this method by treating the sections for 12-48 hours with an aqueous solution of chromic acid, potassium bichromate and glacial acetic acid prepared according to the formula given by Johansen (1940).     

A Rapid Method for Making Permanent Mounts of Nematodes

A rapid method which can be used to mount and clear nematodes and their eggs is presented. Permanent mounts of certain nematodes and parasite eggs have been prepared using a medium consisting of 56 parts of a stock solution of polyvinyl alcohol (“PVA”), 22 parts phenol and 22 parts of lactic acid. The stock solution of PVA is prepared by dissolving 15 grams of PVA in 100 ml. of distilled water. This medium can be used on material killed and fixed in 10% formalin, any concentration of alcohol, alcohol-glycerin or glycerin. Results have been very satisfactory in most instances. An accompanying plate of photographs shows some of the preparations obtained by using this method.     

Dissection, Staining, and Mounting of the Embryos of Conifers

A statement is given of the advantages of this special technic and its place in embryological investigations, including directions for selecting the proper stages in collecting conifer cones and ovules, their methods of dissection from living material and their preservation for later dissection. The choice of dissecting microscopes and dissecting instruments, as well as directions for staining embryos with phloxine which may be combined with slow dehydration in glycerin, or for staining with Delafield's or Heidenhain's hematoxylin which may be followed by the glycerin dehydration are described. Glycerin affords a convenient break for a temporary stopping place in this technic.

Directions are given for transfer from glycerin thru 95% and absolute ethyl alcohol into other solvents such as diaphane solvent, essence of euparel or an easily prepared sandarac solvent. Other mounting media which have been used for conifer embryos are discussed—glycerin jelly, Venetian turpentine and Canada balsam—emphasizing the special advantages found in the media employing sandarac.     

Synthesis of peroxynitrite using isoamyl nitrite and hydrogen peroxide in a homogeneous solvent system

A method for the synthesis of peroxynitrite is described. It involves nitrosation of H2O2 at pH> or = 12.5 by isoamyl or butyl nitrite in mixed solvents of isopropyl alcohol (IPA) and water at 25+/-1 degrees C. Maximum yields of peroxynitrite are obtained after 15 min of incubation at IPA concentrations of 30-70% (v/v). The solutions of peroxynitrite are processed for removal of IPA and isoamyl alcohol by solvent extraction. Unreacted H2O2 is removed by catalytic decomposition on granular MnO(2). The post processed solutions of peroxynitrite are useful in several chemical and biochemical investigations where bolus additions are required. The method as reported is amenable for large scale synthesis as it involves sequential mixing of solvents (water and IPA) to alkali followed by the addition of H2O2 and alkyl nitrite.     

A rapid method for preparing high quality alizarin stained skeletons of adult mice

A simple three-day technique is described for preparing completely cleared and high quality alizarin stained total skeletons of adult mice. Unfixed specimens are partially macerated during staining. Older specimens are heated for 15 min in 1% KOH. A heated solution of benzyl and ethyl alcohol, glycerin, and water is used for final clearing and hardening. This procedure requires about 10 min work per specimen and greatly simplifies preparation of stained and cleared skeletons of adult mice. Another technique, giving slightly better preparations, but requiring 11-14 days, is also described.     

A Rapid Method for Preparing High Quality Alizarin Stained Skeletons of Adult Mice

A simple three-day technique is described for preparing completely cleared and high quality alizarin stained total skeletons of adult mice. Unfixed specimens are partially macerated during staining. Older specimens are heated for 15 min in 1% KOH. A heated solution of benzyl and ethyl alcohol, glycerin, and water is used for final clearing and hardening. This procedure requires about 10 min work per specimen and greatly simplifies preparation of stained and cleared skeletons of adult mice. Another technique, giving slightly better preparations, but requiring 11-14 days, is also described.     

Rapid Removal of Tissue Culture Monolayers by Collodion for Subsequent Staining

Removing cultures from roller tubes before staining eliminates the destaining which often occurs when the cells are first stained and then removed by embedding in collodion. The cells are fixed in situ, dehydrated, and covered with collodion (Merk's flexible) for 10 min. The collodion is poured off, the fluid residue lining the tube allowed to dry for 10 min, and the tube is filled with tap water. The collodion cast containing the cells is loosened and removed, cut into strips, placed on slides and blotted into firm contact. The collodion is then dehydrated and dissolved with absolute alcohol followed by a 1:1 mixture of alcohol and ether. The slides can then be rehydrated and stained by conventional methods.     

Elimination OF Distortion and Poor Staining in Paraffin Sections OF Plant Tissues

Three fixing solutions causing least distortion and bright staining of plant tissues are named. Glycerin dehydration causes less distortion than a series of alcohol concentrations; 95% alcohol removes some of the glycerin, sets the protoplasm and improves the staining. Absolute alcohol causes distortion and should be avoided. Pure chloroform, as a paraffin solvent, is followed by brighter staining but more distortion than are the butyl alcohols. A schedule resulting in minimum distortion is given. The results are shown in photomicrographs. Brightest staining follows the use of C. P. iron alum and hematoxylin. The use of a paper cup for very gradual change from one liquid to another and as a labor saver is described.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

A Comparison of Preparation Techniques in Taxonomic Studies of Longidorus africanus Merny

A comparison was made of ten different techniques for killing, fixing, and mounting Longidorus africanus Merny for microscopic study. The most satisfactory specimens were those killed by Seinhorst''s method, fixed in FAA and mounted in glycerin by the slow method. Specimens killed by "gentle heat," fixed in FAA and mounted in glycerin were also acceptable as were those killed by hot formalin and mounted in glycerin or processed by Baker''s method. Less satisfactory were: nematodes killed by "gentle heat," fixed in formalin and mounted in glycerin and specimens killed by vapor phase perfusion or by Hopper''s lethal stain, both latter groups were mounted in glycerin after fixation in formalin. Killing with cold formalin, gradual heat (60 C for 15 min), or by storage in distilled water produced poorly defined specimens. Nematodes killed by hot formalin, and processed to glycerin by the slow method, maintained their live dimensions. Reduction in length occurred in specimens killed by cold formalin, by storage, or treated with solutions containing acetic or propionic acids. Nematodes processed by Baker''s method increased in size. Other minor modifications occurred in specimens processed by the different methods. Esophageal definition was best in nematodes killed with formalin, hot or cold. There is no correlation between position of the posterior part of the esophagus and position of the onchiostyle.     

Elimination of Fat and Protein Prior to the Selective Staining of Bone

The technic of staining skeletal systems previously described is often unsatisfactory for fetal specimens of Aves, because of the large amount of fat and protein. The writer avoids this by introducing two preliminary steps: (1) The specimen is placed in equal parts of glycerin, 95% alcohol and distilled water, and 10% aqueous pepsin (with a drop of 6N HC1 added) injected into the yoik sac, with 2-3 hours incubation at 40^oC. (2) While in 5% aqueous KOH (with a few drops of 2% H₂O₂), the fat areas are injected with cellosolve; and the specimen is left in this solution until skeletal elements become clearly visible. Staining in alizarin red S then follows.     

The Actinomyces of Potato Scab Demonstrated by Fluorescence Microscopy

The potato scab Actinomyces, like other acid-fast organisms, can be selectively impregnated with carbol-auramin and when exposed to ultraviolet radiation fluoresces bright yellow. The marked contrast of the bright yellow filaments permits ready localization and study of the micropathology of the tissue with a simple fluorescence microscope. The staining technic is done at room temperature and no counterstain is used. The fluorescence technic confirms Lutman's conclusion that the filaments are intercellular and grow within the middle lamellae. After complete removal of the paraffin, the sections are stained 4 minutes in carbol-auramin, (distilled water 97 ml., liquified phenol 3 ml., certified auramin 0.1 g.), washed, destained in a 0.5% solution of NaCl in 70% alcohol with 0.5 ml, HCl (conc.) per 100 ml., washed, and mounted in glycerin.