Elimination Or Reduction of Wrinkles in Semithin Epoxy Sections by Vacuum Drying

Vacuum drying, under appropriate conditions, diminishes the warping and buckling of epoxy semithin sections and enhances visualization with light microscopy. Treatment of sections with chloroform or variations in the drying times or temperatures did not reduce wrinkling.     

A Technic for Preparing Frozen Sections

A combination of the Gram-Pappenheim stains for the examination of gonorrheal pus, cellular exudate and paraffin sections of formalin-fixed tissues has been described elsewhere (Scudder and Lisa, 1931). The crystal violet solution was made stable for the first time by employing phosphate buffers on the acid side of neutrality, and a stable counterstain was prepared for the first time from National Aniline dyes, ethylated methyl green and pyronin yellowish. Original findings were demonstrated by means of color plate I and II (Scudder, 1931) to show gonococci, pneumococci and cells in smears, and formalin-fixed tissue brought down to water in the usual way. A new color plate is published herewith to show the microscopic appearance of cells, Gram-positive and Gram-negative bacteria, higher bacteria, fungi and spermatozoa in the study of genitourinary and gynecological cases. The method has a value in the field of medical jurisprudence. Crystals were well demonstrated, especially those resulting from sulfa drug therapy. The National Aniline methyl green batches numbered NG 10, 11, 13 to 19, and their batches of pyronin numbered NP 5 to 10 were found consistently stable. Earlier dyes were found either too purple or too blue for the technic and the most satisfactory dyes were found to require a ripening time of several days and could be prepared in amounts of from 1 to 4 liters and stored indefinitely without preservatives.     

Freeze Thaw: A Simple Approach for Prediction of Optimal Cryoprotectant for Freeze Drying

The present study evaluates freeze thaw as a simple approach for screening the most appropriate cryoprotectant. Freeze–thaw study is based on the principle that an excipient, which protects nanoparticles during the first step of freezing, is likely to be an effective cryoprotectant. Nanoparticles of rifampicin with high entrapment efficiency were prepared by the emulsion-solvent diffusion method using dioctyl sodium sulfosuccinate (AOT) as complexing agent and Gantrez AN-119 as polymer. Freeze–thaw study was carried out using trehalose and fructose as cryoprotectants. The concentration of cryoprotectant, concentration of nanoparticles in the dispersion, and the freezing temperature were varied during the freeze–thaw study. Cryoprotection increased with increase in cryoprotectant concentration. Further, trehalose was superior to fructose at equivalent concentrations and moreover permitted use of more concentrated nanosuspensions for freeze drying. Freezing temperature did not influence the freeze–thaw study. Freeze-dried nanoparticles revealed good redispersibility with a size increase that correlated well with the freeze–thaw study at 20% w/v trehalose and fructose. Transmission electron microscopy revealed round particles with a size ∼400 nm, which correlated with photon correlation spectroscopic measurements. Differential scanning calorimetry and X-ray diffraction suggested amorphization of rifampicin. Fourier transfer infrared spectroscopy could not confirm interaction of drug with AOT. Nanoparticles exhibited sustained release of rifampicin, which followed diffusion kinetics. Nanoparticles of rifampicin were found to be stable for 12 months. The good correlation between freeze thaw and freeze drying suggests freeze–thaw study as a simple and quick approach for screening optimal cryoprotectant for freeze drying.     

A Rapid lmmunostaining Method for Frozen Sections

A simple and rapid one-step method for demonstrating immunohistochemical markers (leukocyte common antigen, cytokeratin, etc.) is described, which can help define the nature of poorly differentiated neoplasms for diagnosis using frozen section. Microwave irradiation was used to speed immunohistochemical analysis using “Enhanced Polymer One-step Staining” (EPOS) reagents on cryostat sections from a variety of pathologic samples. Reproducible results were obtained using EPOS reagents for leukocyte common antigen and cytokeratin. The overall procedure takes less than 10 min and can be completed during surgery.     

Contraction-Free, Fume-Fixed Longitudinal Sections of Fresh Frozen Muscle

Contraction damage occurring when longitudinal frozen sections of fresh unfixed muscles are thawed on microscope slides has limited histological examination of this tissue mainly to cross sections. Longitudinally oriented sections are advantageous for investigating properties that vary along the length of the muscle fibers. A fume fixation technique has been developed for preventing contraction of thick longitudinal frozen aections. The technique is compatible with histochemical staining of enzymes.     

Cutting Unfixed Frozen Sections for Fluorescence Antibody Studies

A simplified method of section cutting, dispensing with guide attachment on microtome blade, and suitable for serial sections of unfixed frozen tissue of 4μ is described. Essential features are low temperature maintenance (—13°C), critical angle of knife and moistening of slides in cold alcohol or isopentane. This method has been found suitable for fluorescence antibody studies where maintenance of low temperatures is necessary.     

An Adhesive Transfer Method of Cutting and Mounting thin Sections for Autoradiography

A short strip of adhesive transfer cellophane tape is applied to the face of the block to provide backing for the section during cutting and handling. The tape is sealed to the nuclear track plate with the section against the dry emulsion. After exposure the cellophane backing is removed by immersion in water, and the adhesive is dissolved from the section in unleaded gasoline. The section is hydrated and photographic and histological processing are carried out in the usual manner.     

Autoradiography of Mobile, C14-Labeled Herbicides in Sections of Leaf Tissue

Fresh leaf tissue containing a soluble, C¹⁴-labeled herbicide was mounted in cold 1% gelatin on a holder, quick frozen in a cryostat, and cross sectioned at 16 μ with single-edge, stainless steel razor blades. The sections were transferred (without thawing) to cold (—10 C) microscope slides which had been partly covered with double-coated Scotch tape #665. The tissue was freeze-dried in a vacuum desiccator at—20 C then secured to the tape with pressure. Autoradiography was accomplished in a darkroom by covering the slides with dry, nuclear track emulsion films. These films were made by dipping 2 inch diameter wire loops into liquid emulsion, letting the film dry, and applying it by blowing it as it was placed against the tissue. After a 19 day exposure in light-tight boxes at 25-27 C the preparations were processed in the usual manner. The method-was used successfully to trace the movement of soluble, C¹⁴-labeled herbicides in leaf tissue without the loss of labeling material or artifacts caused by its diffusion. High resolution autoradiograms with low backgrounds were obtained.     

Producing Frozen Sections of Calcified Bone

We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision. tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcel-lulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocom-pound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the “sectioning window” is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly “papered” with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide: adhesion is aided by “press-blotting” with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.     

Autoradiography of Mobile,C14-Labeled Herbicides in Sections of Leaf Tissue

Fresh leaf tissue containing a soluble, C¹⁴-labeled herbicide was mounted in cold 1% gelatin on a holder, quick frozen in a cryostat, and cross sectioned at 16 μ with single-edge, stainless steel razor blades. The sections were transferred (without thawing) to cold (—10 C) microscope slides which had been partly covered with double-coated Scotch tape #665. The tissue was freeze-dried in a vacuum desiccator at—20 C then secured to the tape with pressure. Autoradiography was accomplished in a darkroom by covering the slides with dry, nuclear track emulsion films. These films were made by dipping 2 inch diameter wire loops into liquid emulsion, letting the film dry, and applying it by blowing it as it was placed against the tissue. After a 19 day exposure in light-tight boxes at 25-27 C the preparations were processed in the usual manner. The method-was used successfully to trace the movement of soluble, C¹⁴-labeled herbicides in leaf tissue without the loss of labeling material or artifacts caused by its diffusion. High resolution autoradiograms with low backgrounds were obtained.     

Autoradiography of Water-Diffusible Substances in Sections of Whole Baby Rats

Rats, 7 days postnatal which had been injected with a radioactive nuclide, were quick frozen and sectioned in the frozen state. An adhesive cellulose tape (Sellotape) was used to support the section during cutting, through freeze-drying, and attaching to slides. Dehydration of the frozen sections consisted of 1 hr in a chilled desiccator containing silica gel, then at reduced pressure of 2-3 mm Hg until quite dry. The exposed side of the section was sprayed with celloidin dissolved in amyl acetate and allowed to dry. This side of the section was attached to a slide, previously coated with 1% gelatin containing 0.1% chrome alum, by means of an adhesive consisting of 4% gelatin and 5% formalin in 60% glycerol. In applying this adhesive it is mandatory that a border of about 3 mm of bare glass be left outside the adhesive, to allow intimate contact between the sticky side of the tape and the glass. The adhesive was allowed to set for 20 min, the slide immersed in water lor 50 sec, and the cellulose layer of the tape peeled off. The rubber base from the tape was removed with chloroform, the slide dried, and the exposed surface of the section coated with celloidin in amyl acetate, by dipping. After this treatment, the slides could be coated by dipping in autoradiographic emulsion without affecting water-soluble radioactive substances in the tissue.     

Rapid Nissl Staining for Frozen Sections of Fresh Brain

Working with X-ray film autoradiography of soluble isotopes, we needed a staining technique for the localization of nuclei in frozen sections of fresh brain. We have found no Nissl staining method in the literature concerning autoradiography specially recommended for this purpose, nor have we found in handbooks on staining a Nissl method clearly recommended for unfixed, frozen sections of brain. The methods described are intended for paraffin or celloidin sections, and require fixation of brain before sectioning (which must be avoided when working with soluble isotopes). Because autoradiography is a time-consuming method, any technique which shortens time needed for the overall procedure is welcome. Most Nissl techniques described in the literature require long preprocessing of the tissue. We found two rapid methods, described by Humason (1967) and LaBossiere and Glickstein (1976), but their application to frozen sections did not give good results. After trials with several types of techniques, we succeeded in developing two Nissl modifications with slightly different qualities, one of 12 min and the other of 2-3 h. The longer method includes conventional steps in staining; the shorter method does not include fixation or lipid extraction. These methods were applied to 20-60 μm brain sections cut in the cryostat at -10 to -12 C and dried on gelatinized slides.     

Preservation of Vibrio fetus by Freeze Drying

S ummary . Vibrio fetus can be successfully freeze dried using the growth from thioglycollate blood agar. This medium is unsatisfactory for estimating the numbers of surviving organisms after freeze drying and storage. For this purpose a liquid medium containing 0.05% agar has been satisfactory. The long term storage of lyophilized preparations of V. fetus has not been fully investigated.     

Preservation of Bacteria by Circulating-Gas Freeze Drying

Water-washed Serratia marcescens and Escherichia coli were freeze dried in a circulating-gas system at atmospheric pressure. This convective procedure resulted in a substantially higher survival of organisms than could be obtained by the vacuum method of freeze drying. There was little or no decrease in cell viability during convective drying when the residual moisture content was 15% or higher. Below this level, survival declined with decreasing moisture content. A detailed comparison of the convective and vacuum methods indicated that the advantage gained by freeze drying bacteria in air accrues in the early period of sublimation, at which time cells were found to be sensitive to vacuum drying but insensitive to air drying. An explanation for this difference is proposed, based upon the kinetics of water removal in the two processes. In brief, it is suggested that the convective method permits samples to be dried more uniformly; and regional over-drying, which may be deleterious even if transient, is thus avoided in achieving the optimal level of moisture.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

Acid Thionin Stain for Nissl Bodies on Frozen Sections

Using a buffered acid thionin stain with carbolxylene as a clearing agent, a reliable stain for Nissl bodies may be performed on frozen sections of fresh or old formalin-fixed material in a relatively short period of time. The technic is simple: the buffering of thionin makes regressive differentiation unnecessary.