Richard S. Demaree

Four-hour processing of clinical/diagnostic specimens for electron microscopy using microwave technique

A protocol for routine 4-hour microwave tissue processing of clinical or other samples for electron microscopy was developed. Specimens are processed by using a temperature-restrictive probe that can be set to automatically cycle the magnetron to maintain any designated temperature restriction (temperature maximum). In addition, specimen processing during fixation is performed in 1.7-ml microcentrifuge tubes followed by subsequent processing in flow-through baskets. Quality control is made possible during each step through the addition of an RS232 port to the microwave, allowing direct connection of the microwave oven to any personal computer. The software provided with the temperature probe enables the user to monitor time and temperature on a real-time basis. Tissue specimens, goat placenta, mouse liver, mouse kidney, and deer esophagus were processed by conventional and microwave techniques in this study. In all instances, the results for the microwave-processed samples were equal to or better than those achieved by routine processing techniques.

Microwave Techniques and Protocols

Overview of Microwave-Assisted Tissue Processing for Transmission Electron Microscopy Richard S. Demaree, Jr. and Richard T. Giberson Vacuum-Assisted Microwave Processing of Animal Tissues for Electron Microscopy Richard T. Giberson Vacuum-Microwave Combination for Processing Plant Tissues for Electron Microscopy William A. Russin and Christina L. Trivett Basic Procedure for Electron Microscopy Processing and Staining in Clinical Laboratory Using Microwave Oven Ronald L. Austin Specimen Preparation for Thin-Section Electron Microscopy Utilizing Microwave-Assisted Rapid Processing in a Veterinary Diagnostic Laboratory Robert W. Nordhausen and Bradd C. Barr Microwave Processing of Archived Pathology Specimens for Ultrastructural Examination Robert J. Munn and Phillip J. Vogt Microwave Fixation of Rat Hippocampal Slices Marcia D. Feinberg, Karen M. Szumowski, and Kristen M. Harris Microwave Processing Techniques for Biological Samples in a Service Laboratory Lou Ann Miller Microwave-Accelerated Decalcification: Useful Methods for Research and Clinical Laboratories Victoria J. Madden Microwave Processing of Sediment Samples Dawn Lavoie, Janet Watkins, and Yoko Furukawa Microwave Polymerization in Thin Layers of London Resin White Allows Selection of Specimens for Immunogold Labeling Jennifer E. Lonsdale, Kent L. McDonald, and Russell L. Jones In Vivo Microwave-Assisted Labeling of Allium and Drosophila Nuclei Mark A. Sanders and David M. Gartner Microwave-Assisted Cytochemistry: Accelerated Visualization of Acetylcholinesterase at Motor Endplates John P. Petrali and Kenneth R. Mills Microwave-Assisted Immunoelectron Microscopy of Skin: Localization of Laminin, Type IV Collagen, and Bullous Pemphigoid Antigen John P. Petrali and Kenneth R. Mills Microwave Paraffin Techniques for Botanical Tissues Denise Schichnes, Jeffrey A. Nemson, and Steven E. Ruzin Microwave-Assisted Formalin Fixation of Fresh Tissue: A Comparative Study Richard T. Giberson and Douglas E. Elliott Microwave-Assisted Processing of Biological Samples for Scanning Electron Microscopy Richard S. Demaree, Jr

Microwave-assisted immunostaining: a new approach yields fast and consistent results

Advances in microwave technology permitted the development of new antigen labeling techniques. The recent microwave development of a true variable wattage unit designed for laboratory use and an apparatus for dampening standing wave radiation patterns have allowed investigators to better control the conditions within a microwave cavity. Thus, operating limits thought to be endemic to microwave-assisted protocols could be effectively mitigated. Standard protocols for histochemistry call for prolonged incubations and numerous rinses that add considerable time to the procedure. Here, we present microwave-assisted staining protocols for floating rat brain sections and cultured rat hippocampal cells. Acetylcholinesterase (ACHE) histochemistry and immunocytochemistry were conducted inside a specially designed and configured laboratory microwave oven. As a control additional tissue sections were stained on the bench and treated in the same manner as those in the microwave. Labeling was minimal in the control tissue, but specific, high contrast staining was present in the microwave group. Tissues were evenly stained with minimal background, and anatomical structures were easily detected. Also, the differences between lesioned and intact sides of the brain were obvious and agreed with previous observations. Microwave-assisted methods resulted in significantly shorter protocol times (approximately 10-fold) resulting in staining patterns of equal or superior quality to those obtained using conventional methods.

Microwave Processing of Pneumocystis carinii for Electron Microscopy

Although improvements have been made in processing for transmission electron microscopy (“EM), ultrastructural details of many Pneumocystis features frequently are poorly preserved. Improved TEM resolution could better monitor structures altered by drug exposure, development, and pathogenesis processes. Microwave energy has been used to accelerate a number of types of chemical processes. The principles behind its use are not well understood, and there is dispute as to whether it accelerates biochemical processes such as fixation through simple heat or through other mechanisms (12). The use of microwave energy during fixation, dehydration, and infiltration steps significantly reduces processing time for TEM. However, the control and reliability of microwave-enhanced fixation has been problematic and there are disputes about whether there are other advantages to microwave fixation or processing, including enhanced preservation of some types of tissues (1 2). Recent publications have suggested that improvements in microwave power regulation, sample tube configuration, placement of samples in “cold spots” of the microwave oven and temperature regulation (cooling and maintaining low heat) can improve the reliability of microwave fixation and processing (12). For relatively impermeable cells with high levels of hydrolases like Pneurnocysfis, rapid processing with reduced heat may be expected to improve ultrastructural details, so preliminary comparisons of microwave-coupled and conventional fixation were performed. MATERIALS AND METHODS. After ketamine anesthesia and killing by euthanasia and cervical dislocation, lungs of dexamethasone immunosuppressed transtracheally infected rats 131 were dissected and minced in Hank‘s buffered saline. Duplicate lung fragments were placed into Kamofiky’s fixative, one was processed as previously described (1) in 0. 4 ml in 1.5 ml Eppendorftubes and fixed at 4C for 2.5 to 96 hours. Duplicate pieces of tissue were placed into Karnofsky’s fixative and processed in parallel, except that some samples were placed in either conventional or EM microwave ovens and exposed to 15 to 60 seconds of microwave fmtion at 15-37C, in an area of the microwave with less energy, and in the presence oftwo 1-liter beakers of water as microwave dampeners. Additionally, short-term cultured organisms (4) were processed using microwave treatment after overnight incubation in either Hank’s saline or Karnofsky‘s fixative at 4C as previously described (9). Embedding was done using either Epon or Epon-Spur’s 1:l mixture. RESULTS AND DISCUSSION. Approximately 400 trophozoites and 70 cysts were examined from microwave-coupled and conventionally fixed samples. Full microwave processing was rapid ( 3 4 hrs) compared with conventional processing (2 days). However, because each step was monitored, there was more hands-on time for microwave processing relative to controls which used automated dehydration and embedding steps. In total, several thousand cells were examined. In the distinct areas examined for a given sample, there was significant variability in the apparent quality of fixation as monitored by cytoplasmic and membrane integrity and other features, for both microwave-coupled and conventionally-processed samples. Also, a limited number of independent samples were microwave-processed soon after immersion in fixative. With the available samples, it was not possible to determine whether conventional or microwave samples were better preserved but potential differences if any, were slight. In general, near-equivalent ultrastructural details were observed for both normal and microwaveprocessed samples, similar to those previously reported (5,6). Interestingly, trophozoites seem relatively less well preserved than cysts, although the latter have thicker walls. Cultured organisms, even those mailed overnight on ice, displayed comparable ultrastructural details relative to those fixed immediately from rat lung. Typical trophozoites from conventional and microwave-coupled processing are shown (Fig. 1). More detailed blinded studies may allow more definitive comparisons.

Comatricha anomala, a new record for the western hemisphere

warts interspersed with a few isolated patches of reticulum. This ornamentation was beautifully illustrated by Rammeloo (1975) prior to the formal description of the taxon. The collection to be reported on here was made on decayed wood, Lower Bidwell Park, Chico, Butte Co., California, on March 23, 1967 by Dwayne H. Curtis (collection number 657). It has been deposited in the Herbarium of the University of California (UC). Since C. anomala has never been described in English and since the California material differs somewhat from the Belgian material, a detailed description of Curtiss collection is given below. Sporangia (FIG. 1) in small, loose clusters of 10-40, stalked, 0.5-1.5 mm high, sporangium proper ovoid to, more commonly, cylindric, rounded at the base and apex, 0.4-1.25 mm tall and 0.4-0.6 mm diam, bright rusty brown. Hypothallus thin, membranous, shiny, colorless, connecting the individual sporangia of each cluster. Stipe short, approximately one-fourth the total height, ridged, angular or flattened, opaque, shiny, dark reddish brown to, more commonly, black. Peridium predominantly evanescent, but persisting, especially near the apex, in small, irregular, bright rusty brown, iridescent patches. Columella a continuation of the stipe, tapering upward and continuing nearly to the sporangial summit where it merges with the capillitium, sinuose at the tip, colored as the stipe. Capillitium abundant, arising evenly along the entire length of the columella, bright reddish brown, the threads slender, distinctly, but irregularly flattened, lacking large membranous expansions, branching 2-4 times, rarely anastomosing to form a loose, wide-meshed network with numerous short, pointed, free ends, the larger threads bearing sparsely scattered tubercles 1-2 ,um diam. Spores (FIG. 2) bright rusty brown in mass, pale brown by transmitted light, globose, 8.0-9.0 Am diam, minutely but densely warted and bearing 2-5 reticulate areas per spore, these reticulate areas 2.0-2.5 Am diam. Plasmodium unknown. The C. anomala collection from California dif-