A fine structural study of microwave fixation of tissues

Microwave fixed liver and kidney tissues were examined by electron microscopy. It was found that the preservation of fine structure of these tissues by this method is equal to that processed by routine methods. No difficulty was encountered in sectioning microwave fixed tissue blocks. It is obvious that microwave fixation is a faster and more efficient method.     

Microwave fixation of marine invertebrates

Marine invertebrates may be rapidly fixed for histological examination using microwave irradiation generated by household microwave ovens. Ten-second irradiation of whole intact clams gave tissue fixation equal or superior to standard procedures using formaldehyde solutions and eliminated the need for that hazardous chemical. We suggest that invertebrates can be fixed while relaxed in sea-water baths, without having to remove or open the shell, and that invertebrates in bottom sediment cores also may be fixed in situ without being disturbed.     

Ultrafast microwave energy fixation for electron microscopy

We demonstrate that microwave (MW) energy can be used in conjunction with chemical cross-linking agents to fix tissue blocks rapidly for electron microscopy in as brief a time as 26 msec. The optimal ultrafast MW fixation methodology involved immersing tissue blocks up to 2 mm3 in dilute aldehyde fixative and immediately irradiating the specimens in a 7.3 kW MW oven for 26-90 msec, reaching a fixation temperature range of 32-42 degrees C. Ultrastructural preservation of samples irradiated by MW energy was comparable to that of the control samples immersed in aldehyde fixative for 2 hr at 25 degrees C. Potential applications for this new fixation technology include investigation of rapid intracellular processes (e.g., vesicular transport) and preservation of proteins that are difficult to demonstrate with routine fixation methods (e.g., antigens and enzymes).     

Microwave irradiation for fixation and immunostaining of endothelial cells in situ

Using microwave irradiation during tissue fixation and immunostaining reduces sample preparation time and facilitates penetration of fixatives and antibody solutions into the tissues. This results in improved fixation and reduction of non-specific binding of antibodies, respectively. Experimental analyses of endothelial cells in blood vessels in situ have been limited because of the difficulty of tissue preparation. We report here a technique using intermittent microwave irradiation for blood vessel fixation and immunostaining the fixed tissues. Intermittent microwave irradiation during fixation reduced blood vessel contraction and resulted in well preserved morphology of blood vessels, especially the endothelial cells. Microwave irradiation also reduced non-specific binding of fluorescein-labeled antibodies. These microwave irradiation-assisted techniques are useful for analysis of endothelial cell function and for pathological study of blood vessels in situ.     

Microwave fixation: Its potential for routine techniques, histochemistry, immunocytochemistry and electron microscopy

Summary Human tissues, both biopsy and postmortem, and tissues from rodents were fixed by microwaves at various temperatures and compared against formaldehyde-fixed material. Conventional stains, including trichromes, worked well. Red cell were lysed, but white cells were fixed, thus permitting diagnoses of various inflammatory states. Malignant cells were equally well-preserved by the two methods. Histochemical investigations of mucosubstances, lipids and various hydrolases showed no significant difference between the two techniques. Some neurological stains, however, were not as good following microwave treatment. Immunocytochemical localization of IgA, IgM and IgG showed no significant difference after microwave fixation compared to that in tissues fixed with formaldehyde. Microwave fixation did not lead to a greater tissue shrinkage than that obtained with formaldehyde fixation. Both were significantly less than that following treatment with phosphate-buffered saline alone. Electron microscopy gave results which were interpretable, but with damage resembling early postmortem change. Microwave fixation is complete in approximately 1–2 min.The mechanism of fixation appears to be due to denaturation associated with disulphide bond formation and a decrease in solubility of proteins.     

Microwave fixation of the lung

A technique for microwave fixation of inflated rat lung is described. Conventional intratracheal fixation with instillation of fixative into the airways at a constant pressure results in pressure artifacts as well as flushing and disruption of cells and exudates. Microwave fixation fixes these elements in situ without disruption and thus is valuable when evaluating the distribution of inflammatory infiltrates. Exudative pneumonitis was produced in the rat using intratracheal instillations of either endotoxin or silica and comparisons were made between histologic sections fixed using either standard formalin fixation or microwave fixation.     

Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy

Summary It was demonstrated that microwave energy used simultaneously in combination with low concentrations of glutaraldehyde (0.05%) and formaldehyde (2.0%) rapidly preserved light microscopic histology and excellent fine structural details, as well as a variety of cytoplasmic and membrane-bound antigens. Specimen blocks up to 1 cm³ can be fixed in as brief a time as 26 ms using a specially designed microwave device (ultrafast microwave fixation method). The fast microwave fixation method, using a commercially available device, was successfully used to preserve granule-bound rat mast cell chymase which was subsequently detected by a postembedding immunogold procedure. Control of the following parameters is important to the microwave fixation method: (1) specimens with one dimension less than 1 cm; (2) irradiation temperatures lower than 50°C; (3) irradiation times less than 50 s; (4) immediate replacement of the postirradiation solution with cold storage buffer; (5) fixing the specimen within 15 min after it is removed from its blood supply.     

Rapid cold fixation of tissue samples by microwave irradiation for use in electron microscopy

Summary A cold microwave irradiation procedure was developed to fix rapidly and stain various tissues and monolayers for electron microscopy. Because microwave stimulation always produces some heat, melting ice was used to maintain the temperature of the tissue samples, the fixative, and the staining solution at 0 to 4°C. The low temperature also reduced vapour formation, thus minimizing the risk of explosion. The microwave method shortened the total time of fixation and dehydration from the usual 3 h required by the conventional method to 65 min. After microwave fixation, the ultrastructural details of membranes and subcellular structures were excellent.     

Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.     

Potency of microwave irradiation during fixation for electron microscopy

Summary Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl₂ and MgCl₂ led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells.Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration.We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

Formaldehyde fixation and microwave irradiation

Summary Formaldehyde is the most commonly used fixative in pathology laboratories. However, due to time pressures, this fixative is often not optimally exploited. the majority of biopsies are only partly fixed when histoprocessing is started, with adverse effects. This paper reports how formaldehyde fixation is improved, by using 1.5 min of microwave irradiation of tissue previously soaked for four hours in the fixation solution. It is argued that this beneficial effect of microwave irradiation can be attributed to the acceleration of the reaction of formaldehyde to the tissue. Formation of free formaldehyde, by the dehydration of methylene glycol present in the tissue when the irradiation starts, is also enhanced. Five different formaldehyde-containing fixatives were evaluated, using five different working protocols. Spleen was taken as a suitable tissue for these tests. The technique described leads to uniform microscopical results. It is a simple method and is suitable for use in routine laboratories.     

Improved coating and fixation methods for scanning electron microscope autoradiography

A simple apparatus for emulsion coating is described. The apparatus is inexpensive and easily assembled in a standard glass shop. Emulsion coating for scanning electron microscope autoradiography with this apparatus consistently yields uniform layers. When used in conjunction with newly described fixation methods, this new approach produces reliable autoradiographs of undamaged specimens.     

Potency of microwave irradiation during fixation for electron microscopy

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO4 after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO4. Morphometric analysis revealed that microwave enhanced fixation led to a larger mean cell volume, to larger cell surface area, and to larger surface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimicking exocytosis.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Summary Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO₄ after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO₄. Morphometric analysis revealed that microwave enhanced fixation led to larger mean cell volume, to larger cell surface area, and to larger suface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimiking exocytosis.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.     

In vivo regional levels of PGE and thromboxane in mouse brain: Effect of decapitation,focused microwave fixation,and indomethacin

A focused microwave fixation technique was tested for use in determining basal PGE and thromboxane B₂ levels of mouse brain. Focused microwave irradiation (3.5 Kw/0.4 sec) to the head of C3H mice produced basal values of PGE and TXB₂ which were five-fold less than those in animals killed by decapitation. Indomethacin (10 mg/kg) pretreatment blocked the decapitation rise in PGE and TXB₂ levels and gave values similar to focused microwave irradiation. Indomethacin pretreatment combined with microwave fixation did not reduce PG levels more tham microwave treatment alone. When microwave fixation was used, there was no difference in regional (cerebral cortex, whole cerebellum, midbrain, hypothalamus) levels of either PGE or TXB₂. However, PGE levels were significantly higher than TXB₂ in all regions. After decapitation there was a greater increase in TXB₂ than PGE. The cerebellum produced less PGE and TXB₂ after decapitation compared to the other regions. Our results confirm the usefulness of the focused microwave irradiation technique for examining $\text{in vivo}$ basal prostaglandin levels in mouse brain.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds

Summary Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study, I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32–34 s, final temperature between 40° C and 47° C.     

Microwave-assisted staining of mucosal mast cells and granulated intra-epithelial lymphocytes after formalin fixation

Summary Rat jejunum was fixed with either formalin or methanol-formalin acetic acid (MFAA) and stained with Astra Blue or Alcian Blue with or without microwave irradiation. Staining of both mucosal mast cells and granulated intra-epithelial lymphocytes after formalin fixation was considerably improved by microwave irradiation. On the other hand, microwave irradiation slightly impaired staining of mucosal mast cells (MMC) and even more strongly granulated intra-epithelial lymphocytes (GIEL) after MFAA fixation.     

Hydroponic growth and the nondestructive assay for dinitrogen fixation

Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK_a 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.