Rapid contrasting of ultrathin sections using microwave irradiation with heat dissipation

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Journal of Microscopy, Vol. 203, Pt 2, August 2001, pp. 227±230. Received 9 August 2000; accepted 13 December 2000 SHORT TECHNICAL NOTE Rapid contrasting of ultrathin sections using microwave irradiation with heat dissipation F. HERNA Â NDEZ-CHAVARRI Â A* & M. VARGAS-MONTERO² *Facultad de Microbiologõ Âa, Universidad de Costa Rica, San Jose Â, Costa Rica ²Unidad de Microscopia Electro Ânica, Universidad de Costa Rica, San Jose Â, Costa Rica Key words. Electron microscopy, microwave irradiation, staining, ultrathin section. Summary The use of microwave irradiation MWI) to accelerate fixation, dehydration and contrasting staining) for electron microscopy has been applied to the development of rapid methods to process biological samples in electron micro- scopy. A simple explanation is that the reduced time in those procedures is due to heating. In this paper we propose a contrasting method for thin sections that avoids the thermal effects of MWI. Grids with thin sections of mouse kidney, the dinoflagellate Alexandrium monilatum, spermatophores of the fly Archicepsis diversiformis, the bacteria Acinetobacter calcoaceticum and Enterobacter cloacae were placed into Beem capsules and stained with uranyl acetate and lead citrate, while immersed in an ice-water bath, and irradiated for periods ranging from 30 s to 2 min. After each contrasting procedure, the Beem capsule was filled with distilled water to wash the grids under MWI with the same irradiation time as used to contrast. Good results were obtained on irradiating for 1 min and the temperature of the Beem capsule was maintained around 5 8C. Introduction Microwaving facilitates the processing of samples for light and electron microscopy, owing to the capacity of micro- wave irradiation MWI) to cause rapid heating and move- ments of molecules Howood et al., 1990; Kok & Boon, 1992; Login & Dvorak, 1994). This comes about become MWI increases both the amplitude of vibration and the rotation of molecules in the sample Ponne & Bartels, 1995). It affects mainly water molecules, lipids, carbohy- drates and protein side chains Kok & Boon, 1992). As a consequence of increased molecular vibration, molecular interactions also become more frequent and chemical reactions are facilitated Bose et al., 1991). MWI shortens significantly the time needed for fixation, dehydration and immunolabelling Login et al., 1995). Although microwaving results in large temperature increases in the sample Ayappa et al., 1991), it is possible to dissipate excess heat by placing the sample in an ice- water bath as it is being microwaved, and still take advantage of increased molecular vibration to accelerate the chemical fixation process. This has made it possible in our laboratory to successfully process biological samples for scanning electron microscopy in less than 2 h instead of the 8±10 h required using standard methods Herna Ândez & Guillen, 2000). MWI can also be applied to staining methods for light and electron microscopy. Cavusoglu et al. 1998) described a method for contrasting thin sections on electron microscopy grids using double staining, first with uranyl acetate, and followed by lead citrate. They obtained the best results by irradiating the grids for 30±60 s when applying each reactive element, but the temperature increased to almost 40 8C and 70 8C, respectively. The following is a modifica- tion of the process using an ice-water bath to prevent heating. Materials and methods We used mouse kidney tissue, the dinoflagellate Alexandrium monilatum, spermatophores of the fly Archicepsis diversifor- mis, and bacterial suspensions of Acinetobacter calcoaceticum and Enterobacter cloacae. Cardiac perfusion in mice was done using modified Karnovsky fixative solution 2.5% glutar- aldehyde and 2% paraformaldehyde in phosphate buffer, 0.1 mm, pH 7.2) Karnovsky, 1965). Fly specimens and bacterial suspensions were fixed by immersion in the same solution. The marine dinoflagellates also were fixed in Karnovsky fixative diluted as described above, but in sodium q 2001 The Royal Microscopical Society 227 Correspondence to: F. Herna Ândez-Chavarrõ Âa. Fax: 11 506 207 3182; e-mail: [email protected]

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Rapid contrasting of ultrathin sections using microwave irradiation with heat dissipation F. HERNANDEZ CHAVARRIA AND VARGAS MONTERO

Transcript of Rapid contrasting of ultrathin sections using microwave irradiation with heat dissipation

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