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  NDEZ-CHAVARRI A* & M. VARGAS-MONTERO² F. HERNA *Facultad de MicrobiologõÂa, Universidad de Costa Rica, San JoseÂ, Costa Rica ²Unidad de Microscopia ElectroÂnica, Universidad de Costa Rica, San JoseÂ, 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 microscopy. 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 microwave irradiation (MWI) to cause rapid heating and movements 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, carbohydrates and protein side chains (Kok & Boon, 1992). As a consequence of increased molecular vibration, molecular Correspondence to: F. HernaÂndez-ChavarrõÂa. Fax: 11 506 207 3182; e-mail: hchavarr@cariari.ucr.ac.cr q 2001 The Royal Microscopical Society
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 icewater 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 (HernaÂndez & 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 modification 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 diversiformis, and bacterial suspensions of Acinetobacter calcoaceticum and Enterobacter cloacae. Cardiac perfusion in mice was done using modified Karnovsky fixative solution (2.5% glutaraldehyde 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
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R A P I D S TA I N I N G U S I N G M I C ROWAVE I R R A D I AT I O N
cacodylate buffer (pH 7.2). After the first fixation the specimens were washed using the buffer solution employed in the preparation of the respective fixative. They were then post-fixed in 1% osmium tetroxide (1 h at room temperature), washed with distilled water, dehydrated with ethanol and embedded in Spurr resin. After osmium fixation, the microscope specimens, bacteria and dinoflagellates were then embedded in 4% agarose. Ultrathin sections were cut with a diamond knife using an ultramicrotome (Reichert Ultracut) and placed onto 100 mesh grids coated with 0.5% collodion. The double staining (uranyl acetate, lead citrate) procedure was done in a domestic microwave oven (Daytron 2450 MHz, 700 W) set at defrosting (at a power level of 125.6 W calculated according to the formula P mgDT/t from Kok & Boon, 1992). Each grid was placed into a Beem capsule, and a maximum of six capsules, with a grid, were contrasted each time. To reduce the temperature of the ultrathin sections during microwave irradiation the six Beem capsules were placed on a plastic support and then put into a 500 mL beaker containing ice cubes and 300 mL of tap water, which covered the base of the capsules. It took approximately 5 min to equilibrate the temperature in the ice bath to 5 8C. During the contrasting procedure the temperature was also maintained between 4 and 6 8C. The temperature was measured after each irradiation period and ice cubes were added as melting occurred to maintain a constant temperature. Irradiation times of 30 s, 1 min and 2 min were evaluated for each staining and washing procedure. The first staining was done with 100 mL of 4% uranyl acetate in 50% ethanol, followed by washing with 500 mL of distilled water. The second staining was done with 100 mL of Sato's triple lead citrate solution (Sato et al., 1988), and then a final wash with 500 mL of water. Grids with ultrathin sections of each sample were also stained with the standard process, with one exception. In this instance, the first staining was done for 15 min at room temperature, followed by washing, dipping at least 30 times in distilled water, then staining for 5 min with Sato's solution of triple lead citrate and washing again as described for uranyl acetate.
Results Regardless of the irradiation time (30 s to 2 min), the temperature in the ice bath remained at 4.5 ^ 0.8 8C (n 20). The sections contrasted using MWI showed as good a contrast as grids contrasted by the standard method.
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In ultrathin sections of kidney and liver tissues stained under MWI the nuclear membrane and chromatin were well preserved and contrasted with the cytoplasm, and organelles such as nuclear membrane, mitochondria and endoplasmic reticulum were clearly observed (Figs 1 and 2). Figure 3 shows the dinoflagellate (inset shows a whole cell and the asterisk indicates the enlarged area). The cell has many dense chloroplasts, small mitochondria and a pleated amphiesma with thecal vesicles, and a large central nucleus with condensed chromosomes (arrow). In insect spermatophores arrangements of 9 1 9 peripheral pairs of microtubules with a central pair are easily observed in the transverse sections of the spermatozoids (Figs 4 and 5). Bacteria showed good preservation and the structure of the Gram negative wall is easily observed (Figs 6 and 7). In both staining processes, the standard (not shown) and that used by us, there were no precipitates of electron opaque material.
Discussion The microwave oven heats the samples, resulting in the acceleration of chemical reactions and heating, as exemplified in the staining processes described by Cavusoglu et al. (1998). We avoided such thermal increases by immersing the Beem capsules with the grids in an ice bath, as described above, maintaining a temperature close to 4.5 8C. Thus, the shortening of the staining time in the procedure described here is due mainly to the increase in molecular vibration, inducing molecular collisions leading to an increase in the probability of productive molecular collisions, not to an increase in temperature. The lead citrate solution described by Sato does not require treatment with NaOH pellets to avoid lead precipitates. This characteristic was maintained when the Sato's solution was used with MWI. Thus, very few or no electron opaque precipitates appeared on the ultrathin sections stained with this method, using standard methodology or MWI. It is possible to apply this method to thicker sections for ultrahigh voltage electron microscopy or other instruments. Where thick sections are used, it may overcome the problem generated by poor penetration of the stains into resin, which could be improved by irradiation with microwaves.
Acknowledgements The authors are grateful to Dr Jorge D. GarcõÂa, Dr George M. Smith, and to the anonymous referees for their suggestions
Fig. 1. Transmission electron micrographs of thin sections contrasted for 1 min in each stain (uranyl acetate and Sato's triple lead citrate solution) using microwave irradiation in an ice-water bath. The specimens are mouse kidney (Figs 1 and 2), equatorial section of the cingulum depression of the dinoflagellate Alexandrium monilatum (Fig. 3), which appears whole in the inset of this figure, spermatophores of the fly Archicepsis diversiformis (Figs 4 and 5), the bacteria Acinetobacter calcoaceticum (Fig. 6) and Enterobacter cloacae (Fig. 7). q 2001 The Royal Microscopical Society, Journal of Microscopy, 203, 227±230
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and critical review of the manuscript. The study was supported in part by the Vice-presidency of Research of the University of Costa Rica (Project N8430-99-215).
Karnovsky, M.H. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137A.
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