Efectos del secado en punto crítico y secado en terbutanol en el tamaño de levaduras y eritrocitos humanos

Abstract

One of the most frequently repotted problems when using the scanning electron microscope (SEM) to observe biological samples, is the decrease of the specimen size, a problem which has been associated with the chosen method of drying. In the case of critical drying point (CDP), this reduction is calculated at between 20% and 80% but has still not been calculated for the terbutyl alcohol freezedrying method (TFD). The size of Candida albicans and Phaeonnelornyces werneckii yeasts and processed hurnan erythrocytes were studied by SEM using CDP, TFD and phase-contrast. The cells were washed three times with a phosphate buffer and fixed with 2.5% glutaraldehyde in the same buffer at 4°C for 2 hours. They were processed in duplicate over polycationic membranes of poly-L-lysine, dehydrated and dried using CDP and TFD. They were covered with 200 A of gold and analyzed in the SEM. The real size was obtained in the phase-contrast microscope. The results showed that in spite of the morphological variations presented in the studied cells, there was a shrinking of approximately 34.4% and 21.3% in the size of the eryhrocytes processed by CDP and TFD respectively. The variations of specimen size during the SEM process are discussed.