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Ruska 1932) called for intense research for new methods of preparation. 

 As far as biology is concerned satisfactory results were not achieved until 

 ultra-thin sections could be made by routine methods — almost 20 years 

 after the invention of the electron microscope. Several new ways of prepara- 

 tion might be convenient in ordinary light microscopy but ultra-thin 

 sectioning will, no doubt, eventually be the most useful one. 



In response to a request of visitors to the Palynological Laboratory, 

 Stockholm-Bromma, some of the problems met with when using the ultra- 

 thin section technique will be discussed in the following. 



Methods oj preparation 



For studies of ultra-thin sections of non-acetolyzed pollen grains and 

 spores it is, i.a., advisable to consider the question of fixation carefully. For 

 light microscopical work fixation by means of formalin, or freezing and 

 drying is often sufficient. Fixation with osmic acid is, however, to be 

 preferred; in fact, in electron microscopical studies it is, so far, the only 

 reliable fixative. Osmic acid also acts as an electron stain. In acetolyzed 

 exines the staining effect of the osmic acid is negligible and probably of no 

 use. Experiments ought to be made to discover, if possible, other heavy 

 metals or ions suitable as electron stains. In investigations of mammalian 

 tissues a buffered solution of osmium tetroxide is usually used (acetate- 

 veronal buffer at pH 7.2; cf. Rhodin 1954). The same fixative has been used 

 by Turian (1956) and AfzeUus (1955). In the opinion of P. Sitte (personal 

 communication, November 1956), buffering of the solution is not partic- 

 ularly important when fixing plant cells which have not formed a large 

 vacuole, since the buffer might penetrate the cell more rapidly than the 

 osmic acid and thus cause a poisoning of the cell before fixation. Sitte, 

 moreover, emphasized the importance of using an isotonic solution of 1 

 per cent osmium tetroxide by adding sugar or urethan. The slow penetration 

 of osmic acid presents a problem familiar to all who have tried this type of 

 fixation. In pollen grains the fixative probably penetrates into the grains 

 more readily through the aperture membranes than through the wall proper. 

 (N.B. In order to obtain good fixations to meet the high demands of electron 

 microscopy it might be advisable to cut large pollen grains or spores before 

 fixing). Wettstein has obtained very good results by using osmium vapour 

 (cf. PI. IV, V). 



The necessity of using suitable embedding media in the study of thin 

 sections by means of phase contrast has recently been stressed. Ordinary 

 histological techniques have, as a rule, been adjusted to the use of much 

 thicker sections, for which glycerol jelly is often a good embedding medium. 

 Thin sections (about 500-5000 A thick) should preferably be studied in 



