Problems of Osmium Fixation 



G. F. Bahr, G. Bloom and U. Friberg 



Institute for Cell Research and Genetics, and Department of Histology, 

 Karolinska Institutet, Stockholm 



During fixation and subsequent treatment in va- 

 rious fluids great changes in volume of tissue speci- 

 mens may occur. To our knowledge no measure- 

 ments have been reported on the changes of volume 

 during osmium fixation and following embedding 

 according to the general technique applied for elec- 

 tron microscopical specimens. We have therefore 

 considered it justified to present the results of quan- 

 titative measurements on the changes in volume, 

 weight, and specific weight during this procedure. 



The experimental method was based on the prin- 

 ciple of Archimedes. For the weighing arrangement 

 an analytical balance with a sensitivity of 0.1 mg 

 was used. Mostly small pieces of guinea pig"s liver 

 weighing 150-200 mg served as test objects. Other 

 tissues were used for the sake of comparison. A 

 measuring group was comprised of at least 5 speci- 

 mens. Fixation, dehydration, and methacrylate infil- 

 tration were carried out at or 24 C, whereas pa- 

 raffin infiltration was performed at 58'"C. 



Fig. I shows the changes which occur during fixa- 

 tion in 1 % osmium tetroxide in isotonic Tyrode's 

 solution at pH 7.2 over a period of up to 24 hours. 

 A marked and rapid swelling is seen which reaches 

 a maximum of 30 % after 4 hours. Half of the final 

 value is reached after only 15 minutes in the fixative. 

 The weight curve follows closely the volume curve 

 and therefore only the volume curves will be referred 

 to. The specific weight rises during fixation as an 

 expression of the binding of osmium in the tissues. 



Changes during the continued preparation are 



disclosed in fig. 1. The swelling that takes place in 

 the fixative is now transformed — during dehydra- 

 tion — into a shrinkage that almost brings the volume 

 back to the original value. During paraffin infiltra- 

 tion further shrinkage is noted, especially in speci- 

 mens that were fixed at C. Methacrylate, on the 

 other hand, causes only a small shrinkage during 

 infiltration, and can in this respect be regarded as a 

 more favourable medium. During polymerization, 

 however, methacrylate reduces its volume by 20 % — 

 just as does paraffin during solidification — and it 

 seems that this shrinkage is more or less completely 

 transferred to the specimens, as far as we can judge 

 from planimetrical observations on embedded liver 

 pieces. Fig. I also shows that for methacrylate 

 treated specimens the fixation temperature is of 

 httle influence on volume. 



The specific weight that rises during the fixation 

 falls during dehydration and finally rises again during 

 infiltration. 



In another experiment a comparison was made 

 between various tissues (fig. 2). Certain differences 

 in intermediate and final values are to be noted. 

 The principal pattern is, however, the same for all 

 tissues. 



The swelling seen during osmium fixation, is in 

 no way unique for this fixative. In a series of 

 formaldehyde concentrations, ranging from 0.5 to 

 16 % there is a swelling in all solutions, the magni- 

 tude of which is related in inverse proportion to the 

 concentration of the fixative. Maximal swelling is 



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Fig. 1. Volume changes of pieces of tissues during osmium 

 fixation and embedding. 



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Fig. 2. Volume changes of pieces of various tissues during 

 osmium fixation and embedding. 



