Lipids and Osmium Fixation 



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Fig. 3. Volume changes and variations in specific weight of 

 pieces of tissues during formol fixation and embedding. 



120 



o 100 

 > 



80 



1% OSMIUM TET/IOXIDC IN TYRODE 

 WITH ADDITION OF DEXTSAN (Ox) 

 (AND SUCROSE) 



0% 

 3% 



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 Dx 



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12 % Dx 



3 % Dx + _ 

 0.2 M sucRoa 



3 % Dx ♦ 



OSM SUCROSE 



6 % Dx ♦ 



0^5M SUCROSE 



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Fig. 4. Balancing of the volume changes of pieces of tissues 

 during osmium fixation when adding dextran and sucrose to 

 the osmium tetroxide solution. 



reached in 3 hours. Thereafter a shrinkage can be 

 observed, the speed of which is not related to the 

 concentration. 



If formalin fixed specimens are followed further in 

 the preparation procedures it is seen (fig. 3) that 

 they react in a manner very similar to that described 

 for osmium fixed material. Fixation in 4 "o formal- 

 dehyde gives an initial swelling of 18 % after 12 

 hours. Dehydration and paraffin infiltration reduce 

 the volume with 30 "o each. In methacrylate again, 

 the shrinkage is small. The changes in specific weight 

 are unimportant. 



The results obtained indicate that the swelling in 

 the fixatives is due not only to the fixing agents 

 themselves but in a high degree to the aqueous media 

 in which they are dissolved. Liver pieces were 

 suspended in Tyrode's solution of various concentra- 

 tions at C. A swelling is noted in all, even those 

 strongly hypertonic. As expected the swelling is 

 most pronounced in the diluted solutions and in 

 these, maximal swelling is also reached in shorter 

 time. 



Similar results are obtained with Krebs-Ringer 

 media and also with sucrose solutions. 



The addition of small amounts of gelatin to 

 physiological fluids has been recommended in order 

 to reduce their toxical effects on cells. The addition 

 of 0.25 °(, gelatin to Tyrode's solution reduces the 

 swelling significantly with about 20%. 



Fig. 4 shows that a still more pronounced effect 

 is noted with dextran. It is possible to use dextran 

 in much higher concentrations than is possible with 

 gelatin without increasing the viscosity to an im- 

 practical point. It is also seen that by combining 

 dextran with sucrose it is possible to completely 

 prevent the swelling. 



The next question of great interest is if it is possible 

 to reduce the shrinkage during dehydration to some 

 extent. Dehydration in rising concentrations of 

 ethanol brings about a more gradual shrinkage than 

 absolute ethanol, but the end result is but little 

 better. 



In the literature various substitutes for ethanol 

 as dehydrating agent have been proposed. From 

 our results it is obvious that only methanol appears 

 to give better results than those obtained with 

 ethanol. 



The Quantitative Assay of Lipids Extracted from Untreated and 



Os04-fixed Beef Brain 



G. F. Bahr 



Institute for Cell Research and Genetics, Karolinska Jnstitutet, Stockholm 



Although we know today where osmium cannot 

 be expected to be found in a thin section, we really 

 know little about the actual places of osmium reac- 

 tion and deposition (1, 2). There has been much dis- 

 cussion about to what extent osmium is bound by 

 the different constituents of a tissue. Now the main 



interest is focussed on the question whether the 

 lipids or the proteins are demonstrated by osmium 

 deposits. 



Our recent analyses show that osmium fixation 

 renders a fraction of about 2 "„ of the total dry weight 

 unextractable in the \ovm of lipids (fig. I). Skin 



