114 



V. p. GILEV 



The plate of gelatin with the objects included in it 

 is separated from the oil-cloth and cut for rectangu- 

 lar blocks. The blocks are glued up with 40 "o solu- 

 tion of gelatin to the old methacrylic blocks sharp- 

 ened in the form of a truncated pyramid and placed 

 for several hours into a thermostat (37'C) for com- 

 plete drying. 



Immediately before cutting in a microtome the 

 blocks are sharpened in such a way that the square 

 of the section will not exceed 0.2 mm-. The blocks 

 are kept in cans with tight caps. 



Cutting is effected with a glass knife (without 

 fluid). In our work we used a microtome of Danon 

 and Kcllenberger. The long side of the block should 

 be parallel to the sharp edge of the knife. The blocks 

 just dried are cut worse when the weather is dry; 

 in this case it is better to cut them not earlier than 

 in 24 hours after they were exposed to the air. In 

 the case of considerable humidity of the air and 

 blocks, it is recommended to dry them some time 

 before cutting. Position of the knife in cutting gelatin 

 blocks is usually, to some extent, steeper than in 

 cutting methacrylic blocks. The rate of cutting is 

 approximately I section per I sec. It is not difficult 

 to obtain sections 0.03-0.04 /< thick. Usually the 

 sections are formed in the shape of endurable un- 

 disintegrating ribbons. These ribbons are sorted out 

 and individual sections or groups of sections are trans- 

 ferred with the aid of a thin filament into drops of 

 water (or better of 2 % solution of acetic acid) lying 

 on a I.e. Parlodion film that floats on the surface of 

 water (temperature of about 37-40 C) filling a Koch 

 cup. But better results are obtained when the sec- 

 tions are placed into water of room temperature, 

 which is subsequently heated up to 37-40 C. 



Expanding, the sections in most cases lose their 

 bonds with each other and, therefore, not always a 

 series of sections may be obtained. Then the film 

 under each drop is punctured with a thin needle and 

 the sections sink to the undamaged part of the film. 

 The copper grids are placed on the film in such a 

 way that the centre of the grid is above the section. 

 After that from above a microscope slide is placed. 

 Having put it upside down, we withdraw the film 

 with grids.* After drying the specimens are ready for 

 examination (fig. I ). 



For more complete removal of gelatin from the 

 sections the ready grids with the specimens are 

 placed for 2-4 hours into warm water (37-40 C) or into 

 a weak solution of acetic acid. 



The osmium tetroxide that remains in the object 

 after fixation and washing (especially when it is done 

 for a short period of time) interacts with gelatin, 

 after which the latter becomes less transparent, 

 hardly removable from the section and creates a 

 rather strong background that makes some fine de- 





a 



** 



T^ 



, ' -.»'■ 



Fig. 1. Striated muscle tissue of the axo\otl( A mblystoma 

 pitnctotttm). The section has been treated with 4% acetic 

 acid. 



tails of the structure of the tissues less visible. There- 

 fore, it is recommended that before enclosing the 

 objects in gelatin, fixation should be shorter and 

 washing longer. It can be supposed, however, that the 

 presence of the background obliterating the bound- 

 aries of some structures, is connected with the 

 presence of substances which are otherwise elimi- 

 nated or precipitated, while the specimen is treated 

 with alcohols and methacrylates. 



In the case of thinner sections it is recom- 

 mended to examine them without removal of em- 

 bedding medium (gelatin). In this case the sections 

 should be expanded in cold water (about - I to 



^ rc). 



The tissues, embedded in gelatin, can be easily 

 cut with a glass knife for sections 1 fi thick for exa- 

 mination in the light microscope. In this case the 

 blocks should not be too dry and, therefore, are kept 

 in open vessels. The sections are glued up to micro- 

 scope slides with albumen, to which some glycerine 

 has been added. The gelatin is dissolved in warm 

 water. 



References 



1. Brandes, C. H., Mikrokosmos 44, N7, 167 (1955). 



2. FiRMiNGER, H. I., Stain Techno/. 25, N 3, 121 (1950). 



3. Newman, S. B., Borysko, E., and Swerdlow, M., /. 



Research Nat. Bur. Standards 43, 183 (1949). 



4. Palade, G. E., J. E.xptl. Med. 95, 285 (1952). 



5. Richards, A. G., Anderson, T. F., and Hance, R. T., 



Proc. Soc. E.xptl. Biol. Med. 51, N 1, 148 (1942). 



^ This method ofmounting sections was elaborated together 

 with V. I. Birjuzova, one of the workers of our laboratory. 



