The Use of Gelatin for Eniheddini: Bioloi^ical Objects 



13 



Fig. 2. Ultra-thin section of avian tubercle bacillus embedded 

 in Araldite. The organism appears smooth and there is a 

 fine cytoplasmic membrane underlying the cell wall. 60,000. 



Fig. 3. Ultra-thin section of avian tubercle bacillus embedded 

 in Araldite. A spore is developing in the centre of the bacillus 

 and is enclosed in a fine double membrane. 80,000. 



The so-called vacuole is usually filled with a fine 

 network and the central dense structure has a far 

 more luiiform appearance. Instead of the complex 

 mass of threads and granules that are observed with 

 methacrylate we find a smooth thread-like structure 

 with associated dense granules. 



These results with Araldite are only preliminary 

 but seem to us to be promising. Some similar tests 

 have been made with mammalian tissues and there 

 are indications that Araldite will be useful in the 

 preparation of hard tissues, such as adult hairs. 



We would like to thank Dr. R. II. Cilaucrt. of Aero 

 Research Ltd., for his cooperation throughout the course 

 of this work. Some of the electron micrographs were 

 taken in the Cavendish Laboratory, Cambridge, and we 

 would like to thank Dr. V. E. Cosslett and Mr. R. W. 

 Horne for providing electron microscope facilities. 



References 



1. Brifger, E. M., Cossiftt, V. E., and Glaltrt, A. M., 



J. Gen. Microbiol. 10, 294 (1954). 



2. Brifger, E. M. and Glauert, A. M., /. Gen. Microbiol. 7, 



287 (1952). 



3. — Naliire, 178, 544 (1956). 



4. Chapman, G. B., y. Bacteriol. 71, 348 (1956). 



5. Maaloe, O. and BiRf h-Andi rsfn. A., Vlth Symp. Soc. 



Gen. Microbiol. "Bacterial Anatomy", p. 261. (1956). 



The Use of Gelatin for Embedding Biological Objects in Preparation of 

 Ultrathin Sections for Electron Microscopy 



V. P. GiLEV 



Lab. of Electron Microscopy, Acad, of Sciences of t lie USSR, Moscow 



1 HE majority of investigators working in the field 

 of electron microscopic cytology and histology use 

 the method of embedding biological objects in 

 methacrylates (3). 



It was proposed to embed biological objects in 

 polyethylenglycols of high molecular weight which 

 are soluble in water, (1,2, 5). But due to the fact that 

 preparation of ultrathin sections of the objects em- 

 bedded in these substances was difficult, this method 

 did not become wide-spread in electron microscopy. 



The method we oflFer is based on the use of gelatin 

 as embedding medium. It makes it possible to com- 

 pletely exclude treatment of objects with organic 

 solvents and to obtain sections up to 0.03-0.04 // 

 thick and, perhaps, thinner. 



A 10 "o water solution of food gelatin is boiled for 

 several minutes together with beaten-up hen egg al- 

 bumen and activated charcoal. Per 300 cm-' of gelatin 

 solution we take the albumen of one hen egg and 

 3 g of activated charcoal. Then the solution, while 

 it is warm, is filtered first through paper and then 

 through an asbestos bacterial filter. One portion 

 of solution is evaporated in a thermostat at a 



8 — 568204 Electron Microscopy 



temperature of 45 C until there remains half of its 

 content, and the other one until there remain three 

 thirds of the initial volume; in this way we obtain 

 20 "„ and 40 "„ solutions which arc used for impreg- 

 nation and embedding. 



We may prepare 20 ",, and 40 °o solutions of 

 refined gelatin in Ringer solution. 



The solutions should be absolutely transparent. 

 To prevent from rotting some thymol is added. 



The objects (striated muscle tissue of axolotl, 

 Atnblystoina punctatum) were fixed by osmium tetr- 

 oxide (4) at pH 7.4-7.5 during 20 hours at a 

 temperature of TC. After being washed in pipe- 

 line water or in Ringer solution (1-4 hours), the 

 objects were placed for 4 hours into 20 "o solution 

 of gelatin heated up to the temperature of 37°C 

 and then for 15 hours into a 40 "„ solution. During 

 this period of time the pieces of muscle tissue to the 

 size of 1 0.3 0.3 mm are well impregnated with 

 gelatin. Then the 40 "o solution of gelatin with the 

 objects enclosed in it is poured out on oil cloth and 

 dried slowly at the temperature of 37 C to such 

 a state in which gelatin is not brittle. 



