How to Prepare Ullralhin Sections of Tissue Cullures 



V. Dost A L 



Bi'hiins^-Werke, Marhiiii; a. d. La/in. and the Department of Hyj^'icne, Alhert-Liidwii^s-Univer.sitdt, Freihioi,' i. Br. 



Owing to improvements in method, tissue cultures 

 have in recent years become of growing importance 

 for the cuUure of viruses, for the quantitative deter- 

 mination of infectiosity, for carrying out neutrah/a- 

 tion tests, and for the morphological study of multi- 

 plication processes. In addition, they are being used 

 for diagnoses and for the production of vaccines. 



In recent years I have worked on the culture of 

 viruses, especially the poliomyelitis virus and the 

 vaccine virus. The basic materials used were monkey 

 and calf kidneys which I prepared for the examina- 

 tion in the electron microscope. The tissues treated 

 (1,5) in culture bottles grow into a cell outgrowth 

 predominantly in monolayers at the bottom of the 

 container within a few days. 



Various methods as to how to produce ultrathin 

 sections are already known. D. C. Stuart (4) specified 

 a method in which the embedding, i.e. the poly- 



merization, takes place directly at the cell attached 

 to the tube. C. G. Harford, A. Hamlin, and E. Parker 

 (2) have the tissue grow on a formvar foil, after- 

 wards embedding the latter. I chose to make a sedi- 

 ment of tissue cultures which I obtained from full- 

 grown, normal, and infected cultures. 



For preparing the sediment I used such tissues 

 as are normally developed in producing poliomyelitis 

 vaccine. The tissues were partly not infected, partly 

 they displayed a certain state of virus multiplication 

 within the cell. The tissue to be examined by electron 

 microsocpy is processed up to its being embedded 

 in the culture bottle, it is fixed with a solution of I "o 

 phosphate-butTered osmium tetroxide (according to 

 Sjostrand). In order to remove cell detritus, the cell 

 surfaces were previously rinsed with Hanks" solu- 

 tion. The cells were dehydrated with alcohol. The 

 change to methacrylate mixture was via various 



Fig. 1. An ultrathin section of a monkey kidney tissue culture wiiich had been infected with vaccine-virus. The tissue 

 was fixed 48 hrs. after infection. In the photograph the nuclei of two cells are to be seen which distinctly show the 

 double membrane. Furthermore, the cell border is easily recognizable as a double membrane. In the protoplasma 

 various mitochondria with their septa. All over the hyaloplasma there were lots of small, rotund forms; besides, 

 there were some hyaline osmiophilic areas. Siemens Electron microscope UM 100 c, Magnification -28,000. 



