TISSUE CULTURE REAGENTS 307 



METHODS OF TISSUE CULTURE 



Brief outlines for the use of Difco tissue culture media reagents by the common 

 methods of tissue culture are given below. These directions apply generally for 

 any type of animal tissue; however, special investigation may be required to 

 determine the optimal conditions for culturing a given tissue. Proportions of the 

 various reagents given in the discussion may be varied to meet individual re- 

 quirements. 



Slide Culture 



The sterile tissue to be cultured should be placed on a clean sterile glass 

 surface and cut into appropriate sized explants while immersed in balanced salt 

 solution i^pH 7.4). The following sizes are usually suitable for the specified 

 tissue : 



minced tissues 0.5—1,0 mm. 



embryonic tissues 1.0 mm. 



adult tissues 1.5 mm. 



adult skin 2.0 mm. 



Further rinsing of the explants in balanced salt solution may be done if desired. 

 Add one drop of TC Chicken Plasma to a cover-glass, followed by one drop of 

 TC Embryo Extract EEgo- Larger amounts may be used in the same ratio if 

 desired. Mix the two with a spatula and spread over an area having a diameter 

 of 1 cm. Add two or three explants and separate them by about equal distances. 

 Cover with a well slide and set aside to clot. Later, seal the coverglass to the well 

 slide with melted paraffin or a mixture of 5 parts paraffin and 1 part yellow 

 vaseline. 



More diffuse and extensive cell migration may be encouraged by diluting the 

 plasma before using with an equal or double volume of TC Balanced Salt 

 Solution. Incubate the slide culture at 37°C., observe and continue culture in 

 accordance with standard procedures. 



Roller Tube Culture 



Prepare explants as for slide cultures and place them in a small volume of 

 nutrient solution in a well-slide or petri dish. The nutrient may consist of various 

 proportions of TC Balanced Salt Solution, TC Horse Serum, or TC Cord Serum, 

 Human and TC Embryo Extract EE20 (5 parts of Balanced Salt Solution, 3 parts 

 serum and 2 parts embryo extract is satisfactory for general purposes). Place 2 

 drops of whole TC Chicken Plasma (50 per cent plasma is also satisfactory) in 

 a 16x150 mm. tube and rotate until bottom third or half of tube is coated. With 

 a pipette place 4 or 5 explants on the uncoated area near the top of the tube and 

 remove excess nutrient from around explants with a Pasteur pipette. Then with 

 pipette transfer individual explants to plasma coated surface. After about 10 

 minutes, stopper tubes and place them in the roller drum in the 37°C. incubator 

 for 2 hours. The nutrient medium (mentioned above) should now be added in 

 0.5 ml. volumes to each tube and the tubes returned to roller drum in the 

 incubator. Incubate at 37°C., observe and continue culture in the usual manner. 



Flask Culture 



Prepare explants in TC Balanced Salt Solution. Explants may be made con- 

 siderably larger for culture in flasks than for slide or roller tube cultures. While 



