TISSUE CULTURE 355 



passages of a culture Avhen oil immersion observations arc not 

 required. Plasma cultures are more easily cut free of old medium 

 on a mica surface. Afterwards the final sub-culturing can be made 

 on glass coverslips for permanent stained preparations. 



788. Flask Cultures. As many as six small explants can be 

 grown in a single flask, and the larger volume of medium and the 

 ease with which the whole preparation can be washed and the 

 medium renewed, makes this technique suitable for histiotypic 

 growth over long periods of time. The simple pattern of Carrel 

 flask consists of a circular vessel with horizontal upper and lower 

 surfaces, 3-5 to 5 cm. in diameter, and 1-25 cm. in height. A single 

 tubular arm, 3 cm. long and 1 cm. in diameter leads into the flask 

 at an inclined angle. This is fitted with a rubber cap made from 

 a teat or with a special rubber stopper. A second arm placed 

 opposite to the first allows greater freedom in manipulating the 

 explants. 



For the 3-5 cm. flask the following media are recommended by 

 Fischer. Introduce 2-5 c.c. of plasma, 5 c.c. Tyrode solution and 

 1 drop of concentrated embryonic extract (from the first centri- 

 fuging) into the flask. Take up the explants in a pipette and 

 arrange them at equal distances in the medium before it clots. 

 Carrel prefers less solid medium and proceeds as follows : 0-5 c.c. 

 plasma and 1-5 c.c. of 5 per cent, embryonic extract in Tyrode 

 solution are placed in the flask with the explants. After one hour 

 in the incubator, 1 c.c. of extract is added. For fibroblasts and 

 epithelia he recommends 0-75 c.c. Tyrode solution containing 

 0-25 c.c. embryo extract. For leucocytes, 1 c.c. of equal volumes 

 of Tyrode and serum. Cameron (" Tissue Culture Technique," 

 Farrar and Rinchart, New York, 1935) recommends the following 

 procedure for mammalian flask cultures if homologous plasma and 

 embryonic extract are unobtainable. Imbed the explants in a 

 naedium of chicken plasma, chick embryonic extract and Tyrode 

 solution as described above. After clotting, fill the flask with 

 Tyrode and incubate it for one hour. Remove the Tyrode and 

 repeat the process of washing for one hour. Most of the foreign 

 proteins should be eliminated by this washing. Homologous 

 serum, Tyrode solution and embryonic extract (chick) can then 

 be added for the final medium. 



Washing the Flask Cultures and Renewing the Medium. This is 

 done without disturbing the explants unless they have grown to 

 inconvenient sizes. Using a length of fine platinum tubing which 

 is repeatedly sterilised by heating in the Bunsen, and is attached 

 by rubber tubing to an exhaust pump, the surplus medium is 

 sucked from the flask and excess Tyrode solution added. After 

 half an hour in the incubator the Tyrode solution is withdrawn 

 with the suction apparatus and fresh medium, such as Tyrode 

 and embryonic extract or scrum, is pipetted into the flask. 



12-8 



