TISSUE CULTURE TECHNIQUES 235 



4. Rooster plasma obtained frozen in 1 5 ml vials from Microbiological Asso- 

 ciates. 



5. Chick embryo extract. 50% obtained from 9-11 day old chick embryos 

 minced in suitable balanced salt solution. 



(Also have available penicillin-streptomycin mixture to be used as 50-100 units 

 per ml of culture medium as standard prophyllactic) 



All glassware and instruments are to be wrapped in aluminum foil and sterilized at 

 150°C in dry heat oven for one hour. All synthetic media are to be sterilized by 

 filtration through ultrafine porosity fitted glass filter. Plasma and embryo extracts 

 are obtained from the animal source under sterile conditions. 



METHOD: 



Mouse heart fragments may be obtained from various strains in utero. One mm frag- 

 ments are embedded in a soft coagulum consisting of fowl plasma and chick embryo ex- 

 tract on double cover slips on Maximow slides. One drop of nutrient medium (consisting 

 of 90% Eagle's basal medium (EBM) and 10% nondialyzed human or horse serum) is added 

 to each coverslip. This medium is withdrawn and new medium added twice weekly. 



Human neoplastic tissue may be cultured on coverslips inserted into Porter flasks, and 

 may be obtained sterile from the operating room. One ml of EBM nutrient medium is 

 supplemented with 10% nondialyzed serum in each flask. Pleural or ascitic-effusion 

 samples may be centrifuged, supernatant removed, and cells placed in 0. 2 ml aliquots 

 in several Gey roller-tubes or Porter flasks, without plasma. The nutrient medium for 

 these cells is better made of 50% EBM and 50% autologous clear pleural or ascitic fluid. 

 The pH is adjusted to 7. 2 and medium changed twice weekly. 



RESULTS: 



The growth rate is defined as the average distance cells migrated from the explant and/or 

 the average density of the cell population at 72 hour intervals. The extent of outgrowth 

 is defined as the total distance the cells emigrated, expressed in microns. The cell pop- 

 ulation density may be determined by inserting a Howard grid in the eyepiece and esti- 

 mating the number of cells in two representative fields. Cells may be stained with May- 

 Grunwald-Giemsa for best differentiation*. 



Tissue cultures are studied for long-term survival, for growth rate and type, and for the 

 effects of added drugs which might accelerate or decelerate growth. Obviously parallel 

 controls are necessary although they are difficult in that identical conditions for simul- 

 taneous cultures may not be obtained. Since Carrel was able to maintain chick heart 

 cells for many times the life of the species itself, the range of biological possibilities in- 

 this field is limitless. 



REFERENCES FOR TISSUE CULTURE TECHNIQUES 



It is recognized that tissue culture techniques are so specialized and require a back- 

 ground in biochemistry, cytology, physiology and related subjects, that it would be im- 

 possible to provide any student with adequate direction for success within the limits of a 

 single volume, let alone a chapter. However, organ culture in poikilothermic forms is 

 not difficult and those who are successful may wish to continue in the direction of tissue 

 culture experimentation. To aid in such interest there is listed here some of the best 

 current titles in the field, concentrating on techniques, which the ambitious investigator 

 may wish to study. 



* See Hanks et ol, "A Introduction to Cell and Tissue Culture", p. 120, app. IV. (See also sections #26 and #28) 



