TISSUE CULTURE TECHNIQUES 233 



Cell cultures may be maintained on slides which are generally sealed off from the air. 

 Such cultures must be sub-cultured at frequent intervals to supply oxygen and to replen- 

 ish the nutrients. Fresh explants may be maintained in Carrel flasks which are so con- 

 structed as to minimize the probability of contamination, and still allow the transfer of 

 tissues. Test-tube cultures are a modification of the Carrel method, but somewhat more 

 easily handled. In such test tubes coverslips bearing the culture may be protected and, 

 to some extent, observed without contamination. Aggregates of cells or even organs may 

 be cultured, on either solid or fluid media, and generally in depression slides where 

 they may be continually observed if necessary. Possibly the least fruitful (thus far) and 

 yet the most promising material for the future may be the ova and very early embryos of 

 mammals. 



This very brief summary of the major ideas in tissue culture work is meant only to be 

 suggestive to those qualified and experienced students who show aptitude in this direction. 

 The following reference list includes the most recent paper or book by each of the major 

 research workers in the field today. The student is referred to any of the multitudinous 

 sources for detailed information. 



While tissue culture methods are extremely varied with respect to specific tissues and 

 aims, a single procedure will be described here which allows for the fast, undifferen- 

 tiated growth of small fragments of animal tissues, known as explants, placed upon a 

 glass substrate with or without the plasma clot. This procedure was devised by Dr. 

 Jewel Plummer Cobb and Dorothy Walker (Jour. Nat. Cancer Inst. 1958, vol. 21:263- 

 278). 



MATERIALS: 



Instrunients and glassware : 



Porter flasks, approximately 1 3/8 inches in diameter (Karl Schuman, Cliffside, 

 N.J. 681 Jefferson Ave. ) 



Coverglasses, No. 0, size 1 1 x 22 mm. 



Silicone stoppers to fit Porter flasks, sterilized in dry heat oven. (The West 

 Co. , Phoenixville, Pa. ) 



Caratact knives or Bard-Parker blades attached to handles 



Fine curved forceps 



Cornman roller knife (Aloe Scientific Co. , 5655 Kingsbury St. , St. Louis, Mo. ) 



Graduated pyrex pipettes (6) cap. 1. 5 ml. (Bellco Glass Inc. , Vineland, N. J. ) 

 plugged with cotton and attached to 2 ml. cap. amber rubber bulbs (A. A. 

 Henning Co. , 16 E. 23rd. , N. Y. C. ). 



Glass depression slides (6) 



Pyrex petri dishes (6) measuring 150 mm diam. , for instruments. 



Lipless pyrex test tubes (6) measuring 25 x 100 mm (A. H. Thomas Co. , Phil- 

 adelphia, Penn. ) 



Metal test tube rack for slanted large test tubes (Arista Surgical Co. , 67 Lex- 

 ington Ave. , Bethesda, Md. ) 



Touch-o-matic Bunsen burner for flaming under hood (Microbiological Asso- 

 ciates, 4846 Bethesda Ave. , Bethesda 14, Md. ) 



Biological rnaterials : There are several chemically defined nutrient media which, 

 when supplemented with 10% horse or human serum, provide an optimum fluid 

 environment for growth. A few examples of good media follows: 



1. Tissue culture medium 1066, available from Connaught Medical Research 

 Laboratories, Univ. Toronto, Toronto, Canada 



2. Raymond and Parker's Mixture 199, available from Difco Co. , Detroit, 

 Mich, or Microbiological Associates, 4846 Bethesda Ave. , Bethesda, Md. 



3. Eagle's Basal Medium (E. B. M. ) available from Microbiological Associates. 



