246 THE BIOLOGY OF HYDRA : 1961 



an enzymatic digest of lactalbumin, in 90 /f artificial sea water (3) 

 containing 500 units of penicillin and 0.5 mg. streptomycin per ml. 

 Growth can be obtained over a range of Edamine concentrations 

 from 0.4 to 1.5% and artificial sea water concentrations of 40% to 

 100%. Yeasts and molds which are not inhibited by the antibiotic 

 mixture have at times presented difficulties. Mycostatin has been 

 used at a cencentration of 50 units/ml. to free cultures of these 

 contaminants. This antibiotic has not been included routinely be- 

 cause it appears to be somewhat toxic to the anemone cells. 



The cell suspension used for the initial isolations is prepared 

 by mincing the animal in a beaker with a pair of scissors. In 

 some cases, lysozyme ( 1.5 mg./ml. of animal) was added to degrade 

 the mucus that is secreted. Approximately five volumes of arti- 

 ficial sea water is added per volume of minced tissue, and the 

 mixture is stirred briefly. It is allowed to stand in an ice bath for 

 approximately five minutes and filtered through two layers of 

 cheese cloth. The filtered suspension is freed of large tissue frag- 

 ments by centrifugation at 5 for 30 seconds at approximately 1000 

 g. The cell suspension containing very few tissue fragments is then 

 centrifuged as above for 10 minutes. The cells are resuspended in 

 sterile artificial sea water and the differential centrifugation is 

 repeated. The cells are finally washed three more times with 

 sterile artificial sea water. The last washing employs artificial sea 

 water to which has been added antibiotics at the above-mentioned 

 concentration. The cell suspension is diluted to a concentration 

 of close to 3 X 10' cells/ml. This corresponds to 0.100 O.D. at 

 660 m/x in the Beckman Spectrophotometer Model DU and is about 

 equal to 100 [ig. of cell protein/ml. or 5 X 10~^ ml. of packed 

 cells/ml. One-tenth ml. of such a suspension is used as the culture 

 inoculum. Figure 1 shows the appearance of such a suspension. 

 There is great heterogeneity of cell type, and the outstanding con- 

 taminant appears to be fragments of fibrous material from the 

 mesoglea. The two comet-shaped objects in the center of Figure 1 

 are this material. The cells show a size range from 8—2 /x. 



The inoculum is placed in either a 60 mm Petri dish or into 

 a test tube of 15 X 130 mm. containing a piece of coverslip 10 X 20 

 mm. Five ml. of nutrient solution is added and mixed with the 

 inoculum. The test tubes are slanted to allow the cells to settle 



