34 GERLOFF, FITZGERALD, AND SKOOG 



culture media. Four cultures were prepared for each treatment, 

 and, after autoclaving, were inoculated with one milliliter of an 

 actively growing suspension of bacteria-free Coccochloris Penio- 

 cystis. In all operations, precautions were taken to exclude bacteria 

 from the flasks. The cultures were illuminated continuously with 

 fluorescent light of approximately 50 foot candles intensity and 

 kept at approximately 25° C. 



The determination of the amount of growth in cultures of 

 Coccochloris Peniocystis proved to be a difficult problem. The 

 cells are so small that they readily pass through coarse filters, yet, 

 probably because of their gelatinous sheaths, clog fine filters al- 

 most immediately. Colorimetric and turbidimetric procedures 

 were rejected because of errors introduced by variations in the 

 color and degree of flocculation of the algal growths with modifi- 

 cations of the culture medium. It was even difficult to separate the 

 cells from the nutrient solution by centrifugation, because part of 

 the growth remained in suspension or was concentrated at the 

 surface of the solution. The addition of 100 ppm. AI2 (804)3 to 

 the culture medium at the time of harvest and the lowering of the 

 pH to 4.5, however, flocculated the cells so that after centrifuging 

 for twelve minutes at 2700 RPM they collected in a tight mat at the 

 bottom of the tube. In most cases, the 100 ppm. of AI2 (804)3 

 lowered the pH to the desired level. If not, it was adjusted to 

 4.5 with dilute HCl. The centrifuged cells were washed once in 

 distilled water, rinsed into a tared weighing bottle, and dried to 

 constant weight at 63° C. The yields from two flasks were placed 

 in one tared weighing bottle to provide sufficient material for an 

 accurate dry weight determination. 



pH Experiment 



Because it would be difficult to buffer the pH of cultures of 

 Coccochloris Peniocystis at unit intervals between 5.0 and ii.o 

 and still not influence the growth of the algae in other ways, the 

 effect of variations of pH on the growth of this organism was de- 

 termined by frequent adjustments with dilute HCl and NaOH. 

 The Na2C03 usually included in the culture medium was omitted 



