PHOSPHORUS EXCHANGE IN PHYTOPLANKTON 



79 



this method is very accurate for determining uni- 

 algal population sizes, the accuracy agreeing 

 favorably with that found by other investigators 

 (Pearsall and Loose 1937, Pratt 1940, and 

 Winokur 1948). 



PREPARATION OF FILTERS 



A stainless-steel filter (fig. 2), which could be 

 taken apart for the insertion of specially cut 

 Whatman No. 42 filter paper, was used with a 

 vacuum pump. The barium-sulfate precipitate 

 was formed by heating 15 cc. of one-fifth normal 

 H 2 S0 4 and adding 0.6 cc. of normal BaCl 2 . The 

 precipitate was filtered onto the filter paper and 

 rinsed with distilled water before filtrations were 

 made. 



FILTER 

 PAPER 



u 



Figure 2. — Stainless-steel filter. 

 FRACTIONATION OF CELLS 

 The cells were centrifuged from the culture 

 medium and extracted in the cold with 10 cc. of 



cold 10-percent trichloracetic acid (TCA) for 

 about 1 hour. After centrifuging, extraction was 

 repeated with a new volume of TCA. The TCA- 

 insoluble residue was dissolved in HX0 3 and 

 diluted with distilled water for counting. The 

 TCA-soluble fraction was neutralized to phenol- 

 phthalein with XaOH and the inorganic phos- 

 phorus fraction was precipitated with 10-percent 

 CaCl 2 in saturated CaOH 2 . The precipitate, 

 after centrifuging, was dissolved in a small amount 

 of dilute HC1, made to volume with distilled water, 

 and the radioactive disintegrations were counted. 

 The supernatent was also made to volume with 

 distilled water and counted. Thus the radio- 

 active-phosphorus content of the inorganic phos- 

 phorus, the TCA-insoluble phosphorus (con- 

 sidered as protein-bound phosphorus), and the 

 TCA-soluble organic phosphorus (ester phos- 

 phorus) were measured. The protein-bound 

 phosphorus and the ester phosphorus were always 

 combined and reported as the organic fraction. 



EXPERIMENTS 

 PHOSPHORUS ABSORPTION 



Phosphorus in culture medium containing 

 dividing cells can be reduced below a concentra- 

 tion detectable with the colorimetric method of 

 analysis if no other factor limits cell division. 

 Consequently, it has been assumed that phos- 

 phorus completely disappears from the medium. 

 By using radioactive phosphorus, the reduction of 

 phosphorus in the culture medium can also be 

 determined, provided no exchange of phosphorus 

 occurs between the cells and the medium. To 

 accomplish this requires only that a known 

 amount of radioactive phosphorus be added to 

 medium containing nonradioactive phosphorus 

 prior to the addition of cells. Since the cells 

 cannot distinguish between these two isotopes, 

 the same percentage of each should be absorbed 

 in any period of time. Thus, measurements of 

 the radioactive phosphorus absorbed can be 

 converted to represent inorganic phosphorus 

 absorbed, since the initial ratio of the two is 

 known. 



To compare the absorption curves of phosphorus 

 as determined with radioactivity measurements 

 and with colorimetric analysis, culture medium 

 was prepared with enough radioactive phosphorus 

 to give 11,000 counts per minute per 25 cc. of 

 medium and an inorganic phosphorus concentra- 



