Counting was performed by a Packard Tricarb 1600TR liquid scintillation counter.
Although partial hydrolysis of P-containing organic compounds may have led to an overestimate of the tissue content of labeled phosphate, overcoming this problem is a complex procedure (8). However, any overestimate in tissue phosphate due to hydrolysis was trivial, as estimates of the sum of labeled P from all fractions and the medium did not exceed the amount initially introduced into the system (Table 1). Sensitivity of the method is largely influenced by the amplified errors associated with fraction A (see Table 1). The reproducibility of the method is high, as we consistently found no significant difference between the results from duplicate experiments for each time interval (Student's t test, P Ͻ 0.05; data not shown).
Table 1 illustrates how this simple method can be used to track the fate of external inorganic phosphate once it is absorbed by a green alga. We have used this method successfully in a study of bioindicators of eutrophication in an Australian estuary. Washout experiments can be used to further complement the results of this method, providing a more detailed understanding of the flux of P into and out of internal pools.
The method presented here requires a minimum of specialized equipment and takes advantage of disposable products to reduce possible 32 P contamination. We found that with preparation of reagents and plasticware the day before, up to 36 tissue samples could be processed in a day.