concentrations of salts. Different species of algae 

 react differently toward the various amines. The 

 toxicity of TEA is counteracted by increasing con- 

 centrations of Ca for Vol vox globator and by Ca and 

 Mg for y. tertius and Woloszynskia . The toxicity 

 of TRIS for Cyanophora paradoxa can be removed by 

 additions of Mg . and K; Ca and Na are of no use 

 and even in presence of TRIS, they can be dis- 

 pensed with in media allowing optimal growth. 

 Since the amines heighten requirements for the 

 major cations, they may be used to reveal the pre- 

 dominant necessary ions. But the amines can be 

 toxic through other, unknown mechanisms: the 

 toxicity of TRIS (20 mg.%) for Vol vox globator can- 

 not be counteracted by Ca , Mg , Na , K or trace 

 metals . 



Another pitfall of TEA, at least for Volvox , is 

 that minor variations in pH (from 7.0-7.6) are im- 

 mediately reflected in a change in availability of 



trace metals, particularly iron: the same concen- 

 trations of iron act at one pH as if Fe were avail- 

 able in excess (toxic) and at a slightly different 

 pH as if Fe were lacking. A similar inflexibility 

 toward the trace metal mixtures was noted for other 

 algae. The first success was the buffering of the 

 extremely dilute media for Synura at pH 5.0 (Table 

 2) . Buffering these media was particularly diffi- 

 cult yet Imperative because solutions are so dilute, 

 owing to the sensitivity of the organisms to con- 

 centarions of any solute. Histidine adequately 

 solved the problem: it is well tolerated at 15-30 

 mg.%, just enough to buffer the medium effectively 

 at pH 5.0-65 . Much later, following the sugges- 

 tion of Droop (personal communication) , who had 

 found glycylglycine less toxic than TRIS for Oxyr - 

 rhls marina , we tried this buffer for Volvox , Wolo- 

 szynskia , and Phacus . Surprisingly, minor changes 

 in pH no longer affect metal availability in Volvox 



Table 2 . Culture medium for Synura media 



Metal -buffered 



pH-buffered 



EDTA 

 NaN03 

 KH2PO4 

 Mg (as 304=) 

 Ca (as CI") 

 K (as Cl~) 

 Fe (as Cl") 

 Zn (as Cl") 

 Mn (as Cl") 

 Mo (as Na salt) 

 Co (as Cl") 

 Cu (as Cl") 

 Na H glutamate 

 Na acetate. 3H2O 

 pH 5.5 



* a mixture of oC - and p- glycerophosphates, 



89 



