nitrate as Knop did . 



Most algae utilize nitrates except the eu- 

 glenids which, so far as we know, prefer ammonia 

 or amino acids. Ten to 20 mg.% nitrates are in 

 general well tolerated . Ammonia tends to become 

 toxic above 3-5 mg.% in alkaline media except for 

 eurybionts living in polluted waters. Since it is 

 difficult to know priori if an alga needs vitamins , 

 it is advisable to include in the isolation media the 

 three vitamins required by most auxotrophic algae: 

 vitamin B12 .01 jug.%, thiamine 10 )dg .% , and bio- 

 tin 0.05>ag.%. Mineral phosphates should be 

 avoided because they cause precipitates , especial- 

 ly in alkaline media . In a recent survey (Provasoli , 

 McLaughlin, Pintner, (in Provasoli , 1958, p. 294) 

 we have found that glycerophosphate was utilized 

 by all the algae tested. Glycerophosphates have 

 the advantage of forming far more soluble salts with 

 the divalent anions than the phosphates, thus pre- 

 cipitation is often avoided even in slightly alka- 

 line media or in the absence of chelators . Con- 

 centrations of .5-3 mg.% are generally adequate. 



The problem of avoiding precipitates while 

 supplementing the necessary trace-metals was 

 partly solved by Uspenski and Uspenskaja (19 25) 

 by using opportune ratios of citric acid and Fe . 

 But chelation of Fe with citric acid does not prevent 

 precipitates in alkaline media. Hutner (1948) and 

 Hutner et al. (1950) explored the possibility of em- 

 ploying stronger chelating agents to study the es- 

 sentiality and role of trace metals. The criteria for 

 selection of a "good" chelator for studying essen- 

 tiality were that: a) it should form very stable 

 complexes; b) it should be a bulky non-penetrable 

 molecule so that chelation will take place in the 

 external medium and not within the cells; c) it 

 should be photostable and thermostable; d) it should 

 be non- metabolizable and non-toxic for reasons 

 unconnected with its metal-binding properties . 

 Ethylenediamine tetracetic acid (EDTA) met all 

 these specifications. EDTA, or other chelators 

 tried, could not, as hoped, serve to determine the 

 essentiality of single trace metals, but were very 

 effective in providing a non-precipitable metal pool 

 of trace metals, thus approaching the goal of 

 metal-buffering (pM) , and became of general use. 

 No difficulties were found for fresh-water eurybi- 

 onts (Euglena , Ochromonas , etc.) and for some 

 marine fungi (Vishniac, 1955). Enormous quanti- 

 ties of EDTA (50-100 mg.%) were employed at 

 first and the trace metals had to be raised to cor- 

 respondingly high and unphysiological levels prob- 

 ably to prevent the excess EDTA from binding other 

 necessary ions such as Ca and Mg . It was soon 

 found that most organisms (even the marine algae) 

 could not tolerate the high content of free trace 

 metals and free chelator resulting from the equa- 

 tion: K = Civrd . Provasoli et al. (1957) found 



[M] [Y] 

 that artificial synthetic marine media are suitable 



for the greatest variety of organisms when the trace 

 metals pool is low and the trace metals are offered 

 as a mixture slightly overchelated (ratio of chela- 

 tor/trace metals = 1:1 to 3:1) . Since these mix- 

 tures (PI, PII, and TMII are the most widely used 

 for marine organisms) are over-chelated , Droop 

 (1959) has rightly raised the point that when the 

 salinity of the marine media is varied and lowered 

 (by lowering the Ca, Mg , Na , K concentrations) it 

 might be better to employ citrate as a metal buffer 

 because citrate has less affinity for Ca and Mg 

 than EDTA . The free EDTA (of the over-chelated 

 mixture) binds these cations more strongly and will 

 reduce drastically their availability, especially at 

 the lowest salinities. In fact it is possible to em- 

 ploy these over-chelated mixtures even for fresh- 

 water organisms . One can substitute with 1-2 

 ml/100 of PII mixture the metal mixtures which had 

 been experimentally found to compensate for the 

 various chelations employed in the media for Pha - 

 cus pyrum , Volvox globator , V . tertius , and Wolo- 

 szynskia limnetica . But this does not hold for 

 Synura media which are low in Ca and Mg (respec- 

 tively .4 and .05 mg.%) while the other media 

 contain from 2-4 mg.% of Ca and from .4 to 2 

 mg.% of Mg. This seems to confirm Droop's 

 assertion . Experiments with Synura are in progress 

 to see whether increased Ca and Mg will allow the 

 use of over-chelated mixes; this might not be feas- 

 ible because of the very poor tolerance of Synura 

 toward total solids . 



The other important difficulty in fresh-water 

 media is pH buffering. Inorganic phosphate can not 

 be employed because: a) most fresh-water algae, 

 especially those of oligotrophic waters, cannot 

 withstand the concentrations of phosphates needed 

 for buffering (phosphates often become toxic above 

 5-20 mg.% except for organisms living in polluted 

 waters and often for blue-green algae); b) heavy 

 precipitates result because the fresh-water media 

 are generally rich in calcium. Several amines have 

 been employed successfully for buffering in the al- 

 kaline range. Of these the most useful are tris 

 (hydroxy- methyl) aminomethane (TRIS) and triethan- 

 olamine (TEA); ethanolamine is in general more in- 

 hibitory. TRIS is a very good buffer for marine 

 media: most marine algae are not inhibited by 100 

 mg .% and several withstand much higher concentra- 

 tions. However, TRIS and other amines are toxic 

 to certain pathogenic bacteria. MacLeod and 

 Onofrey (1954) found that the toxicity of amines 

 could be counteracted by the addition of K, Ca , 

 Mg , and Na , alone or in combinations. This is 

 probably why Tris is not toxic at useful pH buffer- 

 ing concentrations in media rich in those ions, like 

 the marine media . TEA and TRIS can be employed 

 for fresh-water algae fairly resistant to high total- 

 solids concentrations so that it is possible to raise 

 the cations and counteract the inhibition of the 

 amines without approaching osmotically inhibitory 



