436 Physiology 



Growth in organic media cannot be expected to yield many clues be- 

 cause such materials contain quite a variety of trace elements. In yeast 

 extract, for example, barium, bismuth, boron, calcium, chromium, copper, 

 gold, iron, magnesium, manganese, phosphorus, potassium, silicon, silver, 

 and strontium have been demonstrated by spectroscopic analysis, and a 

 preparation of asparagine has contained as many as 21 trace metals (483). 

 Such difficulties have been illustrated in the investigation of mineral re- 

 quirements in Tetrahymena pyriformis (292). 



For various phytoflagellates, culture media have been prepared from 

 analyzed reagents and used in all-glass culture vessels. Within such limits 

 of accuracy, the composition of the medium is known except for possibly 

 significant contaminations from glassware or other sources. Some of these 

 media contain, as impurities in the salts, barium, cobalt, chromium, cop- 

 per, gold, iron, manganese, sodium, zinc, and other metals, in addition to 

 the intentionally supplied calcium, magnesium, phosphorus, potassium, 

 sulfur, chlorine, ammonium salts, and carbon dioxide. It has been pos- 

 sible, from such starting points, to detect a lew trace-mineral require- 

 ments. The determination of others, merely by selecting different salts so 

 as to exclude particular elements, has been impossible. 



The purification of reagents permits some further progress. The re- 

 moval of certain trace metals has been accomplished with chelating 

 agents, such as 8-hydroxyquinoline, which form metal complexes soluble 

 in chloroform or ethyl acetate (130). In addition, several other methods 

 for purifying important components of culture media are available (229), 

 and a number of purified elements and reagents can be obtained com- 

 mercially.^ 



Additional advantages are promised by the use of non-toxic chelating 

 agents in culture media (226, 229). Such substances as citrate and ethyl- 

 enediamine-tetraacetic acid, which form soluble and fairly stable metal 

 complexes, minimize and tend to eliminate certain technical difficulties. 

 A metal-chelate complex forms a sort of metal "buffer" with an action 

 somewhat analogous to that of pH-buffers. Since the precipitation of 

 metals as hydroxides, phosphates, or sulfates is prevented, it is possible 

 to add quantities sufficient for heavy growth. Metals which are toxic 

 above certain concentrations also can be supplied at levels favoring heavy 

 growth without danger of toxic effects. Aside from such general improve- 

 ment of the simpler culture media, chelating agents can be of assistance 

 in analyzing mineral requirements, rhe addition of a chelating com- 

 pound to a medium containing essential trace metals in minimal amounts 

 may induce a metal deficiency which will prevent growth. Incidentally, 

 materials often supplied as substrates — a-amino acids, glycerol, malate, 

 annnoniiun salts — also may be active enough as chelating agents to induce 



^Johnson, Matthey & Co., Ltd. 1947. Catalogue of standardized substances for spec- 

 trography, chemical analysis and research (London). 



