2 HAROLD R. CURRAN 



insufficiency of K+. The specificity of Mn + + for the sporulation of B. sub- 

 tilis was first shown by Charney and his associates (1951). In both chemi- 

 cally defined and in complex organic media, sporulation was neghgible 

 without added Mn++, although this supplement was not required for full 

 vegetative growth. 



These findings were confirmed and extended by Curran and Evans (1954), 

 who also observed that iron, when used in relatively large amounts, replaced 

 Mn++ in Mn-deficient media. Weinberg (1955) concurred in these find- 

 ings but suggested that the sporogenic activity of the added Fe+ + ( + ) could 

 be largely attributed to appreciable quantities of Mn++ contained as im- 

 purity in CP grade Fe compounds. In agreement with Weinberg, we have 

 since found that a sample of FeCls which in nutrient broth exhibited char- 

 acteristic sporogenic activity for B. subtilis at 200 ;(Ag/ml, when purified by 

 repeated extraction with HCl and with ether, became sporogenically inactive 

 in the same medium at levels of iron in excess of 200 /ng/ml. The spore-in- 

 ducing property of the medium containing purified iron could be restored 

 by small additions of Mn++. 



The careful studies of Grelet (1951, 1952a, 1952b) have contributed val- 

 uable information concerning the minerals required for sporulation by Ba- 

 cillus megatherium. With shake cultures in a glucose-mineral-salts medium, 

 the effect on sporulation was determined for a number of mineral constit- 

 uents. Each constituent of the medium was decreased independently, and 

 its approximately limiting concentration for sporulation determined. Deple- 

 tion of glucose and nitrate in the control medium was followed by rapid and 

 almost complete sporulation; similarly, sporulation remained at a high level 

 with progressive reductions in the NOs^, S04~ ~, CI", Na + , Fe+ + ^ + >, 

 and Zn++. In contrast, depletion of carbon and nitrogen, occurring in con- 

 junction with a deficiency of K + , Mg++, or Mn++, prevented most of the 

 bacilli from sporulating, indicating the essentiality of these minerals for spor- 

 ulation. A deficiency of Ca++ and PO4 reduced the number of spores; 

 and omission of Ca++ from the formula, although without effect on growth, 

 greatly hindered sporogenesis and yielded spores slightly refractile and weakly 

 thermoresistant. Omission of Ca++, but with Mn + + at a concentration 

 equal to that of Ca++ in the control medium, yielded spores about equal in 

 number to that of the control but with only 1/10 as many thermoresistant 

 cells. When both Ca++ and Zn++ were omitted, sporulation did not occur; 

 with omission of Zn++, some Ca++ was necessary for sporulation. 



Brewer et al (1946) described a chemically defined medium for Bacillus 

 anthracis which produced yields in excess of one billion spores/ml. The 

 medium contained a mixture of 18 amino acids, glucose, NaHCOs, gluta- 

 mine, nucleic acid components, mineral salts, and vitamins. Employing a 



