tUE MINERAL REQUlREMENtS ¥0k SPORULATlON 3 



more dilute version of this medium, Brewer studied the effects of Ca++, 

 Fe+ + ( + ), Mg++, and Mn++ ions on sporulation. Each ion was tested in- 

 dividually by varying its concentration, while the other ions were present 

 in approximately optimal concentration, as determined by preliminary tests. 

 In the absence of Mg++, practically no growth occurred; 1.6 ^tg/ml and 

 higher of Mg++ induced good growth and sporulation. A good yield of 

 spores was obtained in the absence of added calcium; however, addition of 

 20 /u.g/ml of Ca++ increased the spore yield about 4.5 times. Inclusion of 

 Fe+ + ( + ) in the formula was not critical for sporulation, although the 

 addition of 0.7 /Ag/ml increased the spore yield about 2.5 times. Added 

 Mn++ was not essential to sporulation, but, as recognized by the authors, 

 enough was probably present as an impurity to provide sporulation require- 

 ments of the organism. Replacement of the K phosphates with Na phos- 

 phates decreased spore yield about 1/3. The addition of Cu++, Zn++, 

 Cd++, and Co++ at levels of 0.1-1.0 /^.g/ml of metallic ions was without effect 

 upon sporulation. 



The influence of mineral salts on sporulation is reflected in the results of 

 experiments involving the use of a nutrient medium both in fluid form and 

 solidified with agar. Thus Roberts and Baldwin (1937) observed that agar 

 in amounts as small as 0.06%, when added to 1% peptone solution, defi- 

 nitely increased spore production of B. suhtilis, and subsequently (1942) 

 that the percentage sporulation in vigorously aerated peptone broth was 

 considerably less than in unaerated medium made slightly viscous with agar. 

 Since sporulation of B. suhtilis is more rapid and attains materially higher 

 levels in shake than in stationary cultures, these results must be attributed 

 in part at least to minerals supplied by the agar — the latter contributes 

 relatively large amounts of Mg++, K+, and Ca++ and biologically signif- 

 icant amounts of Mn++ and Fe+ + ( + ). 



The mineral requirements of thermophiles in relation to sporulation have 

 received very little attention. Ward (1947) observed that sporulation of 

 Bacillus thermoacidurans in proteose-peptone agar was increased by the 

 addition of Li++, Mg++, Ca++, Fe+ + ( + ), Zn + +, or Mn + +. 



The addition of NOs" or NOo" to nutrient agar has been reported to 

 increase the sporulation of Bacillus stear other mophilus (Dahl, 1955). 

 Schmidt's (1950) studies on the same organism, limited to NaCl and CaClo, 

 indicated no clear-cut effect. Subsequently the same investigator observed that 

 the addition of Mn + + to nutrient agar greatly increased sporulation of B. 

 stearothermophilus (unpubHshed data). Sporulation by Bacillus coagulans 

 vdr. thermoacidurans in three peptone-containing agar media was shown to 

 be greatly enhanced by the addition of Mn++, Co++, or Ni++ (Amaha 

 et al, 1956) . 



