68 A. KEYNAN AND M. HALMANN 



^trigger reaction" itself. Therefore, it would be interesting to 

 compare the action of inhibitors on the 'alanine activation' 

 during preincubation at 37° with that of the reaction occurring 

 at 15\ with the idea in mind that inhibitors which prevent 

 germination during preincubation at 37^ but have no influence 

 on events occurring at 15° are true inhibitors of the primary 

 'trigger reaction', while those preventing germination at 15° are 

 inhibitors of other metabolic reactions subsequent to the 

 primary triggering. 



Among inhibitors known to prevent L-alanine activation, 

 D-alanine is active only if added before, together or immediately 

 after addition of L-alanine. In our 'double temperature" experi- 

 ment it was demonstrated that D-alanine prevented germination 

 when added during preincubation at 37°, in the first stage of the 

 experiment, but did not influence events at 15° when added at 

 the second stage. 



Various salts inhibited germination when added during pre- 

 incubation. When, however, these salts were added to spore 

 suspensions after preincubation at 37° during the second stage 

 of the experiment (at 15°), they were without influence on the 

 rate or extent of germination. ED50 of KCl, NaNOs or LiCl 

 when added together with L-alanine was found to be 8 x 10"- M. 

 Some other aspects of the salt inhibhion of L-alanine activation 

 may also be of interest. Salts of divalent cations were more 

 active than monovalent ones: ED50 values were 5 x IQ-^ M for 

 MgCl2 and 6 10^4 M for CaCl-z. The inhibiting action of the 

 salts was initially reversible, since it was abolished when the 

 cells were washed 10-15 min after salt addition. When, however, 

 spores were exposed to the salts for as long as 2 h the germina- 

 tion-inhibiting action could no longer be reversed by washing in 

 a centrifuge. Inhibition by NaCl decreased with increasing 

 amounts of L-alanine. The salt inhibition was therefore com- 

 petitive in respect to L-alanine. 



Other inhibitors, e.g. HgCl-z and octyl alcohol prevented 

 germination at whatever stage they were added, whether 

 at 18° or at 37°. Presumably they block some metabolic 



