196 Journal of Agricultural Research voi. xviii, no. 4 



An examination of the number of spores present per cubic centimeter 

 was made by means of the blood-counting cell, as described by us in 

 another connection.^ Unfortunately it was impossible to disintegrate 

 the clumps, and the reported counts are consequently approximate. 

 In this instance there were about 2,000,000 spores per cubic centimeter 

 of inoculum. The flasks were then heated to 63° C. for 30 minutes for 

 the purpose of killing the spores^ and making the spore wall more perme- 

 able. The contents of each flask were then transferred to Krlenmeyer 

 flasks containing sterile washed sand and shaken vigorously for 5 

 minutes in order to cause further rupture in the spore walls. The in- 

 oculum was then ready to be added to the 200-cc. portions of 10 per 

 cent (by weight) granulated sugar solution in cotton-plugged Erlen- 

 meyer flasks, which had been previously sterilized in the autoclave at 

 15 pounds pressure for 15 minutes. All the flasks had the same polari- 

 zation. Ten cc. of sterile distilled water were added to the control 

 flasks, and 10 cc. and 20 cc. of inoculum were added to the others. After 

 this inoculation both the inoculum and sterile distilled water were 

 heated to 100° for 20 minutes to kill any enzym which might be present. 

 Then this sterilized inoculum was added to the sugar solution as ex- 

 plained above. All flasks were then incubated at 35° to 45° for 3 hours. 

 At the end of this time 50 cc. of solution were removed with a sterile 

 pipette, filtered, and polarized. Reducing sugars were determined in 

 the same sample by the modified Violette method (volumetric). The 

 results are recorded in Table I. The actual polarization values and 

 the average percentage of decrease in sucrose are given in separate 

 columns.^ 



It will be seen from the results in Table I that the flasks inoculated 

 with sterile distilled water and those inoculated with sterilized inoculum, 

 heated to 100° C, had the same polarization (35.7), within experimental 

 error, and the same amount of reducing sugars (0.04 per cent), while the 

 flasks having 10 cc. of spores heated to 63° polarized 34.8, which repre- 

 sents a loss in polarization of 0.9 as compared with the control, or a 

 decrease of 0.23 per cent sucrose. The reducing sugars increased 0.02 

 per cent. The inoculum of spores was polarized alone and exhibited no 

 optical activity. However, where 20 cc. of spores heated to 63° were 

 used, the polarization dropped to 34.1, which was a decrease of 1.5 as 

 compared with the control, or an actual decrease of 0.38 per cent sucrose. 

 The reducing sugars were slightly increased. Where 20 cc. of inoculum 

 were used it is necessary to correct for the added dilution, if we wish to 

 compare these results with those obtained with 10 cc. of inoculum. 



1 KoPELOFF, Nicholas, and KopELOFF, Lillian, op. OT. 



2 Thom, Charles, and Ayers, S. Henry, effect of pasteurization on mold spores. In Jour. Agr. 

 Research, v. 6, no. 4, p. 153-166, 3 fig. 1916. 



' The authors wish to thank Mr. E. C. Freeland, Assistant Chemist, for his kind assistance with these 

 analyses. 



