[Vol. 6 



26 



ANNALS OP THE MISSOURI BOTANICAL GARDEN 



salt were added to the digestion flasks containing 2 gms. man- 

 nite and 30 cc. of cone. H2SO4. After digestion had proceeded 

 for 20 minutes, 10 gms. of Na2S(>4 (anhydrous) were intro- 

 duced into each flask. The time for the solutions to become 

 clear bluish green and the degree of foaming were recorded. 

 The results appear in table v. 



table v 



EFFECT OF VARYING AMOUNTS OF COPPER SULPHATE 



No. 



Amount CuSO< 

 (gms.) 



1 



0.1 



2 



0.2 



3 



0.3 



4 



0.4 



5 



0.5 



Time of clearing 

 (minutes) 



73 

 68 

 78 

 80 

 63 



Order 



3 



2 

 4 



5 

 1 



Foaming 



Decided 



Decided 



Decided 



Moderate 



None 



The differences in regard to time are of minor significance. 

 The differences in regard to foaming are important and in- 

 dicate that as measured by this standard less than 0.5 gm. of 

 CuS0 4 should not be used. The digestion procedure used in 

 the ' ' Final Experiments ' ' was as follows : The sample is di- 



■ 



gested for 20 or 25 minutes with 30 cc. of H 2 S0 4 and 0.5 gm. 

 CuS04. Ten grams anhydrous Na2S0 4 were then added and 

 the digestion completed. The above results were confirmed 

 repeatedly with the slight difference that the digestion of nu- 

 trient mannite or dextrose solutions required a trifle longer 

 than the pure mannite, this probably for the reason that the 

 salts of the solution were slightly inhibitory in action. As a 

 general average the mixtures cleared in 1^ hours ; in all cases 

 the digestion was continued over a low flame for 1| hours 



after clearing. 



METHODS OF INOCULATION 



Two methods of inoculation were used, and 10 cultures 

 seeded by each method were incubated and analyzed. By the 

 one method, a spiral of growth from an agar slant was trans- 

 ferred to 10 cc. of Ashby's solution (plus CaCO^) contained in 

 a 250-cc. Erlenmeyer flask, and the culture maintained on the 



C. One spiral 



shaker for 24 hours in the warm room 



o 



