BY R. GREKj-SMITH. 87 



sponsible for the eoncave depression of these curves and that it may not have 

 had any decided inHiienee in the production of the carbon dioxide. 



It seems that this bacterium is one that has to get accustomed to its environ- 

 ment, for it was slow to produce carbon dioxide fr(mi the tan bark, and it is 

 slow to grow on alkaline media as shown in experiments with increasing alkali 

 where the slopes with a comparatively high alkaline content had to be repeatedly 

 seeded from growths on agar with a little less alkali. It may be that in the 

 tan-bark there is a constituent, e.g., tannin, which prevents the ready growth of 

 the bacteria. 



The addition of sodium hydrate to the Hasks containing the bark was sug- 

 gested by the increased activity of the bacteria in alkaline solution. But there 

 was another reason for its use. On a former occasion (These Proceedings, 1918, 

 p. 162), I showed that the heating of leaf-mould for two hours at 130" 

 resulted in the formation of acid, the barium salt of which was largely soluble 

 in water. The amount jirodueed was e((uivalent to about 31 e.e. of nornuil acid 

 per 100 grams of dry oi'ganie matter. It is probable that a higher temperature 

 will produce a larger amount of acid and that a bark after sterilisation in the 

 laboratory will be more acid than it was before sterilisation. Thus the addition 

 of alkali to neutralise this developed acidity was indicated. 



An attempt to arrive at the amount of acid developed durmg sterilisation 

 was made upon a sample of air-dried tempered bark. Ten gram portions were 

 weighed out, one was treated with 100 e.c. of water, another was sterilised at 

 200° to 176° for two hours, and then treated with 100 c.c. of water. After three 

 days, the liquids were tiltered and diluted one half. Using Sorensen's fifteenth- 

 normal solutions of primary and secondary phosphates and brom-cresol-purple 

 as the indicator, the extract of the air-dried bark had a Pjj. number of 6.8 

 and the sterilised bark of 6.47. Thus an increase in the acidity of the bark fol- 

 lowing the .sterilisation is shown. As the extract of the sterilised bark was 

 brought up to Ph, 6.8 by the addition of 0.1 c.c. of N/100 alkali per 5 c.c, 

 it appears that 100 grams of air-dried bark during sterilisation developed an 

 amount of ionic acidity equal to 4 e.c. of tenth normal acid. This is very much 

 less than had been expected from the experiment with leaf-mould, and seems 

 to show that far too much alkali had been added to the tan bark in the fer- 

 mentation experiment. Still the alkali had been added on account of the liquor 

 reddening litmus paper. 



A fermentation test was made with tempered bark, but it proved a failure. 

 The bark was air-dried, then dried at 130°, and of this dry bark, 40 gi-am por- 

 tions were put into flasks and sterilised at 150° for half an hour, and at 170° 

 for an hour and a half. The flasks received 70 e.c. of water, one containing a 

 suspension of the bacillus. By the second day. the seeded Ha.sk had given oft' 206 

 milligrams of CO2 and the control 121 milligrams. The fluids in both flasks 

 were at this stage shown to contain living bacteria and it followed that the 

 tempered bark had contained some very resistant spores. Cultures from the 

 control flask showed the rod with terminal spores growing at first as translucent 

 colonies and rather gummy. By the time that the impurity of the control had 

 been demonstrated, the amount of CO2 given off was 371 milligrams in the seeded 

 flask and 320 in the control. Five grams of powdered copper sulpliate were 

 added to the control fla.sk and the fermentation was continued. By the seventh 

 day the seeded flask had given ofl" 597 and the control 553 milligrams of CO2, 

 and the control still contained living bacteria. The copper sulphate had been 

 added as a disinfectant, but it would seem that the constituents of the bark had 



