460 STATE BOARD OF AGRICULTURE. 



4 were wholly free from and uninfluenced by micro-organism B, while flasks 

 5, 6, 7, and 8 had in reality been cultures of forty-eight hours standing of this 

 micro-organism. Since the germs had been killed by sterilization, we may 

 conclude that the products resulting from their growth furnished a more favor- 

 able medium for the development of the lactic acid bacteria, causing the more 

 rapid souring of the milk. 



An investigation of acidity in these flask-cultures at intervals of twenty-four 

 hours will also offer conclusive proof and a more intimate knowledge of the 

 changes taking place. At the time of inoculation of these milk flasks with 

 micro-organism A, flasks 1, 2. 3, and 4 had an acidity to phenol-phthalein of 

 22°. flasks 5, 6, 7, and 8, of 2G°. This difference of 4° may be attributed to the 

 action of the products of germ B upon the indicator phenol-phthalein. Tliis 

 has been noted before. The difference is apparent, therefore, and not real. 

 Litmus remained blue and became more densely blue as micro-organism. B 

 continues in its growth in milk. Moreover these four degrees will have little 

 effect upon the striking results obtained. 



Flask-cultures 



1 



2 



3 



4 



5 



6 



7 



8 



The slight differences in the readin.gs in each set at the same hour are easily 

 attributable to experimental error; however, the wide difference in acidities 

 existing between the two sets, flasks 1, 2, 3, and 4 on the one hand, and flasks 

 5, 6; 7, and 8 on the other, clearly indicate that some influence is at work which 

 has the power of increasing the acidity rapidly in flasks 5, G, 7, and 8 over 

 flasks 1, 2, 3, and 4. All are identical in their treatment and conditions, ex- 

 cepting the products which are under discussion. These products alone can 

 account for these results. Heretofore I have called attention to the uniformity 

 in the development of the cultures. This is very noticeable in the study of 

 acidities and such uniformity lends confirmation to the work. 



But one feature more remains in this discussion to bring it up and make 

 it harmonious with my former article for purposes of comparison and that is 

 the consideration of the number of bacteria developed in flasks 1, 2, 3, and 

 4 of one set, and also in flasks .5, G, 7, and 8 of the other. I have already stated 

 that at the beginning, each flask contained 49,800 micro-organisms A. The time 

 for ascertaining the necessary comparison in the number of germs was deter- 

 mined by the lop^ering of the milk in flasks 5, 6, 7, and 8; at this point a com- 

 parison, forsooth, is far more desirable than at any other point. A careful 

 estimate reveals 12,920,000 micro-organisms per c. cm. in flasks 1, 2, 3. and 4: 

 and 517,920,000 micro-organisms per c. cm. in flasks .5, 6, 7, and 8. This esti- 

 mate was made at the end of the first twenty-four hours after the inoculation 

 of the milk with micro-organisms A. The ratio existing may be stated as fol- 

 lows — the number of micro-organisms A in flasks 1, 2, 3, and 4 without the in- 

 fluence of micro-organisms B is to the number of micro-organisms A in flasks 5. 

 G, 7 and 8 growing under the influence of the products of micro-organism B 

 as 27: 15G. It is therefore conclusive that the lactic acid bacterium grows more 

 rapidly in the presence of the products of micro-organism B than when it is 

 not so associated and that milk sours more rapidly when either the living 

 form of micro-organism B or its products are present. This is due to the in- 

 fluence exerted by micro-organism B directly or indirectly upon the develop- 

 ment of micro-organism A. The evidence is three-fold: 1st. It is manifest 

 from the apparent changes in the milk on mere observation. 2nd. It is mani- 

 fest by a study of the development of acid in the dmerent flasks. 3rd. It is 

 manifest by the number of germs actually found to be present in the different 

 cultures. 



In our former article we succeeded in establishing that micro-organism B in- 

 fluences micro-organism A in the souring of milk, in this article we have 



