ACID FORMATION AND ALCOHOL 139 



the presence of sugars. The iodin test is made by adding and 

 mixing successively a few drops of Gram's iodine solution with 

 the media. Amylodextrin and erythrodextrin, if present, produce a 

 purple color; erythrodextrin and achroodextrin, a port-wine color; 

 achroodextrin and maltose, no coloration. The qualitative test for 

 maltose is made with* Fehl ing's solution in the usual manner: Take a 

 few cubic centimeters of Fehling's solution, prepared from equal 

 amounts of solution No. 1 and No. 2, dilute with distilled water, heat 

 to boiling, and then add, drop by drop, some of the previously diluted 

 and filtered starch culture medium. If maltose is present an orange 

 or red-brown precipitate will be formed. If the sugar formed is to 

 be determined quantitatively it must first be ascertained whether 

 it is maltose or dextrose. This can be done by changing the sugar 

 present into an ozazon by the action of phenylhydrazin hydrochlorate 

 and acetate of potash and determining the form of crystallization, 

 the melting point, and the amount of nitrogen present in the com- 

 pound. These are somewhat more complicated, though not difficult, 

 chemical manipulations, the details of which can be found in a 

 text-book on organic quantitative analyses. After the kind of sugar 

 present has been found the amount can also be ascertained by 

 titrating with Fehling's solution. 



Invertin. To demonstrate the formation of invertin in a bacterial 

 growth the latter must be inoculated into a sugar-free bouillon to 

 which a small amount of pure saccharose or cane sugar, which does 

 not possess any reducing power, finally has been added. The presence 

 of invertase is manifested by the inversion of some or all of the cane 

 sugar into invert sugar (dextrose, maltose, etc.), which reduces 

 Fehling's solution. 



Rennet and "Lab" Enzymes. The presence of rennet and "lab" 

 enzymes is ascertained in the following manner : Prepare a sugar-free 

 bouillon and inoculate several tubes with the bacterium to be tested. 

 After keeping the growth a few days in the incubator, heat the tubes 

 for thirty minutes in a water bath at 55 C. This will destroy the bac- 

 teria but not the rennet if any is present. After heating, add about 

 5 c.c. of the contents of these tubes to sterile litmus milk in another 

 set of tubes. Keep for several days at 22 C., and examine every 

 day for ten or twelve days. If there is no coagulation at the end of 

 this time rennet is not present. 



Acid Formation and Alcohol. Acid formation by bacteria is accu- 

 rately determined by inoculating them into 500 to 1000 c.c. of sterile 

 sugar bouillon (see above) of a known definite slightly alkaline re- 

 action. After a number of days the reaction of the inoculated bouillon 

 is again titrated by the aid of a one-twentieth normal solution of 

 caustic soda. The difference in the results of the two estimations 

 indicates the amount of acid formed. If the growth in the flask is 

 very heavy it may be necessary to filter the bouillon through a Pasteur 

 or Berkefeld filter before the final tests can be made. 



