124 SCIENTIFIC REPORTS OF THE AGRICULTURAL RESEARCH 



ported and the whole theory confirmed by- the experiments 

 carried out in the Pusa factory in 1917. 



The main line of experiment lay in the comparison of 

 results obtained in artificially inoculated steeping vats and 

 in uninoculated control vats, making use of pure cultures 

 of various hydrolyzing bacteria isolated from the khazana, 

 or seet water, or from the walls or timbers of the steeping 

 vats of various indigo factories. Isolation of such bacteria 

 was effected by the use of indican agar, on which those 

 bacterial species capable of splitting off indoxyl from 

 indican formed indigo blue colonies, and examination of a 

 large number of samples from various factories has led to 

 the general conclusion that the known yield of a factory is 

 closely and almost directly proportional to the content of 

 such hydrolyzing bacteria in its water supply. 



The first problem to attack was the method of inoculat- 

 ing the water used for steeping in such a way "as to ensure 

 the presence of sufficient numbers of the specific bacteria. 

 It may be said at once that this problem rapidly revealed 

 itself as the basic one of the enquiry as a whole, and its 

 solution is still under investigation. Nevertheless the 

 results of the comparatively limited number of experiments 

 made in the Pusa factory conclusively established the main 

 principle that yield of indigo depended upon bacterial 

 action, and consequently upon the presence of adequate 

 numbers of bacteria of the proper kind. The very first 

 experiment in which a comparison was made between the 

 yields of two vats, one inoculated with a pure culture of a 

 bacterium (laboratory mark In t0 ) and the other untreated, 

 gave an increase of 15 per cent, in the inoculated vat, not- 

 withstanding the unsuitability of the over-mature plant 

 available which should have been cut for manufacture at 

 least a month earlier. Subsequent experiments were not 

 invariably conclusive so far as yield of indigo was con- 

 cerned, but owing to the careful analytical watch, both 

 chemical and bacteriological, kept over every stage of the 

 process, it was not only possible to account for discrepan- 

 cies but to come to definite conclusions as to the underlying 



