INSTITUTE, PUSA, FOR 1917-18 125 



causes of negative results and the feasibility or otherwise 

 of removing them. The general procedure adopted in 

 carrying out each experiment involved chemical examina- 

 tion of the plant before and after steeping, of the liquor at 

 various stages of fermentation and also after blowing and 

 settling, and of the indigo precipitate or mal and lastly of 

 the waste or seet water. Thus a close watch was kept upon 

 the fate of the original indican content of the leaf, and the 

 effect of any method of treatment upon the percentages of 

 this constituent which appeared at various stages either in 

 its original form or as indoxyl or indigotin, was accurately 

 ascertained. This naturally involved a very large number 

 of analyses and as the quantitative estimation of indican, 

 indoxyl, or indigotin is a tedious process and as each 

 analysis was made, during the later period, both by the per- 

 sulphate and isatin methods, it will be realized that the 

 number of experiments was limited by the possible pace of 

 output of the analytical data. 



In the same way bacteriological analysis of the fermen- 

 tation process was carried out for each stage of manufac- 

 ture, but this analysis necessarily began with measurements 

 of the rate of bacterial growth (by plating) in the mass cul- 

 tures used for inoculation some hours before manufacture 

 commenced, in order to discover the best conditions fori 

 securing successful inoculation. Plates were made from 

 the mass culture after various periods of time from the 

 original inoculation, from the khazana (reservoir) before 

 and after the introduction into it of the mass culture, and 

 at intervals up to the time of " watering ' the vats, i.e., 

 running the water from the khazana into the steeping vats 

 containing the cut plant. Fermentation continues after 

 watering for varying periods of time up to some 12 — 15, 

 hours, and plates were made at intervals to determine the 

 rate of multiplication or otherwise of the specific bacteria 

 artificially introduced with the water. The water used at 

 Pusa was drawn directly from the river, and a large num- 

 ber of plates was made to ascertain the bacterial content 

 of this water and the proportion of active hydrolyzers. 



