NATURAL INDIGO (>7 



to flood the vats, and upon the number of indican-spUtting 

 organisms in particular. Use was made of agar plates with 

 which a small amount of indican had been incorporated, and 

 it was found, on adding small quantities of water used at various 

 factories, and at the same factory at various times, that the 

 number of colonies showing up blue, through production of 

 indigo from the hydrolysed glucoside, was related directly to 

 the time necessary for adequate fermentation. This was 

 brought to light in connection with the mahai on Assam estates, 

 where the Behar standard optimum times at various tempera- 

 tures were found to give very poor results. On increasing the 

 time from twelve to twenty-four hours, however, better yields 

 were obtained. More recently mahai has been limited to 

 sixteen or seventeen hours, but, owing to variations in the 

 water-supply, which is derived from small streams instead of 

 rivers or reservoirs as in Behar, the time necessary is variable. 

 This is very decisive evidence as to the preponderating influence 

 of bacteria upon the progress of the mahai. 



Furthermore, there is a change in the proportions of the 

 gases liberated, first nitrogen, which begins to be evolved three 

 or four hours after filling, then hydrogen, and finally carbon 

 dioxide being the principal constituent. It appears that the 

 last-named renders the protoplasm of the cells permeable and 

 the indican diffuses out and is hydrolysed to glucose and 

 indoxyl by bacteria. 



Davis has shown that the final acidity of the vat-liquor is 

 due to carbon-dioxide. No other volatile acids are present, 

 though possibly traces of malic acid, derived from the plant, 

 are in solution. A study of the hydrogen ion concentration by 

 means of indicators has proved that the strongly alkaline 

 river water, Ph = 8-6, found at Pusa is changed during mahai 

 so that an acid reaction is developed, the acidity being at 

 least equal to that found in the leaf of the plant, Ph = 5*6. 

 The yield of indigo is very greatly influenced by the duration 

 of the mahai, as over-steeping results in serious loss through 

 destruction of the indoxyl, so that it can no longer yield indi- 

 gotin. This Davis has shown to be correlated with a high 

 content of carbon-dioxide in the vat-liquor, though it is uncer- 

 tain whether the acidity is in itself the cause of the destruction 

 or whether the carbon-dioxide is merely an indication of the 

 presence of bacteria which destroy it. The possibility remains 

 that the losses due to over-steeping are brought about by 

 enzymes derived from the plant, though the sudden fall which 

 accompanies over-steeping is rather against this view. 



The correct timing of the steeping is therefore of great 

 importance, but no method of gauging this was known till 

 recently. The times given by Bergtheil as average optimum 



