EXPERIMENT STATION BULLETINS. 455 



It yliould be explained here, perhaps, that the strength and persistence 

 of the reduction-activity varied a little with different lots of beetles, and 

 that it always seemed to be best in the case of beetles showing the great- 

 est strength and vitality. 



Before discussing the effect of temperature, gasoline and other agen- 

 cies upon the reductase, it will be best to describe briefly the method 

 used in confining insect tissiie-extract in the absence of air so that a 

 study of its reductase activity under the influence of the various agents 

 and conditions could be carried on in a manner which would permit 

 of comparisons being made. As a rule, it was necessary to use small 

 amounts of the extract and a method was needed by which the air could 

 be excluded as completely as possible — and excluded quickly. After 

 various means had been tried, the following plan was found to serve 

 well. Small glass tubes of the same diameter and of practically the same 

 length were heated and drawn out at each end to a fine capillary bore. 

 The extract to be studied could be quickly drawn up to fill a tube, and 

 then the capillary ends could be sealed at once by touching them in a 

 hot gas flame. Fig. 2, Plate I, is a i^hotograph of such a sealed tube. 

 Methylene blue was the "substrate and indicator'' most often used in 

 making a study of the reduction activity. A standard solution of 0.1 

 gram of methylene blue in 100 c. c. of distilled water was employed, 

 Unless otherwise stated in giving the results, six drops of this standard 

 solution from the methylene blue pipette were used to 4 c. c. of the 

 extract. (In most cases this ratio was found to be best.) The stain 

 was thoroughly mixed with the extract and then the extract was divided 

 into two equal parts — the one part to be treated for a certain time with 

 the insecticide and the other, held as a check. Or, if two equal portions 

 of the extract were taken at first, an equal number of drops of the stand- 

 ard methylene blue was added to each from a graduated pipette at prac- 

 tically the same time. At the end of the treatment period both parts 

 were again shaken until they became a uniform blue. Then, from the 

 treated and untreated stained extracts, tubes were quickly drawn full 

 and sealed. Note was made of the period of time required for each tube 

 to become reduced entireh^ free of the blue color. The time intervals 

 given by this method for tubes of treated and untreated extract thus 

 furnished the means for an instructive study of the influence of insecti- 

 cides (or temperature, etc.) upon the reductase activity of any extract. 

 Methylene blue showed a very slight deteriorating influence upon the 

 reductase, it was found, but in the method this is not to be regarded 

 since the stain was added to the treated (with insecticide) and the un- 

 treated portions of the extract at the same time in equal amounts as 

 explained — all other conditions being the same for both portions of the 

 extract, except the one condition under consideration. 



By this method the influence of temperature, chloroform, ether, alcohol, 

 gasoline, formaldehyde, carbon disulphide, hydrocyanic acid gas, toluol, 

 ammonium formate, sodium fluoride, borax, mercuric chloride, pyreth- 

 rum, to-bak-ine and white hellebore was studied. Representative results 

 are given in Table IV where the ratio between the time required to re- 

 duce methylene blue in the check and the time requird in the case of the 

 treated extract may easily be obtained. 



