EXPERIMENT STATION BULLETINS. 457 



at 58° to R0° C. A low temperature tends to conserve the redncing 

 activity — indeed, it serves, at once, to conserve and to inhibit the reduc- 

 ing power of the extract. No. 31 of Table TV gives a trial with extract 

 kept for three hours at 0° F. When this extract was quickly but gently 

 warmed to room temperature, it reduced methylene blue in 8I/2 minutes, 

 whereas extract that had stood exposed to the air at 70° for three hours 

 required six hours to reduce the color. The checks of fresh extract at the 

 beginning reduced the same amount of methylene blue in one minute and 

 in ll^ minutes. The reducing activity of fresh extract seems to be 

 best at a room temperature of about 70° to 80° P.. but it rapidly 

 deteriorates during the first five to six hours after extraction fat the 

 room temperature) and gradually, more slowly afterward. This is true 

 even when the fresh extract is confined from the air. For example, a 

 certain check of perfectly fresh extract reduced its methylene blue more 

 than six times faster than another portion of the extract (A) which 

 was tested after its confinement from air for IV2 hours from the moment 

 of its extraction. Nevertheless "A" was 2I/2 times faster in reducing 

 its methylene blue than a third portion (B) of the same extract which 

 had been exposed to the air during the II/2 hour period. One might think 

 from this that confinement from air tended to conserve the reducing 

 activity. It is necessary to remember, however, that a dark melanic 

 pigment developed in any extract (as ''B") exposed to the air, due to 

 the influence of the oxydase present, and that a portion (as A) confined 

 from air could develop no pigment. When ''A" and "B" were finally 

 given the reduction-test with methylene blue, then (at the end of li>4 

 hours in this example), the reductase in "A" had only methylene blue 

 to reduce while that in "B" attacked the dark pigment as well as the 

 methylene blue. It seems likely, therefore, that the longer time for the 

 reduction of the methylene blue was required by "B" because of the 

 greater work in reducing the melanic pigment in addition to the methy- 

 lene blue, rather than because its reducing activity had become less than 

 that in "A". 



Mention has already been made of the fact that when fresh extract 

 is exposed to the air, it darkens first, only in a thin film at the surface. 

 Also, if the fresh extract is allowed to stand after it has been stained 

 with methylene blue and shaken so that the color is uniform throughout, 

 it will be but a few minutes until the surface only will be blue — a re- 

 duction of all color in the deeper portions of the container having taken 

 place. After a time, however, the blue coloration will begin — slowly at 

 first and then more and more rapidly — to extend deeper into the con- 

 tainer; in the case of the unstained extract, the dark melanic pigment 

 will gradually form more rapidly as time goes on. It was soon found 

 that this increased rapidity in the reoxidation of the methylene blue 

 and in the formation of the melanic pigment coincided or tallied with 

 the deterioration in the activity of the reductase of the extract on stand- 

 ing. That is to say, when the extract was new and its reductase still 

 remained strong, it visibly held in check the work of the oxydase in the 

 same extract. This proved as well to be the case when alcoholic guaiac 

 or hvdroquinone solution was used in the extract as the as^ent to he 

 oxidized. Now. as may be seen by Nos. 1 and 2 of Table TV. gasoline was 

 found to be quickly and strongly deleterious to the reducing activity of 



