31 
devoid of it. It may be that the leaves tested came from near a barn 
door which was perhaps frequently opened, thus freely permitting the 
destructive action of the sunlight. Therefore in controlling a curing 
crop in the barn leaves should be tested not only from near the wall, 
but also from the more central part of the barn. 
‘The test was at first made in the following manner: The tobacco 
leaf was deprived of the large ribs, and after drying at a temperature 
of 40° C. (104° FEF.) was very finely pulverized. One gram of this dry 
powder was mixed with 20 ¢. c. of water and left to stand for five 
hours in an Erlenmeyer flask at the ordinary temperature. To 10 c. ¢. 
of the filtrate a few drops of an alcoholic solution of guaiac resin were 
added and the mixture was left to stand for ten minutes, with moderate 
shaking. <A blue coloration indicated the presence of oxidase. Since, 
however, the brown color of the tobacco extract is often so dark that it 
interferes with the recognition of a weak blue reaction, it was found 
preferable in such cases to pour guaiac tincture cautiously on the sur- 
face of the tobacco extract, avoiding a thorough mixing. The blue 
color will soon appear as a ring on the surface of contact of both 
liquids. If, however, no blue coloration appears, a few drops of perox- 
ide of hydrogen are added. A blue coloration in this case is due to 
the presence of peroxidase. In order to test for the peroxidase in 
presence of the oxidase in the tobacco extract, it is necessary to heat. 
the liquid for three minutes to 70° C. (158° I.) in order to first kill the 
oxidase before the test for the peroxidase is made. Guaiac solution in 
presence of peroxide of hydrogen is now left to act on the cooled 
solution. The blue reaction for peroxidase sets in at once. 
Some additional remarks may be made on the tests with guaiac 
resin. A solution of guaiacum resin is prepared by dissolving at the 
ordinary temperature 2 grams in 100 ¢.c. of absolute alcohol. This 
tincture must be kept cool and in the dark, and be renewed from time 
to time, since the active principle, the guaiaconic acid, is otherwise 
gradually oxidized. Old tincture will soon turn bluish on addition of 
hydrogen peroxide alone. Attention must be paid to the reaction of 
the liquid to be tested, which must not be alkaline but neutral or slightly 
acid, and also to the quality of the resin, since worthless kinds exist in 
the market. A normal resin must yield an intense blue color with 
highly dilute nitrous acid. Only the interior parts of transparent pieces 
of guaiacum resin should be selected for preparing the tincture of 
guaiac. It is of no particular advantage to use the guaiaconie acid, 
instead of the resin itself. 
An attempt has been made to disqualify guaiacum resin as a reagent 
for oxidizing enzyms on the ground that nitrous acid, ferric chloride, 
and other oxidizing mineral compounds also yield a blue reaction. 
Such objections are groundless when fresh organized tissues are under 
investigation. Other oxidizing influences are easily recognized by 
control tests, which the careful worker will not omit to make. The 
