THE TOXICITY TO FUNGI OF VARIOUS OILS AND SALTS. 17 
In a few instances where the preservatives were low in toxic 
properties more than the specified 3 c. c. was necessary in order to 
secure the higher concentrations, and in these cases it became nec- 
essary to take into consideration the excess of preservative, and, 
considering it roughly as having the specific gravity of water, to 
reduce the agar by just this amount, in order that the combined vol- 
ume might not exceed 20 c. c. 
The concentrations to be used in the first series x of experiments on 
a given preservative were governed largely by the judgment of the 
investigator. The results thus obtained usually determined between 
what limits it was necessary to continue the work. The experiments 
were then carried on between these points, usually to within an accu- 
racy of about 10 per cent for the actual and total inhibition point 
for each preservative. Thus, if growth stopped at 0.5 per cent or 
below, the tests were carried to the nearest 0.05 per cent; if between. 
0.5 and 1 per cent they were carried to the nearest 0.1 per cent, and 
so on up to the highest concentrations employed, usually 40 per cent, 
which would thus be tested, to the nearest 4 per cent. This 40 per 
cent concentration is equivalent kran injection of 24.9 pounds of 
the preservative per cubic foot, and it was thought unnecessary from 
a practical standpoint to go above this point. 
After the proper quantities of preservative had been placed in 
50-c. c. glass-stoppered bottles, these were sealed and sterilized in 
exactly the same way as the agar bottles and along with them. 
As a few experimental weighings before and after sterilization indi- 
cated that no loss occurred, even of such volatile substances as are 
contained in the lowest fractions of coal-tar creosote, the method 
may be considered safe. 
After sterilization, both the agar and preservative bottles were 
heated on the water bath, then transferred to a sterile culture case 
(PI. I, fig. 2), where the hot agar was poured into the preservative 
bottle and thoroughly mixed. In some cases one or two sterile glass 
beads were added to the preservative bottles to facilitate the mixing. 
These were removed later. 
The agar-preservative mixtures were then poured into sterile Petri 
dishes 100 mm. in diameter and 10 mm. deep. After cooling, each 
plate was inoculated at the center with a weft of mycelium 5 or 6 
mm. square, cut from a Petri-dish culture (PI. I, fig. 3) 2 to 3 weeks 
old of the fungus desired, either Fomes annosus Fr. or Fomes pinicola 
(Sw.) Fr. The dishes thus prepared were then placed in an incu- 
bator and held at approximately 25° C. for periods varying from 4 
to 10 weeks, usually from 4 to 6. 
1 A series consists of a set of progressively increasing concentrations of a given preservative, tested at 
the same time against the action of a single fungus. 
88340°— Bull. 227—15 3 
