Fungi on Culture Media. 117 
plates, as uniform as possible (i. e. containing the same depth of the same 
medium), was inoculated with the fungus to be tested. When these cultures 
had reached a certain age a second batch of plates of the same size was 
prepared, and one of the second batch, still without inoculation, inverted 
over each culture of the first batch, the lids being removed. Similar unin¬ 
oculated plates were kept unexposed as controls. After one to two days’ 
exposure all the plates of the second batch were removed and their lids 
restored as rapidly as possible, after which they were inoculated with the 
same or another fungus, and the growth in a given time, usually two days, 
determined. 
As an illustration the following experiment may be quoted. The 
staling was produced by ten-day-old cultures of Sphaeropsis on potato 
agar. The exposed plates had shallow layers of potato agar. After three 
days’ exposure, the exposed and control plates were inoculated with 
Sphaeropsis. The diameter of the new growths in two days were as follows : 
On unexposed plates (eleven colonies) . 1-57 cm. (max. 1-65, min. 1*5). 
On exposed plates (twelve colonies) . . 0*42 cm. (max. 0-5, min. 0-3). 
This staling effect soon disappears if the exposed plants are allowed to 
remain for some time with their lids off before inoculation. Also, the growth 
on the exposed plates tends to recover its normal rate as time goes on, 
probably by gradual escape of the staling gas. Again, the staling effect is 
more pronounced if the exposure is continued after inoculation. 
A large proportion of these experiments were carried out before the 
nature of the staling substance was recognized. Cultures on potato agar, 
where the evolution of ammonia is small and not readily recognizable 
as such by its smell, were employed, and so a large number of control 
experiments were made to eliminate other factors. Two of these may 
be mentioned. A certain amount of drying of the exposed plates takes 
place during the process, but tests showed that this was of no importance. 
Also the carbon dioxide evolved by the cultures plays no part. Plates 
exposed in 40 per cent, carbon dioxide show no signs of being stale after 
removal from the gas. The absorbed carbon dioxide is apparently dissipated 
with great rapidity. 
Deep plates of a given medium are more slowly staled by this treat¬ 
ment than shallow ones, as is illustrated by Table III. 
Table III. 
Plates of Potato Agar exposed to Cultures of Sphaeropsis on Potato Agar. 
Unexposed, deep 
,, shallow 
Exposed (two days), deep 
,, ,, shallow 
Growth of Sphaeropsis in 
First two days. Second two days. 
1-91 2.54 
1.94 2.37 
J '/ 7 
1.10 
2 '35 
2-07 
These figures also illustrate recovery from staling. 
