Balls . — • Temperature and Growth. 
575 
The possible Limiting Factors in Standard Culture. 
The essential points of the standard culture experiment are as 
follows : — • 
Minute fragment of mycelium develops hyphae in a relatively large 
volume of fresh, rich medium for twenty hours at a temperature which 
is not higher than 20° C. 
A few of these hyphae are then removed, placed in the special damp 
chamber described above, and provided with a large excess of pure oxygen 
for their respiration. 
The temperature of the chamber is not raised above 20° C. until growth 
has begun in the hypha under observation. 
The temperature is then raised at least i° C. in every four minutes. 
All these precautions have proved to be considerably in excess of 
what is needed for any one of them. 
In such a culture the possible limiting factors are somewhat as 
follows : — 
1. Amount of available oxygen. 
2. Rate of diffusion of oxygen through wet semi-permeable 
membranes. 
3. Amount of carbon dioxide present 
4. Rate of diffusion of carbon dioxide. 
5. Amount of available food material. 
6. Rate of diffusion of food materials. 
7. Rate of chemical change in the protoplasm, limited by temperature. 
8. Water-supply. 
9. Percentage of ‘ # 5 in the cell sap. 
10. Rate of outward diffusion oi i x\ 
This arrangement is not quite logical, because the amounts depend on 
the rates of diffusion, but it serves as a convenient mode of statement. 
1. The amount of oxygen. The same results have been obtained with 
air in the chamber as when oxygen was employed, except in two early 
experiments when a very small air bubble was used for the respiration 
of a large mass of mycelium. In order to eliminate any possibility of this 
factor being limiting I afterwards used pure oxygen in the chamber for 
all experiments, though I believe it to be quite unnecessary. 
2. Rate of diffusion of oxygen . This can hardly be a limiting factor 
at any time, for the use of pure oxygen has no effect on the stopping point, 
nor (as far as one can judge) on the rate of growth, though the partial 
pressure under these conditions is five times as great as in ordinary atmo- 
spheric air, and the rate of solution in wet membranes is correspondingly 
increased. 
