Embryo and A leurone Layer of Hordeum . 805 
These somewhat tedious operations, affording by far the chief oppor¬ 
tunity for reinfection of the material, were carried out with special care and 
precautions, the apparatus, dissecting instruments, and the operator’s 
hands being, just prior to the commencement of the operation, thoroughly 
sterilized by passage through the flame of a Bunsen burner. 
It is of interest to note that the successful sterilization of the grains 
turns on the discovery by A. J. Brown 1 of the existence of a semipermeable 
or selective property possessed by one of the integuments of the seed. The 
steeping of intact uninjured grains in the reagents mentioned is thereby 
rendered possible, as neither the copper sulphate nor absolute alcohol 
penetrates into the interior of the grain. 
Method of establishing and conducting cidtures. The sterilized material 
was quickly transferred to the liquid or semi-solid culture medium contained 
.in small Petri dishes ; either a definite number (5 or some multiple of 
5) of objects (embryos and aleurone layer fragments) were placed on the 
surface of the medium, scutellar or inner surface respectively downwards, or 
a definite number of inner endosperms and endosperms were embedded in 
a thin stratum of semi-solid culture medium of just sufficient depth to cover 
them completely. 
Other methods of establishment of the culture having reference to special 
experiments are described in the sections dealing with these experiments. 
The cultures thus established were conducted under absolutely sterile 
conditions) at the ordinary temperature of the laboratory (15 0 to 18 0 C.), for 
periods of time varying in duration from 3 to 68 days. 
In the case of cultures on liquid media the sterility of the culture 
medium was, at the termination of the culture experiment, controlled by 
removing a small quantity (0-5 c.c.) and introducing it into a series of 
tubes containing various nutrient media-—dextrose-wort, dextrose-maltose 
yeast water, &c.)—and subsequently incubating these tubes at 30° C. for 7 to 
si days. 
Such inoculations invariably furnished negative results, affording in these 
instances ample confirmation of sterility of the culture already indicated by 
macroscopic inspection. 
The subsequent employment of semi-solid gelatine and agar medium 
rendered this system of control unnecessary, and, moreover, provided more 
favourable conditions for the display of the vital activity of certain of the 
objects under culture. 
Numerous series of petty cultures were also instituted with embryo and 
aleurone layer fragments for the purpose of ascertaining whether or not the 
scutellar epithelium and aleurone layer, as stated by Brown and Morris and 
that of an equal number of aleurone layers removed by cutting in the manner described above. The 
difference in no case amounted to more than 4 to 5 %, the former value being the nearer. 
1 Ann. Bot., xxi, p. 79 (1907). 
