( 149 ) 
In the first place it was necessary to subject their distribution in 
nature and their isolation to an investigation, beeause the literature 
thereon offers but very deficient data. The best way to attain this 
object seemed to try whether the method of “accumulation” gave 
in this case, as in so many others, any definite result, and that for the 
following reasons. 
The character of this way of experimenting is the cause, that many 
biological properties of the species there by accumulated may be 
predicted ; 
it renders it possible, in a simple way, directly and with certainty 
to isolate from nature a determined species; this is of special 
interest imasmuch the cultures of most bacteria, by being kept in 
the laboratoria, change their character to such a degree as to become 
irrecognisable, so, that the descriptions, found in bacteriological literature, 
according as they are made after newly isolated or long kept material, 
may be wholly different ; 
it teaches us to recognise the sought- for species in the different 
varieties occurring in the material used for infection, as these varieties 
are bound to corresponding culture conditions; 
the identification and synonymy of the bacteria, which are always 
extremely difficult, even in case we possess good descriptions, made 
of freshly isolated cultures, are much facilitated by good “accumu- 
lation experiments”; 
these may, moreover, be controlled by anyone, and render the 
investigator independent from material isolated by others. 
For the arrangement of my experiments I have followed the 
example given by Dr. H. H. Gran’) of Bergen in his researches in 
the Bacteriological Laboratory at Delft on denitrifying sea bacteria. 
By exclusively using nitrate as source of nitrogen in the culture 
liquid, which was contained in a cotton-plugged flask, so that the air 
could freely enter, he succeeded to restrict considerably the number 
of developing species of bacteria, when taking fresh sea-water for 
infection, bringing the denitrifying species to vigorous growth. He 
furthermore selected, as source of carbon the caleiumsalts of organic 
acids, by which the prejudicial alkaline reaction, which appears in 
bouillon in consequence of the decomposition of the alkalinitrate, 
was avoided. Mostly calciummalate was used, which is a very 
good bacterial food, and has moreover the advantage of solving only 
to 0,8°/, at 25° C., so that it can be added to an excess, whence, 
as the salt is oxidised, a new quantity is solved. 
1) Studien über Meereshacteriën 1, Bergens Museums Aarbog 1901 N°, 10, 
