925 
from other organisms is easily shown by plate cultures, and that they are 
capable of further multiplication is also easily demonstrated by transfer- 
ring them to media inoculated with a satisfactory symbiotic organism. 
Multiplication will then be renewed. 
Upon a few occasions we have succeeded in obtaining some slight 
multiplication by transferring the organisms from such pure cultures 
to other sterile media, but the reproduction in all cases has been very 
limited and it always entirely ceases after a few transplants have been 
made, whatever the source of the amceba or whatever the medium em- 
ployed ; nor have we been more successful in using various special media, 
some of which contained various enzymic and other organic extracts. 
Details of all these experiments would be tedious and unprofitable, but the 
following brief summary indicates the scope and extent of this line of 
work: 
(1) Ameebe were grown from a head of cabbage, and in order to demonstrate 
whether or no an extract of this cabbage would be a medium for amoebie free 
from bacteria, the cabbage was rapidly macerated with sterile water and filtered 
through a porcelain filter. The result was negative. Again, such an extract, 
inoculated with the bacteria in symbiosis with the ameebe, kept for twenty- four 
hours and then filtered, would not nourish the amcebe. 
(2) Amoebe from liver abscesses and the intestine would not thrive in various 
filtered extracts from human and animal livers, although these proved excellent 
culture media for bacteria. 
(3) Various extracts from, and emulsions of,.bacteria failed to prove satis- 
factory media even where the symbiosis between the ameebe and the living bacteria 
of the same stem was all that could be wished. Some encouragement in this 
direction was obtained in the following way: 
Amebe growing in symbiosis with Spr. cholerw seemed at first to be splen- 
didly nourished by the cholera vaccine prepared by autolytic digestion according 
to R. P. Strong’s method.? When the living bacteria were also present even in 
small numbers, this substance certainly exerted a most favorable influence upon 
the amebe, but with bacteria free cultures such effects were very transitory. 
(4) Liver and other animal tissue extracts obtained in a sterile manner 
furnished no better results than the filtered products. 
(5) Various preparations of blood and the serous effusions of man and animals 
proved unsuccessful. 
(6) Gelatin cultures of Spr. cholere after liquefaction, killed by heat or 
chloroform, were not satisfactory media for the ameebe which were propagating 
with the living Spr. cholere of the same stem. 
IV. SYMBIOSIS AND SYMBIOTIC ORGANISMS. 
EXTRANEOUS OR SAPROPHYTIC SYMBIOSIS, 
Ameebe in water, soil, on vegetables, or in any other location outside 
the animal body, have never been found except in association with other 
microorganisms. 
The variety of such organisms is very great, including bacteria, yeasts, 
* Publications of the Bureau of Government Laboratories, Biological Laboratory, 
Manila (1903), No. 16. 
48040——2 
