ale eg Sabie 
924 
avail against the protozoa in the encysted state, and this latter phase of 
their life cycle is in reality the most dangerous one, even if it is not the 
only period in their existence when they are a serious menace. 
PURE CULTURES. 
Pure cultures of amebex, which will continue to propagate in media 
free from other living microdrganisms, have not been obtained. That 
this fact is due to difficulty or inability to free the amoebe from 
other associated organisms without injury to the protozoa, has been stated 
by others, and we formerly tentatively concurred in the belief, but this 
position must now be abandoned. ; 
Perhaps the easiest and simplest method is to get the protozoa in 
culture with some delicate bacterium and then place the cultures aside 
until all the bacteria are dead. Bacteria grow very poorly on media 
suitable for amoebx, and some of them, such as Spr. cholera, pneumococci 
ete., die very quickly under such surroundings unless frequent trans- 
plants are made. This process may be hastened by still further reducing 
the amount of nutritive substance in the amcebe medium. 
Other methods by which we have succeeded in obtaining amoebw pure 
and free from bacteria on artificial media are as follows: 
Sometimes, in old, encysted cultures, the bacteria which are present may be 
destroyed by heat and the amebe still retain their viability. This is shown by 
transplants when they are in this condition made to media which contain a 
satisfactory symbiotic bacterium. The bacteria in an old, encysted culture may 
sometimes be destroyed, without killing the amaba cysts, by the careful use of 
certain antiseptic solutions, such as benzoyl-acetyl peroxide, succinie peroxide 
acid, formalin vapor, chloroform, ete. 
In some instances success in separating and obtaining living ameebie, free from 
bacteria, can be obtained by using the plate methods described in our first paper 
for securing ameba together with a pure culture of bacteria, For this purpose 
the original method is only altered in the particular that the bacterial rings, 
after being made on the plate and the organism allowed to develop, are then 
killed by exposing the plates to a temperature below the melting point of the 
agar. The plates are then inoculated in the usual manner within the ring, with 
the mixed cultures. Occasionally, under these condition’, the amcbe not only 
grow through the dead bacteria but also leave the living ones behind them, and 
in this manner may be found encysted at a suflicient distance from living bacteria, 
to allow of their being removed with a platinum loop. 
Amebe free from bacteria may also often be obtained by the injection of 
the mixed cultures into the livers of monkeys or subcutaneously, and then inocu- 
lating subsequent animals with the contents of the abscesses thus produced until 
finally an abscess is obtained in which the amobw are found to be bacteria free. 
The most satisfactory method is the one which consists in allowing the bacteria 
to die out of a culture, leaving the encysted amoebxe behind. 
In considering the above-mentioned means of separating living ameebe from 
their symbiotic organisms in cultures, it must always be borne in mind that the 
amebe encyst under such conditions and do not multiply. 
By these methods numerous working cultures of amecbee, in large num- 
bers and free from bacteria, may be obtained. That the amcebex are free 
