284 SELLARDS. 



taminating organisms were eliminated. It was a comparatively simple matter 

 to obtain an apparently pure culture of bacteria, but more extended observations 

 often revealed the presence of contamination. Thus minute quantities of a 

 culture when plated out for development of single colonies frequently showed 

 nothing but B. prodigiosus, while in the course of a few weeks, other bacteria 

 might appear in the original culture, although it had been carefully protected 

 from contamination. It is to be noted that the culture of prodigiosus lost its 

 power of producing pigment on the amoebje agar, but this property was at once 

 regained on the first transplantation to ordinary agar. In the isolation of 

 amcebae with Vibrio cholerce, a culture was readily obtained which showed only 

 cholera colonies when small quantities were inoculated on ordinary agar. How- 

 ever, the inoculation of a large quantity of growth into a highly active anti- 

 cholera serum resulted in the destruction of practically all of the cholera 

 organisms and a spore-forming bacillus developed in the serum. 



The stock cultures of amoebae were always kept in test-tubes. Bacteria were 

 added at practically every transfer and the inoculations were made in the same 

 manner as for the purification of the cultures; that is, the entire surface was 

 inoculated with B. prodigiosus, but only the base of the slant with amoebae. 



Technique of cultivation. — The majority of the various media for amoebae 

 depend upon the same principle, namely the reduction of the nutrient material 

 to such a minimum that only a scanty development of bacteria can take place. 

 Thus, there are the dilute liquid media such as the hay and straw infusions; 

 the solid medium of Beyerinck(4) is prepared with agar which has been repeat- 

 edly extracted with water; the alkaline agar of Musgrave and 01 egg (5) contains 

 0.03 to 0.05 per cent of beef extract and no peptone. An agar based upon the 

 last formula was used for maintaining the stock cultures of amoebse. The 

 principal difference consisted in the reactions of the media, the final product 

 when ready for inoculation being neutral to phenolphthalein. The following 

 formula was used: Agar-agar 2.5 per cent, Liebig's beef extract 0.05 per cent, 

 normal sodium hydroxide 2 per cent. This medium, without clarification, was 

 sterilized at 7 kilograms pressure per square centimeter for three-fourths of an 

 hour. After sterilization it was found that the reaction of the medium was 

 neutral to phenolphthalein, the alkali presumably having combined with the 

 organic material in the agar. Furthermore, under these conditions of temperature 

 and duration of heating, the quantity of alkali which was sufficient to render 

 the medium permanently alkaline was slightly in excess of the amount which 

 was sufficient to prevent the agar from solidifying at room temperature. 

 Nevertheless, although the reaction of the medium was ultimately neutral, 

 to phenolphthalein the addition of the excess of alkali gave a better medium 

 for the cultivation of amoebae than was afforded by the use of agar which was 

 just neutral or slightly alkaline before sterilization. 



On agar media of the ordinary composition with an acid reaction such as are 

 used for bacteria, no growth of amoebae could be obtained, although some investi- 

 gators have used such a medium successfully. (6) Under certain conditions, when 

 it was desired to obtain amoeba-free cultures of bacteria which were associated 

 with amoebae, ordinary agar was used, the first transplantations being free from 

 amoebae. 



With the development of the methods for the cultivation of amoebae, solid 

 agar media have largely replaced the liquid media which were used by the earlier 

 workers. Although straw infusions and similar fluids have been used in ob- 

 taining amoebae for study, their isolation and propagation has been carried out 

 chiefly on solid media. It has even been stated that, with many amoebae, direct 



