THE CULTIVATION OF ENDAMCEBA HISTOLYTICA 173 



beads. Fifty cubic centimeters of Locke's solution (see below) are then 

 added and the mixture broken up by shaking. Test tubes are then filled 

 with sufficient of the mixture to produce slants of from one to one and 

 one-half inches in length upon coagulating by heat. The tubes are then 

 slanted in an inspissator and heated at 70° C. until the egg mixture is 

 solidified. They are then autoclaved at fifteen pounds pressure for twenty 

 minutes. The tubes are then covered to a depth of one centimeter above the 

 egg slant with a mixture composed of eight parts of sterile Locke's solu- 

 tion and one part of sterile inactivated human blood serum, and incubated 

 to determine sterility. 



Medium No. 2. Locke-Egg-Albumen, or L.E.A. Medium. Drbohlav modi- 

 fied the above medium by using crystallized egg albumen instead of human 

 blood serum. A one per cent solution of the crystallized egg albumen in 

 Locke's solution was employed and this was sterilized by passage through 

 a Berkefeld filter and then added to the tubes containing the egg slants as 

 described above for the L.E.S. medium. 



The initial reaction of these media varied from pH 7.2 to pH 7.8 and 

 needed no adjustment. 



The Locke solution used had the following formula: 



NaCl 9.0 gms. 



CaCU 0.2 gm. 



KCl 0.4 gm. 



NaHCOs 0.2 gm. 



Glucose 2.5 gms. 



Distilled water looo.o cc. 



The solution is sterilized in the Arnold or the autoclave. The human blood 

 serum must be passed through a Berkefeld filter after adding the Locke 

 solution, and sometimes a second filtration is necessary. 



E. histolytica may be cultivated without difficulty upon either 

 of these media and subcultures should be made every other day 

 from the sediment at the bottom of the tubes, obtained by a sterile 

 pipette. The most favorable temperature for incubation was found 

 to be 37° C. 



Kofoid and Wagcncr's Medium (1925). 



These investigators have modified the L.E.S. medium of Boeck and 

 Drbohlav by adding to the egg slant ten cubic centimeters of Locke's solu- 

 tion containing 0.5 cubic centimeter of defibrinated rabbit blood. They state 

 that this medium is not as favorable to the growth of bacteria as the Boeck 

 and Drbohlav media, thus favoring the growth of the amoebae, and that 

 transfers should be made every forty-eight hours. 



Dohcll and Laidlazifs (1926) Media. These investigators recom- 

 mend the two following media in the cultivation oi E. histolytica: 



Medium No. i. An egg-serum medium made as recommended by Boeck 

 and Drbohlav but with Ringer's solution instead of Locke's solution. Ringer's 



