572 PROTOZOA 



Locke's solution — sterilised by filtration, if necessary. The 

 medium thus prepared and warmed to the required temperature is 

 inoculated with the material containing the organisms to be 

 cultivated. This is introduced at the junction of the solid and 

 liquid medium at the bottom of the tube by means of a sterile 

 capillary pipette, and it is in this region that the organisms 

 chiefly develop when incubated at 37° C. 



The medium has an initial reaction of pH7-2 — 7-8, which is suitable 

 for the growth of amoebae. However, owing to the growth of acid- 

 forming bacteria in the presence of the glucose of the Locke's solution,* 

 the medium soon becomes too acid for continued growth and sub- 

 cultures have to be made every two or three days. 



Many modifications of this medium have been devised by Boeck 

 and Drbohlav, one being the use of ordinary blood agar (or N.N.N.) 

 for the solid medium, and for the liquid, dilute egg white (the white of 

 one egg to a litre of Ringer's solution). 



1120. For the cultivation of Entamoeba histolytica and some 

 other entozoic protozoa, valuable improvements have been made 

 in the constitution of this L.E.S. medium by Dobell and Laidlaw 

 (Parasit., xviii, 1926, p. 283). Also Dobell {Parasit., xx, xxiii, 

 xxvi). They recormnend making up the medium with Ringer's 

 solution f and the introduction of solid rice starch to replace the 

 carbohydrate (glucose in the Locke's solution) of the L.E.S. 

 The very small grains of this form of starch are rapidly ingested 

 by the amoebae, which multiply enormously and often finally 

 store up glycogen in their protoplasm, and encyst. 



Blastocystis, which causes much trouble in most cultures of entozoic 

 protozoa, can be eradicated by the presence of the starch, provided 

 that there are no starch-splitting bacteria present, and these, the 

 authors find, can usually be eliminated by adding flavine (1 in 20,000) 

 to the culture. The starch should be dry and sterilised by heating it, 

 loosely packed in small tubes, to a temperature of 180° C. It is intro- 

 duced into the medium by a platinum loop or spatula, and falls in a 

 little heap to the bottom of the tube, and the amoebae are inoculated 

 on to its surface. 



After trying many different forms of the double mediiun, they find 

 that the richest and most prolonged growths of amoebae are obtained 

 on the one having coagulated horse serum as the solid constituent and 

 Ringer egg-white as the liquid, together with rice starch. 



To prepare this medium, " HS re + S," suitable volumes of whole 

 horse serimi (sterilised by filtration) are placed in tubes, with all aseptic 

 precautions, and set in the form of ordinary slants by heating in an 

 inspissator at 80° C. It is most important not to overheat, for the 



* Owing to the numerous slight modifications now given in the 

 formulae for these fluids, it is necessary to state that the Locke's solu- 

 tion used by Boeck and Drbohlav contained 9 grm. NaCl, 0-4 grm. KCl, 

 0-2 grm. CaClg and NaHCOg, 2-5 grm. glucose in a litre of distilled water. 



t The Ringer solution used by Dobell and Laidlaw contained 9 grm. 

 NaCl, 0-2 grm. KCl, and 0-2 grm. CaClg in a litre of distilled water. 

 For other fluids, see § 1430. 



