258 FAMILY: AMCEBID^ 



only free-living non-parasitic forms. Miisgrave and Clegg (1904, 1906) 

 made extensive observations in Manila. They thought they had culti- 

 vated the amoebae of the human intestine and isolated them from the 

 water supply. They also stated that it was possible to produce dysentery 

 in monkeys by injecting cultures of these amoebae. The w^riter (1907), 

 using the same medium, attempted to obtain cultures of E. muris of mice 

 and E. coli of man, but succeeded in growing only free-living amoebae. 

 He pointed out that the amoebae obtained in culture by Musgrave and 

 Clegg in no way resembled E. coli, which they claimed to have cultivated. 

 It appeared that what actually happened was that cysts of free-living 

 amoebae were constantly passing through the intestine of man and animals, 

 and that it was these which were responsible for the cultures obtained. 

 Walker and Sellards (1913) again investigated the claims made by Mus- 

 grave and Clegg, and showed that the writer's explanation was undoubtedly 

 correct. By causing individuals to ingest the cysts of the amoebae which 

 appeared on agar plates, they were able to isolate the same amoebae a 

 few days later by smearing agar plates with the faeces. Fantham (1911a) 

 gave a description of E. coli based entirely on agar cultures of free-living 

 amoebae. 



The amoebae which appear on agar plates after smearing them with 

 the faeces of man or animals are usually small forms which are rarely 

 more than 10 to 20 microns in diameter. They are actively amoeboid, 

 and live by ingestion of bacteria which grow at the same time. It is 

 necessary, if good cultures are to be obtained, to have a medium which 

 is not too rich in nutrient material, so that the bacteria do not overgrow 

 the amoebae. The medium used by Musgrave and Clegg is very suitable, 

 and consists of agar 20 grams, sodium chloride 0-5 gram, extract of beef 

 (Liebig) 0-5 gram, water 1 litre. The solution is then made 1-5 per cent, 

 alkaline to phenolphthalein. About 10 c.c. of the medium is warmed till 

 liquid, and poured into a Petri dish, where it is allowed to set. On this 

 medium with a low power of the microscope the amoebae may be seen 

 spreading across the surface beyond the edge of the bacterial growth. 

 Multiplication is rapid at laboratory temperature, and in a few days a 

 plate will contain thousands of amoebae. In the central and older parts 

 of the culture the amoebae encyst in spherical cysts. Subculture is readily 

 effected by transferring small portions to fresh plates. It is possible, by 

 using a finely-drawn-out glass filament with a rounded bead at the end, 

 under a low power of the microscope, to transfer a single isolated amoeba 

 to a new plate, and thus to obtain a perfectly pure culture of a single 

 species. 



It has already been mentioned that cultures of amoebae often appear 

 in stale stools, and care must be taken not to confuse them with Endolimax 



