178 OAKUISON AND LEYNES. 



NORMAL DEVELOPMENT. 



Tlie ova ol' raniijoiiivnis were first developed to tlie free miracidial 

 stage by Manson in 1880, who after shaking tlie sputum with water and 

 renewing tlie Avater daily for about a week found the motile mii'acidia 

 developed in from four to six weeks after the sputum was expectorated. 

 The favoi'able results of such a method wei'e entiix'ly in liarmoiiy with 

 what was known oi the develoj^inent of the ova of other digenetic trema- 

 todes and in our hands the method has never failed throughout a series 

 of several hundred such cultivations of the ova, provided two important 

 requirements were complied with, namely, that the washing of the ova 

 be thorough and that it be not too long delayed after the sputum is 

 expectorated. 



Sedimentation. — The method we have used for eleai'ing the ova from 

 the sputum or fgeces is as follows : 



The specimen (sputum or faeces) is placed in a tall museum jar, holdinff 

 about 2,500 cubic centimeters and about 10 centimeters in diameter. Such a jar 

 while holding a good volume of water confines the sediment to a comparatively 

 limited area. Tap water is run into the jar as violently as possible in order 

 to break up the mucoid or solid parts of the specimen and the jar allowed to stand 

 until the ova have sunk to the bottom, which usually happens in from one to 

 four or five hours, depending upon the specific gravity of the solution. 



If the specimen be sputum, after standing for an hour or two, the water is 

 decanted ort' to as close to the sediment as possible and the jar refilled and 

 allowed to stand until the following day, when the water is again changed. If, 

 after sedimentation has taken place, the water is perfectly clear it is poured oflf 

 and the sediment transferred to a bottle of half a liter capacity or less and 

 not again disturbed, unless the water it contains becomes clouded or covered 

 with a scum by the excessive growth of bacteria or other organisms. 



Faeces are more difficult to wash satisfactorily. In the case of stools with 

 a large amount of soluble matter, we have frequently used 10 liters or more of 

 water in order to get the first solution sufficiently light to allow the ova to 

 settle and it was found to be necessary to change the water repeatedly until the 

 faecal character of the stool apparently disappears. When once thoroughly washed, 

 the ova from faeces develop as well apparently as those from sputum, but we 

 have frequently failed to get them sufficiently and quickly clean and both for 

 this reason and also because of their bulk and of the larger amount of sediment 

 remaining faecal specimens have been little used in our experimental work. 



Conditions of temperature and light. — Cultures of the ova, including 

 those used as controls for experiments, regularly have been kept un- 

 covered, out of direct sunlight, upon a laboratory shelf, where the 

 temperature varies from 25° to 34° C. 



Since thoroughly washed ova kejDt under these conditions developed 

 satisfac-torily and as under no vai'iation from these conditions was develop- 

 ment nioie constant, more rapid, or apparently more healthy, we have 

 used the maximum development so obtained as the standard for com- 

 parison and considered it, at least for the laboratory, as normal. 



Time required. — Manson found developed miracidia in from four 



