182 BACTERIOLOGICAL EXAMINATION OF WATER. 



1-250 by taking a known volume of it in the pipette as before 

 and adding four equal volumes of broth. The 1-250 dilution 

 is converted into 1-500 by taking a portion of the dilution and 

 mixing with it an equal portion of broth. In this manner the 

 serum is diluted with broth in the following proportions: 1-5, 

 1-25, 1-50, 1-250, 1-500. 



A twenty-four hours agar culture of the suspected organism 

 is now rubbed up with broth in a watch-glass until a perfect 

 emulsion is made. The end of a pipette is now placed in the 

 emulsion, which is allowed to run up the tube until a column of 

 any desired length, usually one-third of the tube, is obtained. 

 The upper limit of the column is marked as before, and the 

 culture is then blown out into a clean watch-glass ; a 

 column of fluid of equal length obtained by the same pipette 

 from the serum diluted 1-5 is now added, and the whole 

 thoroughly mixed. In this way a mixture of serum and culture 

 is obtained, the dilution of the serum being 110. The mix- 

 ture of serum and culture is finally drawn up into the pipette 

 until it is about half filled ; the point is then rapidly sealed in 

 the Bunsen flame. The same procedure is followed with the 

 other dilutions of the serum, and in this wav tubes containing 

 serum, mixed with an equal volume of culture, are obtained in 

 the following dilutions, viz., 1-10, 1-50, 1-100, 1-500, 1-1000. 

 For ordinary diagnostic work these dilutions are most useful, 

 but any other dilutions desired can be readily obtained by 

 applying the same principles. The tubes thus obtained are 

 preserved at the temperature of the laboratory and examined 

 at the end of two hours, and again at the end of twenty-four 

 hours. The results obtained at the end of two hours by the 

 naked eye can be confirmed in the hanging-drop by snipping off 

 the end of the capillary tube and blowing out a drop of the 

 mixed culture and serum on to a cover-glass and examining it 

 in the ordinary way. The capillary tube should be sealed up 

 again and preserved for twenty-four hours. If it is preferred, 

 a drop of the mixed serum and culture from the watch-glass 

 can be at once put up in a hanging-drop and kept under obser- 

 vation for two hours. In my own work I have found that in 

 the hanging-drop so prepared the clumps of bacteria often settle 

 to the bottom of the drop by the end of two hours, and it is 



