528 METHODS FOR INVERTEBRATES. 



a few drops on a slide. Allow this to set. Place on the microscope 

 and focus the upper surface with an inch objective. Tap out on to 

 filter paper some of the liquid in the capillary tube, and then, whilst 

 looking through the microscope, gently touch the film with the fine 

 end of the tube. A small volume of the suspension of cysts will run 

 on to the jelly and will spread out in an area which is quite visible, 

 and which occupies only a small portion of the field. If no cyst is 

 present, or if there should be more than one, place another drop on' 

 a fresh film and repeat till a single cyst is obtained on the film. The 

 method is simple, and with practice one can make half a dozen such 

 cultures in an hour. Place the slide film surface downwards above 

 water in a petri dish (this is conveniently done by resting on two 

 corks), and cover the dish. Examine day by day, till numerous 

 amoebae are found, and then allow them to encyst. From this 

 culture, prepare cultures in test tubes containing the special agar 

 sloped as for bacteriological work. Allow these to grow for a week 

 till covered with cysts. This may be observed with a low-powered 

 lens through the wall of the test tube. Prepare a 3 per cent, solution 

 of hydrochloric acid in sterile distilled water and cover the jelly film 

 in the test tube with this solution and replace the plug. Allow this 

 to act for twenty-four hours. Pour off the acid, and fill up with 

 sterile water, and again pour off, using aseptic precautions through- 

 out. With a platunim loop scrape some of the emulsion of cysts off 

 the tube on to the surface of a fresh sterile tube of agar. Then 

 add a loopful of a very dilute emulsion of the selected bacterium 

 and rub gently over the tube. In the course of twenty-four to forty- 

 eight hours the amoebae will excyst, and multiply, and will generally 

 be found to be in pure culture with the bacterium added. Should 

 they not be so on bacteriological analysis, allow to encyst and again 

 treat the cysts with the acid and proceed as already described. It 

 will be found that the best organisms wherweith to grow amoebae are 

 those naturally occurring in water, one of the best being Bacillus 

 fluorescens non-liquefaciens. When pure mixed cultures have been 

 obtained, the stock cultures should always be kept in test tubes, as 

 the petri dish method over water, although excellent for impure 

 stock cultures, is very liable to allow contaminations to occur. By 

 means of the method described, amoebae have been obtained in pure 

 mixed culture with the following bacteria, and with the results 

 detailed below. 



NAME OF BACTERIUM. EEMARKS. 



B. fluorescens liquefaciens . . Excystation rapid, good growth. 

 B. fluorescens non-liquefaciens . Excystation rapid, good growth. 



