300 Dr. T. Goodey. Further Observations on 



soaked up the last traces of liquid. Fresh filtered liquid was then poured 

 into this side of the trough until it rose above the level of the top of the 

 glass partition. The plasticine was again removed, allowing a flow of filtered 

 liquid into the side containing the protozoa. The electric current was 

 reversed and again switched on, this time causing the protozoa to swim 

 back into the fresh filtered liquid, in which a large number were obtained. 

 By this means the protozoa were washed in one lot of bacteria-free liquid 

 and finally secured in a comparatively clean condition. Bacterial counts 

 showed that the original mixed culture contained about 36 million bacteria 

 per cubic centimetre, whilst the liquid in which the protozoa were finally 

 obtained contained only 300,000 bacteria per cubic centimetre. Thus a very 

 considerable reduction in the numbers of bacteria was effected, although it 

 was not possible to rid the protozoa entirely of bacteria : a result scarcely 

 to be expected seeing that large free-swimming ciliates might easily carry 

 over bacteria on the surface of their bodies. The requisite quantity of this 

 liquid containing " bacteria-free " protozoa was then taken and used for 

 moistening one of the samples of partially sterilised soil. 



.Another sample of treated soil was inoculated with a large number of 

 amceba cysts free from bacteria, obtained by the following method of Cropper 

 and Drew.* A single cyst of a small limax amceba, obtained originally 

 from soil, was picked off from the surface of an agar plate by means of a 

 capillary pipette. The cyst was lying in an area free from bacteria, having 

 moved out to the clean agar in advance of the growing bacteria. This cyst 

 was next transferred to a fresh agar plate and an emulsion of Bacillus 

 fluoresceins non-liquefaciens in sterile distilled water was added. After 

 incubating at 20° C. for a few days the plate was examined, and it was 

 found that the single cyst had given rise to a large number of amcebse. Two 

 or three more fresh agar plates were then inoculated from this culture of 

 amoeba? and B. fl. non-liq., and were left in the incubator until the amoeba? 

 had all encysted. A 2-per-cent. solution of hydrochloric acid (2 per cent, 

 of the ordinary pure reagent) was then added to the cultures and allowed 

 to act for two days. It was then poured off and the surface of the cultures 

 washed with several changes of sterile distilled water so as to get rid of all 

 traces of acid. This treatment killed off the bacteria but left the amoebae 

 cysts uninjured, as was proved by making sub-cultures of the cysts on to 

 fresh agar plates. On these, bacteria failed to appear, but when a suspension 

 of B. fl. non-liq. was mixed with the cysts and the mixture inoculated on 

 to nutrient agar, the amceba? excysted and readily multiplied. Quantities of 

 the cysts were obtained by gently scraping the surface of the agar, and 

 * ' Researches into Induced Cell-reproduction in Amcebse,' p. 83. London, 1914. 



