EXPERIMENTS WITH FORMALIN 83 



treated by formalin were then placed with a small 

 quantity of a pure culture of B. fluorescens liq. 

 (which had been isolated from the tap water) on a 

 sterile 2-per-cent. agar slope, and, after a few days, 

 amoebae had escaped from the cysts and were 

 actively multiplying. This might reasonably have 

 been regarded as a "pure mixed culture," as the 

 bacteria in fixed films looked morphologically pure, 

 but to remove any doubt the culture was plated 

 out both by pouring and spreading, and to our 

 surprise three types of colony appeared. Petri- 

 dish cultures were therefore made to see if a 

 loopful of cysts could be picked off with one of 

 these types alone, but the subsequent analysis 

 showed that only one of them had been eliminated, 

 namely B. flavo coriaceus. This bacillus grows 

 very slowly, forming minute pin-head colonies, 

 which may easily be overlooked. Recourse was 

 again had to formalin, but this time no cysts could 

 be recovered which would grow when placed with 

 pure cultures of different bacteria. Even if only 

 washed with the formalin for half an hour, and the 

 reagent removed with repeated washings, no cultures 

 could be obtained. The uncertainty with formalin 

 is due to the fact that it is impossible to say 

 whether you have killed the cysts or merely 

 rendered them so resistant that the bacterial pro- 

 ducts which cause excystation are unable to pro- 

 duce this effect, and if excystation is delayed too 

 long it will not occur at all. 



Mercuric chloride was then tried in various 

 strengths (0-005 to 1 per cent.), and from one plate 

 cysts were placed in culture with B. f. liq., but 

 again the supposed pure culture consisted of the 



