28 Killing of Botinftis Spores by Phenol 



the temperature indicated in each experiment, and kept stirred; and 

 samples were withdrawn at intervals for examination. The volume of 

 the mixture was not less than 10 c.c, usually about 20 c.c, and the 

 samples were of 0-5 c.c. volume, except when the suspension was very- 

 dense, in which case smaller samples were taken. Each sample was 

 immediately dropped into a centrifuge-tube containing 8 c.c. of distilled 

 water (in experiments with high concentrations of phenol 10-5 c.c. were 

 used), and rapidly centrifugahsed. The supernatant fluid was removed 

 till about 0-25 c.c. remained, and in this the deposited spores were stirred 

 up to form a suspension. From the latter a standard platinum loopful 

 was transferred to a coversHp, on which had been placed a standard 

 loopful of Czapek's fluid, and the two drops well mixed. The covershp 

 was then placed on a van Tieghem cell, and this incubated at 26° C. 

 for 22 to 24 hours. Two to four such cells were prepared from each 

 sample. After incubation they were examined with a Zeiss C objective 

 and No. 6 eyepiece, and the proportion of spores that had germinated 

 ascertained by counting successive fields. This is readily done, the 

 spores capable of germination producing in the time allowed hyphae 

 or germination tubes which are easily seen. As a rule, the whole drop 

 was counted, and to get rehable figures it is necessary to count several 

 hundred spores from each sample. Four or five hundred spores are 

 enough, but in most cases six or eight hundred, and sometimes more, 

 were counted. It is sometimes stated that more spores germinate round 

 the edges of the drop than in the centre (2). I was unable to convince 

 myself that this occurred under the favourable germination conditions 

 used in these experiments, but it is advisable to count all spores in the 

 whole drop, in order to avoid any error from this source. 



The method gives uniform and satisfactory results. The first experi- 

 ment given below is set out in detail to illustrate the amount of variation 

 obtained in duphcate sHdes. The variation does not exceed the standard 

 error of sampling. But to get consistency the technique must be carried 

 out with rigid uniformity and scrupulous attention to details. All shdes, 

 pipettes, coverslips, etc. are, of course, cleaned and sterihsed. There 

 would seem to be great opportunity for contamination in mixing the 

 drops on the sHps, but as a matter of experience contamination is a 

 rare occurrence — I estimate it at less than 2 per cent, of all preparations 

 made. The bottom of the van Tieghem cell (of which the rings were 

 fixed with beeswax) was covered with Czapek solution, the covershp 

 laid on without vasehne or other cement, and the whole cell placed in 

 a covered glass dish containing distilled water to prevent drying. In 



