322 
Transactions of the Royal Society of South Africa. 
Methods. 
Motility. —Ill determining the presence or absence of this character a 
hanging-drop preparation from a 4 to 6 hours bouillon culture, or the 
*' condensation " fluid of a sloped-agar cultuie of the same age, was examined. 
Tested in this way the presence (or absence) of motility proved to be a 
definite character. 
Liquefaction of Gelatin. — This was tested for by making stab inoculations 
in ordinary nutrient gelatin and incubating the tubes at 22° C. for two weeks. 
Indol Froduction. — The presence or absence of this property was deter- 
mined by testing a ten-days peptone water culture of the strain in question 
with Ehrlich's reagents, paradimethylamidobenzaldehyde and persulphate of 
potassium, according to the usual procedure. 
Voges and, Proska uer Reaction. — For this test a 2 per cent, peptone water 
solution containing 1 per cent, glucose was inoculated and incubated at 37° C. 
for three days, when a solution of potassium hydrate was added and the 
tube allowed to stand at room temperature for several hours. A 
positive reaction was indicated by the development of a red fluorescence. 
Fermeyitation Test. — The basis of the medium used for testing sugar 
fermentations was a 2 per cent, peptone solution with 0'5 per cent, sodium 
chloride. To avoid the possible decomposition of the sugar in the medium 
by overheating in the process of sterilisation, the different fermentable sub- 
stances were added to the ah-eady sterilised medium in the form of sterile 
watery solutions. The proportion of sugar in the medium was 1 per cent. ; 
neutral red (0*25 per cent, of a 1 per cent, watery solution) was added as 
an indicator of acid production. The medium was distributed in Durham's 
tubes (for the observation of gas production), and placed at 100° C. in the 
Koch's steriliser for ten minutes on two successive days. This short final 
sterilisation, while not acting deleteriously on the sugar, was sufficient to 
ensure complete sterility. 
I have observed that for the proper appreciation of gas production 
fluid media must be used, as shake or stab cultures in solid agar media are 
open to fallacy. With certain specimens of peptone water agar (without 
meat extract), it was found that gas production might result on inoculation 
with B. coli, i. e. even in the absence of sugar, and this was especially 
marked if the agar was not freshly prepared. 
Thus, a shake culture of B. coli communis made in peptone water agar 
immediately after preparation showed no gas production in the medium ; the 
same medium a week later was again inoculated and on this occasion a 
considerable number of gas bubbles appeared in the medium after twenty- 
four hours' incubation ; a fortnight later inoculation resulted in an abundant 
production of gas throughout the medium. No acid formation was, however, 
noted. 
