SULPHIDE PRODUCING ORGANISMS 
By E. N. COUTTS, M.B., Tor. 
Johnston Colonial Fellow 
The bacterial method of sewage treatment having brought into prominence the 
action of the septic tank, it was thought that a contribution to the study of the 
bacteria which give rise to the black colour of the sludge might not be without 
interest. 
Substances examined. Sludge of septic tanks, faeces, black mud and water, and 
decaying vegetable matter were examined for the presence of these bacteria. 
Methods. For the isolation of the organisms, some of the material was added 
to a flask containing 100 c.cm. sterile water ; I c.cm. of this dilution was added to 
a second flask, etc. Plates were made of nutrient agar with i c.cm. of each dilution, 
and incubated at 37° C. Further, tubes of sterile ferro-peptone medium (peptone 
water containing tt-i c.cm. saturated solution tartrate of iron per litre) were at once 
inoculated with original material, and incubated at 37° C for twenty-four hours. 
Dilutions of these cultures were then made, plated, and incubated as before. 
By the latter method the stronger sulphide producers were more readily obtained, 
as they multiply very rapidly, apparently suppressing other bacteria. 
The colonies thus obtained, differing in so far as macroscopic and microscopic 
appearances indicated, were testeci for sulphide producing properties. And one might 
mention here that while differences in appearances of colonies are certainly of service 
in separating different bacteria, yet the same organism may present several strikingly 
different appearances in its colonies according to age, situation, or other unknown 
conditions. 
The following method of testing for sulphide formation was ascertained by 
experiment to be the most valuable. A solution of ten per cent, peptone in tap- 
water is made alkaline by the addition of 20 c.cm. normal potassium hydrate per litre. 
A minute quantity of flowers of sulphur is added, and 1 c.cm. saturated solution of 
tartrate of iron per litre. It is then at once poured into tubes before precipitation of 
the iron can occur. In the neck of each tube is suspended between the plug and 
the glass a strip of filter paper. Sterilization is done for twenty minutes on each of 
three successive days in a steam sterilizer at ioo° C. On inoculation with the 
various colonies the tips of the filter paper are moistened with sterile solution of lead 
acetate, and so hung as to be secure from contact with the medium. Incubation is 
