A New Method of obtaining cultures from Single Bacterial Cells.

WHILE investigating variations in agglutinability, occurring among certain strains of paratyphoid bacilli, it became desirable to obtain cultures derived from single bacterial cells. The technique at first adopted was that devised by Mutch (1919), but the results obtained were not satisfactory. Certain modifications of this method were attempted, but were no more successful. The main difficulty, and one which appears to us to have been far too little emphasised in all the methods hitherto described, has been encountered in obtaining satisfactory conditions for the microscopical observation of the bacillary suspensions employed. The difficulty of accurately observing hanging-drop preparations, and still more of controlling manipulation of them by microscopical observation, would seem to us to have been insufficiently realised; and the ease with which faulty interpretation of microscopical appearances may introduce fatal errors into work of this kind has not, perhaps, been fully taken into account. For this reason we first turned our attention to the problem of securing really satisfactory conditions for microscopical observation, and adopted the dark-ground method of illumination for this purpose. Using this technique, with a relatively granule-free medium for suspension, and examining thin films between a slide and a cover-slip, it is possible to pick out a single bacterial cell, lying apart from any other organism, with a certainty quite unobtainable in the observation of hanging-drop preparations by directly transmitted light, or of organisms situated on the surface of any solid nutrient medium. The conditions of this method of observation, on the other hand, entirely preclude subsequent isolation of an individual bacterial cell by mechanical means, while the experience met with in attempts to examine large series of such preparations, until one was found which contained a single bacterium only, did not offer any inducement to continue work along these lines. For these reasons we definitely abandoned all attempts to obtain the desired results by methods such as those described by Barber (1908, 1911, 1914, 1920), Mutch (1919), Hort (1920), Hewlett (1918), Malone (1918), and others; and tried to discover a solution of the difficulty along entirely new Journ. of Hyg. xx 16 NOVEMB R, 1921 ]NO. 3 VOLUME XX