The isolation of single bacterial cells

Abstract

The writer has three reasons for introducing what, to many workers, is a somewhat threadbare subject: (l) In the minds of not a few people with whom the author has discussed the matter, the impression still persists that the technique of the isolation of single bacterial cells is extremely difficult, involving months, if not years, of practice for its successful acquisition. Heller (1921) stated that she found the Barber (1914) method "wasteful of time, material, eyesight and nervous energy" in the isolation of anaerobic bacteria. There is some justification for saying that a wastage of time and energy occurs when, after the successful isolation of ten to twelve bacteria or spores, not one germinates. This point, the germination of the single germ, especially when it concerns the anaerobes has not received the same amount of attention that the actual isolation has. The latter is a simple mechanical process, the former one over which the operator has not the same degree of control. (2) A method of isolation, to be of real practical value and to enjoy a constant and wide application, must be simple and devoid of tedium and time wastage. Preferably should it be such that the worker would prefer to employ it rather than use plate or shake methods of isolation. Again, the learning of the technique should not require months of practice nor should it be of such a nature that only those people with "hands" can easily acquire it. Reyniers (1933) described a method by which beautiful micro-pipettes may be made mechanically. The writer gained the impression, wrong, he hopes, that the making of the apparatus would be one requiring considerable skill and time. Doubtless, however, to see the designer himself at work would dispel this idea. The method, used by the writer for over four years, has proved to be so simple that three colleagues, after half-an-hour's practice in pipette-making have settled down and isolated (with successful germination) single germs of Cl. welchii and B. anthracis. (3) The third reason advanced for the presentation of this note is in the nature of a plea for the more extended use of the single cell isolation method as a routine measure. The saving of labour and of time involved in obtaining definitely pure cultures and the feeling of assurance in having these pure cultures greatly outweighs the only disadvantage (in the writer's opinion) of the expenditure of the money for the micromanipulator. But even a machine costs no more than a good microscope and where the outlay is impossible a locally-made apparatus or the modification used by Malone (1918) may be employed. Finally, there is the possibility that less would be heard of bacterial mutation if cultures were first purified by the single-cell method. The making of Micro-pipettes. On the making of satisfactory pipettes depends the success of isolation. The writer was quite unable to make an efficient article by the "tiny flame method" as described by Barber (1914) and Chambers (1922). That this method gives highly satisfactory results there is no doubt; never having had the procedure demonstrated to him and the use at these laboratories of "paraffin gas" (Mansfield system) are doubtless the reasons for the failure. The electrical heater described (Mason 1933) solved the difficulty. The two elements, arranged in the form of a V, may be made broad or narrow. If narrow, the type of pipette most desirable for isolation work is easily prepared. It is advantageous that the micro-portion should not be more than 1.5 to 2 cm. long and that it should leave the thick portion of the hand drawn capillary sharply. A long tapering micro-pipette trembles easily and when raised against the cover slip in the process of isolation "gives" considerably, necessitating much adjustment of the control screws of the machine. Such a tapering pipette is liable to be pulled when the breadth of the elements is 3.0 cm. or more. Elements of 2.0 cm. give satisfactory results.