A Technic for the Inoculation of Bacteria and Other Substances Into Living Cells

Abstract

The method here described is an outgrowth of the technic for the isolation of single micro-organisms as described in previous papers.1 The new feature makes possible not only the segregation of one or more micro-organisms but the injection of them as well as of measured doses of fluids into the protoplasm or vacuoles of living cells. In order to accomplish this, pipettes have to be constructed of such a fineness as to minimize the injury to the cells injected and of sufficient rigidity to pierce the cell wall. Further, an injection force has to be employed sufficient to overcome cell pressure, capillarity, and any obstruction in the pipette. The first requirement was met by modifying the method of drawing pipettes, and the second by the use of the expansion of mercury as a source of power for injection. Each phase of the method and each part of the apparatus will be described in detail, and, for the most part, in the order that one would follow in carrying out the process. The pipette holder {ph, Fig. 4) is the same as that described for the isolation method. It consists essentially of an attachment to the stage of the microscope which holds the pipette, and by means of screws allows an up-and-down and a lateral movement of the pipette (sv and si, Fig. 4). A double pipette holder, which is described below, may be used in place of the simpler form. Any mechanical stage may be employed which allows a considerable range in both directions. The glass box is prepared as for the isolation method. Two convenient forms have been used, the smaller 60 mm. long by 25 mm. broad by 16 mm. high and a larger one 70 mm. long by 36 mm. wide by 16 mm. high. Water is kept in the bottom of the box and, further to insure moisture, the sides are lined with wet filter paper. A number 2 cover-glass