﻿IX, B, 4 Barber: The Pipette Method 349 



in outside diameter — the same dimensions as for an isolating 

 pipette, although it is better to have the wall of the capillary 

 slightly thicker (about 45 microns in thickness). 



The pipette should now be filled with mercury to a point well 

 within the capillary. This is done by heating the arm at about 

 point s (fig. 17) sufficiently to vaporize some of the mercury 

 and expel all air from the capillary. Dip the capillary into 

 mercury, and it will fill as the mercury in the arm condenses 

 and cools. If air bubbles remain in the arm or loop, they may 

 be worked out. Freeing from air is facilitated by heating the 

 arm and sealing the tip while the mercury is vaporized at the 

 point heated. On cooling, a vacuum will be left at the end 

 and the air may be more easily worked out. When the capillary 

 is opened again, it may be necessary to refill it with mercury. 

 The pipette should be as free from air as possible, although 

 a small bubble does not prevent its successful use. Time will 

 be saved by making two or three pipettes at a time. One may 

 be carried through a stage of the pi'ocess while another is cooling. 

 After they have been finished to the stage described, they may be 

 set aside until a time convenient for use. 



When ready for the inoculation, the cells into which substances 

 are to be injected should be placed in a shallow hanging drop 

 near the center of a large cover which is sealed over the isolating 

 chamber, as for ordinary isolation. Sufficient moisture must be 

 supplied. If the cells or filaments are in pure culture, as a fish 

 mold in agar or broth, it is well to have a round or oval paraffin 

 barrier under the cover. This is traced on the cover by means 

 of a coarse bent pipette containing melted paraffin. The fungus 

 may be cultivated within this barrier a day or two before in- 

 oculation. The barrier protects against contamination with 

 bacteria.^^ Animals, algse, or fungi not in pure culture may be 

 placed under the cover with no special precautions. A hanging 

 drop containing the organisms to be inoculated should be put 

 under the cover glass, and near it a hanging drop of sterile water 

 should be placed. These should be as near as possible to the 

 cells to be inoculated. If a paraffin barrier is used, they may be 

 placed just outside of it. Lines should be drawn on the cover, 

 preferably with India ink, to serve as guides to different parts 

 of the preparation. When all is ready, the isolating chamber is 

 placed on the stage and the outer edge brought to focus under 

 the low power. 



" For a description of the method of cultivating fish molds for inoculation, 

 see This Journal, Sec. B (1913), 8, 373. 



