﻿IX, B, 4 • Barber: The Pipette Method 353 



possible opening is made ; if bacteria, a larger opening is needed ; 

 and if yeasts or spores of fungi, one still larger. As a rule, 

 the opening should be as small as the nature of the work will 

 permit. If desired, a point with an opening scarcely exceeding 

 1 micron in diameter may be made. To determine whether a 

 fine point is open or not, one has only to touch it to a droplet 

 of condensed moisture. If the point is open, the liquid will 

 instantly enter by capillary. 



With the tip in the hanging drop of water, lower the cup and 

 allow the mercury to expand sufficiently to expel the air from 

 the pipette. As the pipette was supplied with mercury with 

 the loop partially cooled in ice water, the room temperature 

 will furnish sufficient heat for the expansion of the mercury. 

 When mercury appears at the tip, raise the cup enough to stop 

 the pressure. If some mercury comes out, no harm is done, 

 except when in very tapering pipettes a plug of mercury some- 

 times clogs the tip. Now bring the tip into the hanging drop 

 of bacteria or other substance to be inoculated. If the mercury 

 column has retreated, apply a little pressure to bring it to the 

 tip again. Raise the cup until sufficient negative pressure has 

 formed to draw in the dose required. The size of the dose can 

 be regulated roughly by the extent of the fall of the mercury 

 column. Then lower the pipette and, as quickly as possible, 

 move to the organism to be inoculated. Bring the tip under it, 

 and raise the point until it penetrates the cell wall and reaches 

 the vacuole or the part of the cell desired. Now apply pressure 

 gradually until the dose is seen to come out and enter the cell. 

 Then stop the pressure "at once by raising the cup containing 

 the ice water until it surrounds the loop. 



In order that the dosage may be regulated and that mercury 

 may not be driven into the cell, the tip of the pipette should be 

 unobstructed when it pierces the cell wall. Substances may 

 successfully be injected in spite of a clogged tip, but so much 

 force is often required to overcome the obstacle that injection 

 takes place with a rush and mercury is apt to follow the dose. 

 Further, both for the regulation of the dosage and the prevention 

 of clogging, pressure in the pipette should be nearly in equili- 

 brium at the time of the entrance of the point within the cell. 

 Positive pressure may not be a disadvantage if one does not care 

 to avoid ejecting some of the contents of the pipette outside of 

 the cell, but a too strong negative pressure causes the pipette 

 to take up liquid surrounding the cell and increases the danger 

 of clogging when the point enters the protoplasm of the cell. 



