264 



HANDBOOK OF PHYSIOLOGY 



NEUROPHYSIOLOGY I 



metal-electrolyte surface area, has plent)' of problems 

 of its own. But in addition to the important role it 

 has played in peripheral nerve (see Chapter III), 

 muscle and ganglion studies, the micropipette has 

 provided most of our present detailed knowledge of 

 the physiology of single nerve cells in the central 

 nervous system. This is partly because these electrodes 

 have been made small enough to penetrate single 

 nerve cells without destroying them, thus permitting 

 measurement of the potential across the cell mem- 

 brane (figs. iB and 65). For this reason and because 

 the use of this type of electrode appears to hold much 

 promise for future investigations, more space will 

 be devoted to its description. 



Ling & Gerard (42) are commonly given credit for 

 introducing micropipettes, probably because they 

 were the first to record action potentials from inside 

 single muscle fibers with these electrodes. However, 

 Graham & Gerard (30) had previously recorded the 

 resting membrane potential of frog muscle fibers with 

 similar fine electrodes filled with saline, and many 

 workers before them have used coarse pipettes for re- 

 cording electrical potentials as in the use of small 

 calomel half-cells and agar bridges [references to this 

 work are listed by Svaetichin (52)]. 



CONSTRUCTION. Ling & Gerard (42) made micro- 

 pipettes by hand, drawing i to 2 mm glass tubing 

 down to a fine tip in an oxygen-gas microflame. 

 Alexander & Nastuk (4), Livingston & Duggar (43) 

 and others have devised machines for pulling pipettes 

 similar to that shown in figure 3. This machine heats 

 the glass tube over a short length until it is soft enough 

 to be drawn out by the pull of the electromagnet. 

 Increasing the heater temperature or the length of the 

 heater coil produces a longer, gentler tapered section 

 on the pipette. Figures sA and B show the size and 

 one way of mounting glass micropipettes. Another 

 method of mounting supports the tip of a glass micro- 

 pipette on a I mil tungsten wire (59). This method 

 has been used to permit recording of intracellular po- 

 tentials from muscle where movement would dis- 

 lodge a rigidly mounted pipette. 



FILLING. The size of the tips of such micropipettes 

 ranges upward from a few tenths of a micron (fig. 

 2B). The problem of filling them with an electrolyte 

 becomes more difficult as the size of the tip is reduced. 

 Above about 5 m the pipettes can be filled with a 

 syringe by expelling the air from the tip. Boiling for 

 several hours submerged in the electrolyte is satis- 

 factory for all but the smallest tips although it appears 



FIG. 3. Vertical micropipette puller. Upper clamp fixed; 

 lower clamp pulled down by solenoid, gently at first when 

 glass begins to .soften, hard just before pipettes separate. Tips 

 are drawn down out of heater coil which is turned off any time 

 after pull is completed. (Developed at National Institutes of 

 Health, Instrument Section.) 



to enlarge them somewhat. N. Tasaki (55) has de- 

 vised the most satisfactory method of filling. The 

 pipettes are immersed in methyl alcohol in a chamber 

 which is gradually evacuated until the alcohol boils 

 vigorously for a few minutes. The low \iscosity and 

 low boiling point of the alcohol permit the finest tips 

 to be filled quickly and without damage. The alcohol 

 can then he replaced by the desired electrolyte by 

 dififusion in about two days. Preboiling and filtering 

 the electrolyte reduces the formation of air bubbles 

 and clogging by foreign particles. The micropipettes 

 are best stored in alcohol or water and are transferred 

 to the desired electrolyte a few days before they are 

 needed. 



A micropipette can be filled with a variety of differ- 

 ent electrolytes. Considerations to be taken into ac- 

 count are electrical conductivity, similarity' of cation 

 and anion mobilities, the possibility of damage to cells 

 through the diffusion of the electrolyte out of the 

 pipette and the purpose for which the pipette is used. 



