is just over the condenser. By gently moving the needle from side to side locate 

 the position of the needle under the microscope and center its point. With the 

 point continually under observation slowly rack the moist chamber forward. Stop 

 the movement as soon as the edge of the cover slip comes into view, and determine 

 the relative positions of needle point and cover slip. If the point is above the 

 cover slip, lower it by means of the coarse adjustment until it is definitely be- 

 neath it. If the point is below, raise it slightly. The fine screw should not be 

 used for these preliminary adjustments. Now rack the moist chamber completely back. 

 Mount a needle of type B in the other holder, and slowly rotate the point inward. 

 With the naked eye approximate this point as closely as possible to the other point. 

 THIS REQUIRES GREAT CARE. Under the #lb, find and check the position of the second 

 needle point with respect to the first. Be certain that both points are in the same 

 plane. Rack the moist chamber forward over the needles. Change to the #3b objective. 

 Center the needles with respect to the microscope field under this magnification. 



Making and Use of Micropipettes 



1. Making Micropipettes. Using six inch lengths of glass tubing draw a supply of 

 capillaries as outlined above. Always use chemically cleaned glass of at least 4 mm 

 bore and with thin wall. Have a supply with the ends fused to keep the interior 

 clean and dry. To make a pipette, take a capillary and break off the fused ends. 

 As in the directions for making microneedles, heat and pull the capillary tubing 

 over the microburner. Examine under the microscope. The kind of pipette point will 

 again depend on the amount of heat, and rapidity and smoothness of pull. A fairly 

 wide variety of pipettes will undoubtedly result. They may be roughly classified 

 into four categories. (See figure 7). 



B 



> - d 



D 



Ti 



A. Coarse quick taper, usually of little value. 



B. Coarse gradual taper, may be used for injecting viscous 

 fluids in large cells. 



C. Fine quick taper, pest for general use. 



D. Fine gradual taper, best suited for fluids of low viscosity. 



2. Bending the Micropipettes. Bend the micropipettes using the same technique as 

 for microneedles. The two most convenient angles are 45° and 90°. Make a dozen 

 micropipettes with shafts about three inches long, place these in your pipette 

 block according to the classification given above, and present to the instructor 

 for inspection and criticism. 



3. Setting up Injection Apparatus. Clamp the metal injection tube to the base-plate 

 by means of the metal square attached to the tube. The end carrying the hypodermic 

 needle should be toward the syringe holder. Fasten the syringe in its holder. The 

 length of tubing between the square metal clamp and the hypodermic needle will be 

 found to exceed the distance between the clamp and the syringe holder. This length 

 must be reduced by careful bending or coiling until the metal tip of the syringe 

 can be securely fitted into the hypodermic needle without strain. Next fasten the 



