EXPERIMENTAL EQUIPMENT AND PROCEDURES 



Into a small hot flame. If the capillary end is curved, the point of heating 

 should be on the opposite side of the curve, ^^^hen this point is soft, hlow gently 

 into the ruhher tubing and the melted glass will bulge outward. The size of the 

 heated area will determine the size of the bulge. Break off the glass bubble down 

 to the wall of the pipette and smooth off the edges in a micro-burner. V/ith dia- 

 mond point cut off the closed tip of the capillary end so that the aperture will be 

 be about 1 mm. in diameter. Cut a short piece of thin-walled rubber tubing and 

 slip it (from larger end) over the pipette to the point where it covers the lateral 

 hole. Heat the broad end of the pipette until very soft, press down on metal sur- 

 face to give a ridge to hold the rubber nipple. Add rubber nipple to this end, 

 and the pipette is ready for use. 



This latter pipette is of special value in the transfer of small pieces of 

 tissue under solution. With nipple, suck in solution until the capillary end is 

 filled almost to the lateral hole. Bemove fingers from nipple and place a fore- 

 finger over the rubber- covered lateral hole. With the capillary point under the 

 solution, force out a small amount of the contents of the pipette by gently press- 

 ing on the lateral rubber cover with the forefinger. Then, by releasing the 

 gentle pressure a small amount of fluid, or tissue, may be drawn up into the 

 pipette and held there. This tissue may be oriented at any place by slight pres- 

 sure on this lateral membrane. The capillary end must hold solution (not drip) 

 even when held vertically with the point suspended. Practice the use of this 

 pipette with small objects. 



7, Glass bridges : These are small pieces of cover glass, thinnest grade, used to 

 hold transplanted tissues in place for 15+ minutes while they "take" or heal onto 

 the host. Using a diamond point pencil and ruler, cut thin cover glasses into 

 strips of about 2-5 mm. wide and 5-10 mm. long. With forceps run the edges of 

 these glass strips through a micro- flame to make them smooth. To put a slight 

 curve in some of the pieces of cover glass, grasp the edge of a piece with forceps 

 and bring the center of it over a micro-burner with a 1 mm. flame. The weight of 

 the cold end will bend the cover slip slightly when the center is heated. The 

 height of the bridge should be determined by the size of the embryo to be used as 

 host. Frequently flat cover slips will prove to be adequate^ especially when the 

 host is held in a depression. The bridges should be sterilized in 70^ alcohol 

 over cotton and kept thus in a covered stender dish. 



There is a refinement of this type of bridge (or Briicke) recently described 

 (Schultze, 1958). The cover itself consists of transparent Pyralin of less than 

 2 mm. thickness cut into rectangular blocks of 1/8 inch by 5/8 inches each. Holes 

 are drilled through each end of the block with #70 or #71 wire gauge drill. The 

 edges of the block are smoothed off with fine file and then dipped in very weak 

 balsam to increase transparency. Take care that balsam does not fill the holes. 

 Small safety pins, (preferably gold plated to prevent rusting) are straightened 

 out and then given three right angle bends as shown in the diagram. The pins are 

 then forced through the holes, to the extent of 5-6 mm. Such a graft cover may 

 be pressed down onto the graft exactly as desired, the pins anchoi-ed in the Permo- 

 plast and the Pyralin allowing constant observation of the graft. In most trans- 

 plantations such an elaborate Briicke is not necessary. 



OPERATING DISHES 



Most operations will be carried out in Syracuse dishes or salt cellars. Since em- 

 bryonic tissues often adhere to glass, the base of the Syracuse dish is lined with one or 

 another plastic substance. The fol ''owing are satisfactory: 



a. Permoplast - American Art "lay Co., Indianapolis, Indiana. 



b. Beeswax to which has been added lampblack, to take glare off background. 

 0. Eainbow Wax - American Art Clay Co., Indianapolis, Indiana. 



d. White refined beeswax. 



e. Pieces of cellophane or Pyralin. f. Agar, 2% or more concentrated. 



The Permoplast is probably best for delicate operative procedures, as it is easily molded 

 without iieating. However, under solution it tends to fragment and it may be necessary to 

 compensate for this by melting It with about 20^ low melting point paraffin. 



