wheel. The first trial demonstrated that the par- 

 ticles of emery and glass would completely plug 

 the small bore of the glass tip. A thin-walled 

 flexible rubber tubing was attached to the large 

 end of the tip. An effort was made to dislodge 

 the particles by blowing through the tubing dur- 

 ing the grinding process. But blowing by mouth 

 did not keep the bore free. It was found neces- 

 sary to make a connection to an air or oxygen 

 pressure tank in order to keep a flow of air or 

 oxygen tliat would prevent the entrance of par- 

 ticles into the glass tip. The progress of grind- 

 ing was checked under the microscope and was 

 considered finished when there was a beveled, 

 smooth, sharp tip. 



When the grinding was finished the tubes were 

 cleaned with alcohol and ether, and dried with 

 air. 



Method for taking blood from the dorsal 

 aorta of the 48- to 72-hour embryo 



Eggs that are presumed to be fertile are taken 

 from the cool room where they have been held 

 at a temperature of about 55 degrees since they 

 were laid. They are placed in the incubator 

 with a record of the hour and date. In these 

 studies the age has been taken as 3 hours less 

 than the total time held in the incubator. When 

 the embryo has reached 48 hours, incubation 

 age (51 hours actual time in the incubator), it is 

 removed, opened carefully, and slid into a bowl 

 of warm saline or Ringer's solution. Sugiyama 

 (1926) followed Sabin's suggestion and in- 

 creased the salt content of Locke-Lewis solution 

 to L04-percent NaCl for an embryo on the second 

 day of incubation, to LO-percent for an embyro 

 on the third day of incubation, and to 0.9-percent 

 for an embryo on the fourth day of incubation 

 and older. These improvements in technic are 

 useful if the embryos are to be held for study over 

 a period of time. Wliere the whole procedure 

 can be completed within a few mimites, the use 

 of 0.85-percent or 0.90-percent solution of so- 

 dium chloride alone produces no ill effects. 



Prior to opening the embryo, rings from filter 

 paper were cut. These had an outside diameter 

 slightly larger than the margin of the area vascu- 

 losa, and the inner diameter was slightly less 

 than this margin. The saline was removed from 

 the bowl until the embryo lay above the level of 

 the surrounding fluid. The vitelline membrane 



FiGUKE 412. — A tripod to hold an infrared lamp, 

 used to warm slides and to drive off moisture be- 

 fore the smear is made and to dry the blood 

 after it is spread. 



was lifted off and the filter ring was placed over 

 the embryo, which usually adhered readily to 

 the surface. With fine curved scissors and for- 

 ceps the embryo was cut free from the yolk sac, 

 washed in saline, and lifted to a Syracuse watch 

 glass. It was then placed under a low-power 

 dissecting microscope. 



In the meantime clean microscope slides were 

 drying and warming under the infrared lamp 

 (fig. 412). The slides should feel warm, not 

 hot, to the back of the hand or the cheek. A 

 flexible, thin ruljber tubing of the type used for 

 taking blood in erythrocyte counts was attached 

 to the glass cannula, and under the low power 

 of the dissecting microscope the cannula was 

 guided into the dorsal aorta. The heart nmst 

 be beating and the blood flowing in order to ob- 

 tain a satisfactory preparation. A slight posi- 

 tive pressure is set up with air from the mouth 

 as the tip approaches the moist surface of the 

 embryo. If this is not done capillary attraction 

 tends to draw saline and embryonic fluids into 

 the tube and these fluids quickly distort the 

 embryo blood cells. 



226 



