466 ON THE ORIGIN AND EARLY DEVELOPMENT 



METHOD OF OPERATION. 



Description of the details of the operations must be left for the account of the 

 individual experiments, since a number of modifications were adopted in each case. 

 All of the operations were carried out under the binocular microscope inclosed in a 

 warm chamber heated to incubator temperature. The operations were performed 

 on chicks taken from the incubator to the warm box, where the shell was swabbed 

 with cotton or cloth saturated in alcohol. As soon as the alcohol had evaporated 

 a hole was pricked in the shell with a sterile needle. Into this opening the point 

 of a pair of sharp forceps was inserted and the shell carefully picked off over an 

 area about 6 mm. in diameter. A little warm sterile Ringer's solution was then 

 dropped on the shell membrane, thus making the dissecting away of this membrane 

 from the yolk much easier. Immediately after removing the shell the yolk sags 

 away from the opening and it then becomes necessary to add, gradually, enough 

 Ringer's solution to bring the embryo up to a level with the opening. If it is added 

 too rapidly it may cause the embryo to rise out of the opening and result in a tear 

 in the yolk membrane. If the region of operation is covered by amnion this mem- 

 brane must be opened with forceps before proceeding. After the operation is 

 finished the amnion can be "sutured" by pinching the edges together, whereupon 

 it heals and continues to develop in a normal manner. 1 



The instruments used differed according to the type of operation performed; 

 small iridectomy scissors were used when a portion such as the tail or wing bud was 

 removed, while well-sharpened needles or a small knife with a triangular blade 

 proved most useful in cases where it was necessary to dissect away somites or 

 body- wall. In all cases sharp edges and fine points to the instruments are essential. 

 Moderate aseptic precautions were used. The Ringer's solution was boiled and 

 allowed to cool to a temperature of 37 to 39 C. before using. Needles and mica for 

 the window were flamed, while more delicate instruments, such as the small forceps, 

 knives, and scissors, were simply dipped in alcohol and allowed to dry. 



After completing the operation more Ringer's solution was dropped into the 

 opening. This helped to exclude air and to prevent sticking of the yolk-membrane 

 to the ragged edges of the shell. A thin piece of mica was then placed over the 

 opening and the edges sealed with a warmed mixture of beeswax (4 parts) and 

 resin (6 parts). The egg was then returned to the incubator. The sticking of the 

 yolk-membrane to the shell can be prevented if the egg is kept with the window 

 side down, so that the embryo will float to the opposite side. It is also advisable 

 to rotate the egg gently several times during the first few hours after the operation. 

 The condition of the embryo (whether alive or dead), its stage of development, and 

 hints as to the success of the operation can be observed from day to day by rotating 



1 A decided improvement in the teehnic of operating has been developed since this work was done. It was found that a 

 considerable mortality is caused by the fact that, when the hole is made in the shell, the air is gradually forced out of the air- 

 chamber until the chamber is obliterated. As a result a slight increase in the temperature of the egg after closing causes such 

 an increase in pressure as to stop the heart-beat. If this is prevented by the simple expedient of immersing the ej;g during the 

 operation in water at incubator temperature to a depth sufficient to cover the air-chamber, the air remains in the chamber, the 

 chick does not sink away from the opening, and the mortality during the first 4S hours after operation is reduced nearly to 

 zero. It is well to roll the egg slowly before operation, in order to make sure that the embryo is freely movable, and also to 

 candle it in order to control the mobility and to determine the line of latitude on which to make the opening. 



