Chicken embryo 



INJECTIONS 



167 



mercial India ink and dilute it with about 

 ten times its volume of ilistilled water, to 

 each 100 milliliters of which has been 

 added one droj) of ammonia. The use of 

 tap water, pai'ticulai'ly if slightly acid, 

 tends to agglomerate the carl)on jiarticles 

 and thus to make injection impossible. 

 Next make a series of hollow glass needles 

 of a size suitable for the injection. For 

 these take short lengths of ordinary, soft- 

 glass, 3-millimeter tubing, heat them in a 

 flame until they are soft, remove them 

 from the flame, and draw them out to 

 about 1 millimeter in diameter; then re- 

 turn them to the flame, pulling sharply, 

 when they are soft so as to secure a fine 

 tip. This tip should be almost exactly the 

 size of the Cuverian sinus of the embrj'o 

 which is to be injected. It is simplest to 

 prepare two or three dozens of these little 

 injection needles, approximatelj' the right 

 size and then to select the best. One needle 

 may be used for the entire batch but the 

 exact size is vital to the success of the 

 operation. In case the worker is unac- 

 quainted with the Cuverian sinus, he is 

 recommended to look at the body of the 

 embryo just along the side of the heart, 

 where he will see that a large anterior and 

 a large posterior vein come together to 

 form the cross piece of a T, the stem of 

 which passes downward towards the heart. 

 This stem is the Cuverian sinus. 



Now secure two stender dishes, one 

 filled with the diluted India ink, and the 

 other with phj'siological saline. Syracuse 

 watch glasses, one for each embryo to be 

 injected, should be placed in a dish of 

 water at about 40°C., so that they may be 

 warmed through, and a small beaker of 

 any fixative, with an eye-dropper type 

 pipet in it, should be at hand. Two pairs of 

 fine forceps will be required, as well as the 

 large scissors and blunt-nosed forceps used 

 to remove the embryo. The injection is 

 best made under a wide-angled dissecting 

 binocular which is set up to transmit light 

 through the embryo. 



Now remove an embryo from the yolk 

 and, after washing it in the manner de- 

 scribed in Chapter 20, transfer it to one of 

 the warmed watch glasses containing 

 physiological saline. Drain out most of the 

 saline so as to leave the embryo and 



its surrounding extra-embryonic areas 

 stranded on the bottom, and transfer the 

 dish to the stage of the binocular micro- 

 scope. A needle of the required size is now 

 selected and attached to a short length of 

 rubber tubing, terminating in a mouth- 

 piece of the type sold for use with blood 

 pipets. Dip this needle under the surface 

 of the injection medium and suck until a 

 consideraljle portion of the glass tube is 

 filled. Remove the needle from the injec- 

 tion medium and transfer it to the dish of 

 normal saline. Now give a very slight suck 

 on the tube in such a manner as to fill the 

 fine capillary portion of the tube with 

 saline without di'awing in enough to dilute 

 the India ink in the body of the tube. This 

 is the most vital stage in the proceedings, 

 for if the fine capillary is left filled with 

 India ink, this ink will flow out as soon as 

 the needle touches the heart and the 

 operator's view w'ill be obscured. 



After each injection is done, return the 

 injection tube to the dish of normal saline 

 and again refill the capillar}' tip with 

 saline. 



The only dissection of the embryo that 

 is necessary is to split a hole through the 

 pericardium. This may be readily done by 

 tearing with two pairs of fine forceps, one 

 held in each hand. Utmost care should be 

 taken not to break any blood vessels in 

 doing this, but a few minutes' practice on 

 some embryos will be far more instructive 

 than any amount of description. Now pick 

 up the injection needle and take the 

 mouthpiece at the end of the rubber tube 

 between the teeth. 



Provided the mouthpiece is kept in the 

 teeth and the mouth kept open, no pi'es- 

 sure can possibly be applied before it is re- 

 quired. Assuming the operator to be right- 

 handed, so that the tube is in his right 

 hand, the embryo is now turned so that 

 its anterior-posterior axis is at about 45° 

 to the operator with the head lying, as it 

 were, to the northwest. The tip of the in- 

 jection needle, which it is to be remem- 

 bered is filled with saline and not with 

 India ink, is now applied to the heart just 

 at the bend. A sharj), shoi't stal) is used to 

 drive the needle through until the end of 

 the needle lies free in the cavity of the 

 ^•entricle; a very slight and careful pressure 



