352 IMMUNOGENETICS 



aimed slightly toward the animal's head and toward the midline. It is useful to wet 

 the syringe before use with an isotonic citrate solution (2 per cent sodium citrate, 

 0.5 per cent NaCl). 



In mice and rats, small quantities of blood may be obtained by incision of a vein 

 in the tail; particularly in mice, it is preferable to make a shallow cut with a razor 

 blade across the ventral surface of the tail, rather than to use one of the lateral tail veins. 

 Rapidity of bleeding can be promoted by warming animals under an ordinary study 

 lamp, before bleeding. Hemolysis of the serum can be avoided if the dry tubes into 

 which the blood is allowed to drip have been coated with silicone. Many believe that 

 a more convenient technique than tail-vein incision or cardiac puncture in the mouse 

 or rat is to obtain the blood by Halpern's method from the orbital sinus. 1283 Hema- 

 tocrit determinations can be made directly in the tubes used for bleeding, without 

 transfer, if heparinized tubes are prepared. Cardiac puncture is also convenient in 

 mice and rats; mice are bled under Nembutal anesthesia and rats under ether or 

 urethan anesthesia. The approach to cardiac puncture described for the rabbit also 

 works for the mouse and the rat; shorter and smaller needles are used (for the mouse 

 | inch, 26 gauge; for the rat, \ inch, 24 gauge). Many find it easier to bleed a 

 mouse by cardiac puncture, using an approach immediately under the tip of the 

 sternum and directing the needle straight forward and almost horizontally. With 

 this approach a 22-gauge, 1-inch needle is proper. Guinea pigs and other small 

 mammals are also conveniently bled by cardiac puncture ; we have bled, for example, 

 opossums, monkeys, and raccoons easily this way. When considerable quantities of 

 blood are to be obtained from the mouse, particularly when blood from homogeneous 

 populations is to be pooled, mice are often sacrificed, and blood is collected in a beaker 

 after decapitation. 



If the plasma is to be used, or if test red-cell suspensions are the main objective 

 of the bleeding, the blood is collected in an anticoagulant. Several of these are used: 

 for example, heparin, versene (0.1 M, pH 6 to 7, in 0.85 per cent saline), oxalate, or 

 citrate solutions. When, however, it is a test serum that is required, the blood is 

 collected in a dry (preferably silicone-coated) tube, and is allowed to clot. The clot 

 should be freed from the wall of the test tube after it has formed ; for most samples, it 

 can simply be shaken free after it has been permitted to stand undisturbed for an hour 

 after bleeding, or the clot may be rimmed with a wooden applicator or glass rod. In 

 most bloods the clot then shrinks, squeezing out the serum; after an hour or two the 

 serum is poured from the clot, the free cells are centrifuged out, and the clear serum is 

 decanted or pipetted into storage bottles. 



Better yields of serum are obtained from mice if the blood is collected into a tilted 

 petri dish, so that the clot forms as a film over half of the plate. The clot can then be 

 freed from the glass surface over most of the area, and the plate tilted so that the serum 

 as it is squeezed out of the clot runs down to the clear side of the plate. With the 

 small quantity of blood obtained from an individual mouse it is very desirable to keep 

 the plate covered during the process, to avoid drying. For most practical laboratory 



