350 TISSUE CULTURE 



a paraffin coated glass cannula is then inserted into the artery 

 and tied lightly with the proximal ligature. The blood flow is 

 controlled by light pressure with the finger on the most proximal 

 part of the exposed artery. After a few drops of blood have 

 escaped the remainder may be collected in sterile, ice-cooled, 

 paraffin coated tubes, fitted with corks or rubber stoppers. As 

 each tube is half or three-quarters full, return it to a freezing 

 mixture. The anaesthesia should be light throughout this pro- 

 cedure. 



Centrifvige the blood at 2,500 revolutions per minute for eight 

 to ten minutes, using ice in the centrifuge buckets in summer. 

 The supernatant plasma is then removed with a paraffin coated 

 pipette to paraffined storage tubes fitted with cork or rubber. 

 After forty-eight hours in paraffin coated tubes the plasma will 

 remain fluid even in clean glass tubes. It should always be stored 

 in the refrigerator where it keeps liquid for at least a month or 

 more. No anti-coagulant is necessary in preparing fowl plasma. 



In an emergency, paper centrifuge tubes may be used. They 

 should be immersed in paraffin wax at 120° C. and allowed to cool. 

 If half an inch of sterile paraffin oil is placed in each glass or paper 

 collecting tube, the blood remains sealed off from the atmosphere 

 during centrifuging. In this way " true " plasma without loss of 

 CO2 and consequent change in _pll is obtained. 



About 50 c.c. of blood may be taken from a fowl if the wound is 

 to be sutured and the bird allowed to recover for subsequent 

 removal of its blood from the intact carotid artery on the other 

 side. A rapid method of taking small quantities of blood from 

 the heart of the fowl is described in detail by Lewis {Archiv. f. 

 exper. Zellforsch., vii, 1928, p. 82), 



783. Mammalian Plasma. Using ordinary laboratory equip- 

 ment, an anti-coagulant is essential in the preparation of mam- 

 malian plasma. Heparin (see Craciun, Bull. Johns Hop. Hosp., 

 xxxviii, 1926, p, 327) is the most useful anti-coagulant, since it 

 withstands autoclave sterilisation and does not appear to affect 

 the growth of cultures. Fischer uses 0-1 per cent, heparin in 

 Ringer solution, and Cameron recommends diluting this con- 

 centration, after sterilisation, with Tyrode solution, 1-5,000. 

 Most workers prefer to expose the heart under aseptic conditions 

 and to draw off the blood from the ventricle with a glass syringe. 

 Either sterilise the syringe in paraffin oil and transfer the blood 

 to a paraffin coated tube containing heparin (1 c,c. of 0-01 per cent, 

 heparin is sufficient for one rat, rather more for a guinea-pig), or 

 take up the heparin in a dry sterilised syringe before drawing the 

 blood. The rest of the technique is exactly similar to that 

 described for the fowl. Small amounts of blood can be obtained 

 by heart puncture through the sterilised skin of the lateral 

 thoracic wall with subsequent recovery of the animal. 



