560 APPENDIX 



A complication to bleeding from the tail is that the cut surface is frequently reopened 

 after bleeding by contact with box or bedding, leading to unnecessary loss of blood. 

 If a series of counts are to be made on the same animal, it is well to start near the tip 

 of the tail and move proximally. 



When repeated samples are to be taken from the same individual, and minimal 

 loss of blood is important for maintenance of homeostasis, small samples of blood 

 may be taken from a sinus in the inner canthus of the eye. This site has two advan- 

 tages. Irrelevant loss of blood can be avoided by starting blood flow with the tip of 

 the collecting tube. The tip of a Harshaw pipette or a capillary hematocrit tube 

 is inserted into the network of capillaries in the medial side of the eye socket and 

 rotated slightly. Blood is collected directly into the collecting tube, and post- 

 treatment bleeding can be prevented by holding the eye closed for a brief period. 

 Anesthesia is not necessary. This site can also be used for sterile collection of large 

 volumes (0.5+ ml.) of blood for serum. 135 



Blood may be collected from newborn mice by decapitation or, for smaller quanti- 

 ties, from a cut through the tissues of the neck and jugular vein. When applicable, 

 the latter method is preferable, since there is less contamination with tissue fluids. 

 This same site is suitable for collection of 1-2 lambda of blood from 15 + -day fetuses. 



Quantitative determinations of the erythron. — Three basic determinations for charac- 

 terizing the flowing blood are the number of erythrocytes per unit blood volume, the 

 proportion of the volume of packed red cells to total blood volume ( = hematocrit 

 percentage) and the amount of hemoglobin per unit blood volume. 



The classical method for determining erythrocyte number (erythrocytes/mm. 3 ), 

 involving collection of an exact volume of blood in a Thoma pipette, 200 x dilution 

 with isotonic Hayem's solution, and counting in a Neubauer counting chamber, 

 may be used for mice. For further details of methodology the reader is referred to 

 Wintrobe's Clinical Hematology. 1391 Special adaptations are required to use this 

 method for the small volumes of blood available in fetuses and newborn mice. 1102 

 These include extra calibrations on the Thoma pipette, coating of the inside of the pipette 

 with glycerin or silicone, and modification making the tip of the pipette more 

 pointed to facilitate collecting from a small area. 



A recent improvement especially valuable in murine hematology is use of the 

 Coulter electronic cell counter 859 (Scientific Products Co., Evanston, 111.). In this 

 system, cell counts depend on interruptions of an electric current by cells in a known 

 volume of fluid passing through an aperture. Very high dilutions (1/50,000) of 

 blood in 0.9 n filtered NaCl solution are used, and the number of cells in 0.5 ml. 

 of diluted blood is recorded electronically. (Corrections must be made for coinci- 

 dence due to two or more cells passing through the aperture simultaneously.) Agree- 

 ment between successive samples from the same animal is excellent, and extensive 

 tests have been made of the reliability and accuracy of the method. 127 - 859 Although 

 initial investment is high, the savings in labor and time make possible much more 

 extensive programs than could otherwise be undertaken. The great value of this 



