Exercise XVI 



BLOOD AND CIRCULATION 85 



will now see the two former bands replaced by a 

 single broad absorption band centering at about 

 565 m/i. This is the absorption band of reduced 

 hemoglobin (Hb). The hemoglobin can be re- 

 oxygenated by shaking vigorously; the former 

 bands re-appear. This cycle can be repeated over 

 and over again; indeed, this is how hemoglobin 

 functions in the body. 



Carboxyhemoglobin 



A preparation of diluted blood through which 

 carbon monoxide has been bubbled will be 

 available. Examine its spectrum. Add sodium 

 dithionite. What happens? 



Spectrophotometers are available with which 

 these absorption spectra can be accurately 

 measured. Working in groups of 2 to 4, measure 



the absorption of each of the above solutions 

 at 5 m^u intervals throughout the visible range 

 (400 to 700 myu). Plot the optical density against 

 the wavelength. 



Optical density or extinction is the most useful 

 measure of absorption of light. If the intensity of 

 light of a given wavelength entering a solution is 

 /o, and the intensity of light that emerges is /, 

 then ///o is the fraction transmitted, or trans- 

 mittance. The fraction absorbed is (I — ///o). 

 The optical density or extinction is logio/n//, i.e., 

 the logarithm of 1/transmittance. It has the 

 special virtue of being proportional to the con- 

 centration of pigment, and to the depth of layer. 

 For example, on doubling either the concentra- 

 tion of hemoglobin or the depth of layer meas- 

 ured, one doubles its extinction at all wave- 

 lengths. 



EQUIPMENT 



Per student 



lancet, sterile, disposable 

 microscope slides 

 cover slips 

 2 small test tubes 



Per 2 students 



frog 



cork board 



box of pins 



compound microscope 



dissecting microscope 



slide of mammalian lung 



slide of mammalian artery and vein 



Per 8 students 



bottle 70% ethanol 

 absorbent cotton 



dropping bottle 0.9% sodium chloride solution 

 dropping bottle methyl alcohol 

 differential staining solutions for blood cells* 

 wax crayon 



blood typing sera: anti-A and anti-Bf 

 mammalian blood J 



carboxyhemoglobin solution: the carbon monoxide 

 complex of hemoglobin is prepared easily by bub- 

 bling a gentle stream of carbon monoxide through 

 defibrinated blood diluted 1:100 with water. This 

 must be done in a hood! 



Per laboratory 



beef hearts 



bottle sodium hydrosulfite (dithionite) Na2S204 



spectrophotometer (e.g., Bausch and Lomb "Spec- 



tronic 20") 



hand spectroscope 



*Red and blue stains as provided by Scientific Products Co. (Division of American Hospital Supply Corp., 

 1210 Leon Place, Evanston, III.; branch offices in many cities. Wright's stain may be used instead. 



tAnti-A and anti-B serum can be obtained from most medical supply houses or from Hyland Labora- 

 tories, Los Angeles, Cal. 



jFresh mammalian blood, most easily obtained from a slaughter house, can be defibrinated by shaking 

 vigorously in a bottle with glass beads. Clotting can also be prevented by the addition of 0.1-0.2 ml 10% 

 potassium oxalate for every 10 ml of blood. 



