22 ' Fixation (chap. 1) 



Fixation by Perfusion 



Perfusion (forceful flooding of tissue) is of advantage only for a tissue 

 that requires rapid fixation but is not readily accessible for rapid re- 

 moval. A prime example is the central nervous system. Many organs are 

 not adequately fixed by this method because the perfusion fluid may be 

 carried away from rather than to the cells. 



Special equipment necessary for perfusion includes (1) a glass cannula 

 which fits the specific aorta to be used; (2) rubber tubing to connect the 

 cannula to the (3) perfusion bottle. 



When the animal is dead or under deep anesthesia, cut the large ves- 

 sels in the neck and drain oiu as much blood as possible. Expose the 

 pericardium by cutting the costal cartilages and elevating the sternum. 

 Cut the pericardium and reflect it back to expose the large arteries. 

 Free part of the aorta from the surrounding tissue and place a mois- 

 tened ligatvire behind it. Make a small slit directed posteriorly in the 

 wall of the aorta and insert the moistened cannula. Bring the two ends 

 of the thread together and tie the cannula firmly in place. Cut open the 

 right atrium to permit escape of blood and other fluids. 



Precede fixation with a small amount of saline (50-100 ml.). Fill just 

 the rubber tubing leading from the perfusion bottle to the cannula. 

 (Separate the saline from the fixative with a clamp near the attachment 

 of the rubber tubing to the bottle.) Fill the perfusion bottle with fixa- 

 tive (500-1000 ml. depending on size of animal). The fluid should be 

 warmed to body temperature. The saline precedes the fixative to wash 

 out residue blood before it becomes fixed to the vessel walls. If a forma- 

 lin-dichromate fixative is being used substitute 2.5% potassium dichro- 

 mate for normal saline. 



When ready to start perfusion, with bottle at table level open clamp 

 on rubber tubing. Gradually raise the bottle to increase pressure of 

 fluid. Continue to raise the bottle gradually until at a height of 4 to 5 

 feet enough pressure is exerted to force out most of the blood. After 

 5 minutes, open the abdomen and examine the organ to be perfused. 

 If the surface vessels are still filled with blood and the organ has not 

 begun to take on the color of the perfusing sohuion, it is possible that 

 the perfusion has failed. But sometimes stubborn cases may require 

 10 to 30 minutes to perfuse. When blood color is absent, perfusion is 

 complete. 



Observe these suggestions: (1) the cannula used should be as large 

 as possible to permit as rapid a flow as possible. This aids in washing 



