264 MANUAL OF MICROBIOLOGICAL METHODS 



membrane. Under good light the membrane is grasped in an area away 

 from the better lesions by sterile forceps. With sterile small scissors 

 (curved nail scissors are good) the membrane is cut loose at the periph- 

 ery. If the operator is steady and gentle, the membrane disk can 

 be removed intact. The membrane can then be floated in sterile saline 

 in a petri dish. If the dish is held over a dark background and at the 

 edge of a desk-lamp shade, lesions are usually visible. A better method 

 is to view the membrane under a dissecting microscope by both trans- 

 mitted and reflected light. It is well to be alert for several changes in 

 the membrane. The amount of edema, presence of lesions, perivascular 

 changes, condition of the blood vessels, hemorrhage, and membrane luster 

 are some of the things one should look for. The beginner could profit- 

 ably spend considerable time examining normal membranes and mem- 

 branes inoculated with various menstrua containing antibiotics, sterile 

 tissue, and other substances in order to become well acquainted with a 

 range of nonspecific changes. 



Following allantoic, amniotic, and yolk-sac inoculations, it may be 

 necessary to harvest these membranes as well as the fluids. If the 

 contents of the egg are evacuated into a sterile petri dish, individual 

 membranes may be pulled away from the remaining material. With a 

 little practice in learning where to grasp the membranes, whole mem- 

 branes can be disengaged readily rather than picking the membrane out 

 by pieces. If one wishes to remove as much contaminating fluid as 

 possible, the membranes can be rinsed quickly in several changes of cold 

 sterile saline. Any of the above membranes may be ground with an 

 abrasive and saline to prepare a suspension for passing. The suspension 

 should be cultured for bacterial sterility. Stained smears may be 

 made from them, or they may be fixed, sectioned, and stained for 

 observation. 



Embryo. If the embryo is to be collected, it may be removed after 

 opening the amniotic sac. If the embryo is to be removed intact, care 

 must be exercised, for it is very fragile. 



Determining infectiuity titer. Certain investigative problems and 

 diagnostic procedures require the worker to have some concept as to the 

 potency of his viral preparation. Infectivity titers of some viruses can 

 be determined in embryonated eggs. The viral preparation is serially 

 diluted in a protective diluent as mentioned above. Using a separate 

 pipet for each of the preliminary dilutions 10-fold decreases are usually 

 employed, since finer increments not uncommonly result in a scattered 

 end point. Several factors may influence the end point. As mentioned 

 earlier, freshly harvested virus gives a more reliable end point. Prep- 

 arations with very high titers require careful treatment. If the antic- 

 ipated titer is high, it is advisable to dilute into the proper range by 

 larger increments such as 100-fold decreases. For example, if the antic- 



