260 MANUAL OF MICROBIOLOGICAL METHODS 



Allantoic sac. When considerable numbers of embryos are to be 

 inoculated, e.g., in determining infectivity titers, the allantoic sac may be 

 inoculated quickly and simply. Two holes are punched in the egg, one 

 over the air sac and one slightly eccentric from the embryo and toward 

 the blunt end. A ^-^-in. 27-gauge needle is introduced at an acute angle 

 from the surface of the shell. This procedure avoids injuring the embryo 

 and usually places the inoculum in the allantoic sac. Practice using 

 methylene blue will permit you to appraise your technic. Drilling a 

 small hole with a 27-gauge needle and drilling the hole over the air sac 

 both contribute to prevent leak-back of the inoculum. One-tenth milli- 

 liter of inoculum is commonly used for the allantoic sac. With proper 

 care large amounts up to 2.0 ml may be used. 



Inoculum. The preparation of inoculum for embryonated eggs is 

 important and frequently requires considerable judgment. Inoculating 

 too much virus may encourage lower titers than anticipated. This is 

 especially true if the virus is not from a fresh harvest. It is held that 

 inactive viral particles compete for cell receptors but do not grow. If 

 this is true, one can see why it would be wiser to dilute the inoculum 

 considerably. Too great a dilution may take the virus concentration 

 beyond a minimal infective dose. Until the peculiarities of a particular 

 strain are known, it is probably best to employ at least two dilutions, e.g., 

 10-1 and 10-^ 



Diluent. Dilute virus may be quite unstable. For this reason it is 

 usually desirable to use a menstruum protective for the virus such as 

 10 per cent serum in saline. One must never cease to consider the 

 menstruum as often as he considers the virus suspended therein. For 

 example, antibodies and inhibitors may be present in any serum or 

 body fluid. Buffered salt solutions are usually preferable to ordinary 

 saline. Some viruses are precipitated by phosphates. Calcium ions are 

 necessary for the receptor-destroying enzyme to function properly; 

 citrate, however, interferes with the complement-fixation test. Equal 

 parts of hormone broth and saline have served well as diluting fluids in 

 some virological laboratories, although this diluent should not be used in 

 the hemagglutination test. No matter which diluting fluid is employed 

 in infectivity titrations, the inoculations had best be done quickly start- 

 ing with the most dilute viral preparation. 



Tissue Preparation. Material obtained from infectious diseases fre- 

 quently presents several problems when it is prepared for inoculation into 

 embryos. Since viruses grow in intimate contact with body cells, it may 

 be advisable to disintegrate the host cells by grinding the exudate or 

 tissue with an abrasive. Freezing and thawing, sonic vibration, or dis- 

 integration in an homogenizer or blender may free a greater number of 

 viral particles. The use of antibiotics may circumvent the problem of 



