268 MANUAL OF MICROBIOLOGICAL METHODS 



pended in 3.0 ml of salt solution to facilitate handling. If 4.5 ml of 

 balanced salt mixture are deposited in each 50-ml flask, 0.25 ml of embryo 

 suspension will give about the right proportion. If a large-bore pipet 

 is used and the embryo suspension shaken just before use, one can with- 

 draw a representative specimen. 



Several salt-solution formulas have been used successfully. The 

 formula for Simms and Sanders' (1942) salt solution follows: 



Solution A G. per liter 



Sodium chloride (NaCl) 160 .00 



Potassium chloride (KCl) 4 . 00 



Calcium chloride (CaCl2-2H20) 2.94 



Magnesium chloride (MgCh-GHaO) 4 . 06 



Solution B 



Sodium bicarbonate (NaHCOa) 20 . 20 



Disodium phosphate (Na2HP04) 4 . 26 



Dextrose 20.00 



Phenol red 1 .00 



Solution A is sterilized by autoclaving; solution B by filtration. Before 

 use 50 ml of solution A and 50 ml of solution B are added to 900 ml of 

 sterile, glass-distilled water. Stock solution B is best refrigerated until 

 needed. Reagent chemicals should be used throughout. 



Too much dilution of the culture by the addition of inoculum is unde- 

 sirable. Approximately a 1 : 10 dilution is satisfactory. If 0.5 ml of 

 inoculum is added to the culture described above, this will be achieved. 



Incubation. Temperature and time of incubation will vary with the 

 virus employed. From 35 to 37°C usually suffices for most viruses. The 

 more rapidly growing viruses will produce satisfactory yields in 2 days. 

 Slower growing viruses require longer. 



Transfer. The virus may be serially transferred to new media by 

 pipetting 0.5-ml quantity of the culture. Preparation, inoculations, 

 harvests, and transfers can best be done in a cubicle to avoid air con- 

 tamination. Antibiotics in subtoxic concentrations may be used judi- 

 ciously in some cases to help combat bacteria. Bacteria should be 

 excluded, if possible, rather than killed or inhibited after admission. 



Practically all the precautions urged under the discussion of embryo- 

 nated egg propagation of viruses are urged here. Inoculated controls 

 held at 4°C and uninoculated controls held at the experimental tempera- 

 ture should be employed. If there is no propagation in the former and 

 no evidence of virus in the latter, the worker may feel secure. One still 

 has to prove growth in the experimental cultures, however. This proof 

 may be accomplished by checking an increase in some reliable function of 

 the virus or by extinction dilution. In the latter case, if the virus is 

 serially diluted from one culture to the next for a sufficient number of 



