VIROLOGICAL METHODS 261 



bacterial contamination in the embryonated egg. It must be remem- 

 bered, however, that certain viruses are also susceptible to antibiotics. 

 Filtration through filters of proper porosity to withhold bacteria but not 

 viruses would seem ideal. Some viruses are held back by bacterial filters, 

 and organisms of the pleuropneumonia group pass these filters quite 

 readily. Some of the latter organisms grow quite well in the chick 

 embryo. Not uncommonly it is wise to treat material containing an 

 unknown virus by more than one method prior to inoculation. Penicillin 

 and streptomycin in concentrations of 100-500 units per milliliter are 

 frequently employed antibiotics used to discourage bacterial growth. 



Postinoculation incubation. After the inocula have been appropri- 

 ately prepared and the eggs inoculated, they are ready for sealing, mark- 

 ing, and incubation. Small apertures such as punch holes may be sealed 

 by swabbing with sterile melted paraffin or nail polish. A small piece of 

 gummed tape may be used for this or for closing windowed eggs. Win- 

 dowed eggs are best incubated in a rack which does not allow the window 

 to deviate from its upright position lest the contents be spilled or con- 

 taminated. Punched eggs may be incubated in almost any position. A 

 convenient rack for handling punched eggs can be made simply and for 

 low cost. A double row of IJ^-in. holes drilled l}i in. deep and 2 in. on 

 center in standard 2- by 4-in. lumber prepares such a rack. Material 

 adaptable to repeated autoclaving is best where highly infectious material 

 is used. Eggs may be placed in the holes small end down. Scrubbing 

 with iodine, punching, inoculating, sealing, labeling, and incubating may 

 all be done without removing the embryos from such a rack. 



Incubation temperature. The incubation temperature employed will 

 vary depending upon the virus to be propagated. Most commonly this 

 range is small, 35-37°C. For well-adapted viruses altering the tempera- 

 ture from an optimum of 37 to 35° C results in less growth but not a com- 

 plete failure. Temperature for isolation may be more critical wherein 

 one may fail sometimes at 37°C and succeed at 35°C. It is not uncom- 

 mon to find experienced virologists employing two temperatures for 

 initial isolation of a virus. Some incubators vary in temperature from 

 one shelf to another. It is well to keep a thermometer on each shelf and 

 examine them daily. Temperatures may vary with line load, with a full 

 or an empty incubator, and for other reasons. Generally speaking, 

 incubation temperatures are more critical for viruses than for bacteria 

 and must be controlled carefully. 



Periodic inspection. The time of incubation will vary with the agent 

 under investigation. Previously described well-adapted viruses have the 

 time of incubation well worked out. This may vary from 1 to 7 days for 

 different viruses and wdll vary also depending on what the investigator 

 desires. Unidentified viruses must be handled differently. If the inocu- 



