APPENDIX H 
Comments on: 
(I ) This fraction should be measured both at the time of lysis and 
at 24 hours after lysis with the culture maintained under optimal growing 
-conditions. The denominator in both cases is the titer of phage at 
the time of lysis . The numiber of fragmient -containing bacteria should be 
measured in an appropriate manner with justification by reconstitution 
experiments. The test at 24 hours is intended to represent a worst 
possible case, which should not normally arise. The requirement for 
a 2 4-hour test applies only to the host used for propagation, but not 
necessarily to the host used in procedures at the scale of individual 
plaques. 
+ 
As an example, such a test might measure the number of gal 
bacteria formed after infection of permissive host bacteria (lacking 
the gal base sequence homology with the cloned fragment) with a phage 
+ 
vector containing a gal (model cloned DNA ) fragment. Demonstration 
+ 
that authentic gal colonies could be detected at the frequency measured 
in the test would constitute appropriate justification. 
(2) The Working Group does not wish to specify at this time non- 
permissive conditions that must be met in this test for all cases. 
The two tests suggested in current applications: "survival in raw 
sewage" and "survival in tap water, " though useful, refer to survival 
in nature and are therefore more appropriately considered in the context 
of EK3 certifications. 
For the present, the safety factor characteristic of any particular 
host can be considered independently for each application. An example 
of the application of this principle is the host for a current A EK2 
Appendix H — 2 
