APPENDIX H 
II. INSTRUCTIONS TO INVESTIGATORS CONCERNING DATA TO BE 
SUBMITTED ON HOST -PLASMID SYSTEMS PROPOSED FOR 
EK2 CERTIFICATION 
I. Survival 
(A) Time -course experiments must show that survival of the 
-8 
host -plasmid falls to 10 or lower within 24 hours of incubation 
in vitro under non -permissive conditions (i.e., conditions that have 
been demonstrated to obtain in the mammalian intestinal tract). 
(B) The above loss of survival must depend on at least two 
phenotypic traits which contribute independently to the overall contain- 
-8 
ment (i.e., either one alone must be demonstrated to produce 10 
survival within 24 hours under non-permissive conditions). At least 
one trait must be non-revertible, and the reversion rate of the others 
-8 
must be shown to be equal or less than 1x10 per cell per generation 
II. Transmission 
Although only non-conjugative plasmids may be used in EK2 system 
the flora of the intestinal tract include bacteria that harbor conjugative 
plasmids; transfer of the latter to an EK2 cell may result in the 
mobilization of the EK2 plasmid* for transfer to a third, recipient 
bacterium. 
The probability of this occurring will be the product of four 
independent probabilities: 
*We will refer to the separate components of the EK2 system as the 
"EK2 plasmid" and the "EK2 cell. " 
Appendix H--6 
