Dr. Donald S. Fredrickson 
September 23, 1978 
Page 7 
tures which is usually by shaking and/or aeration and the processing of these cul- 
tures by centrifugation. It is also well known that substantial aerosols can arise 
by aeration of cultures during growth and similarly in the filling of centrifuge 
tubes, in the removal of supernatant fluids and in the suspension of sedimented 
cells. This is particularly true when using cultures and/or suspensions of one 
liter or more. Thus, even if centrifugation is done in sealed rotors or using 
safety centrifuge cups, it is still important to load and unload centrifuge rotors 
using a biological safety cabinet. There are also water baths and other means to 
preclude distribution of aerosols arising from aerated growth of microorganisms 
containing recombinant DNA. These issues should be clarified since some labora- 
tories around the country operating under P2 containment do not have biological 
safety hoods and others do. Certainly if such hoods are not necessary, it will be 
a substantial saving of equipment monies that could be used for other purposes. 
10. Section II-D. Biological Containment (p. 33075). I noticed a few grammatical 
or typographical errors in this section (organelle DNA rather than organelle vector, 
0 replaced by X and conjugative plasmid rather than conjugation-proficient plasmid) 
but a few more substantial problems are enumerated below. 
11. Section II-D-1. Levels of Biological Containment (p. 33075). In this section, 
it states "Any combination of vector and host which are to provide biological con- 
tainment must be constructed" (my underlining) which seems at variance with the 
statement in the Decision of the Director to Issue Revised Guidelines that wild-type 
organisms might be allowed as components of HV1 systems. The above wording would 
certainly seem to preclude the use of wild- type host-vector systems as would the 
requirement in Section I I -D-2- b- ( 1 ) dealing with approval of HV1 systems wherein 
it states that an investigator must submit "data on any mutations which render this 
organism less able to survive or transmit genetic information". The intent of NIH 
should, therefore, be clarified. In resolving this issue, it should be noted that 
even prototrophic "wild-type" E_. col i K-12 strains are not equivalent to wild-type 
E_. col i strains recently isolated from nature since the K-12 strain has acquired 
mutations during its long maintenance under laboratory conditions causing LPS and 
K antigen syntheses to be defective. These laboratory-acquired mutations certainly 
contribute to biological containment. Thus in deciding on requirements for HV1 
systems, one should not discount the importance of laboratory-acquired as opposed 
to induced mutations and should use the term "wild-type" in reference to the usual 
phenotype associated with strains of the species recently isolated from nature. 
12. Section II-D-l-a-(l) EK1 (p. 33075). Most host components of EK1 systems used 
in recombinant DNA research have mutations in addition to those acquired during 
K-12's laboratory sojourn that confer nutritional requirements, cause recombination 
to be defective, etc. and thus are not wild-type prototrophic strains of £. col i 
K-12. Indeed, it is known that certain of these mutations very much diminish sur- 
vival in the intestinal tract or in other environments and/or reduce the likelihood 
for transmission of recombinant DNA. While the inclusion of such mutations in any 
newly developed host strains to be employed as components of EK1 systems should not 
be mandatory, it might be wise to encourage it. I make this suggestion because, 
during the International Congress of Microbiology Meeting in Munich this past week. 
[A-314] 
