iriU/v- lb 
- 6 - 
B\ P3 Physical Containment 
1. Assay of £. c ol i lysates, demonstrated to be free of viable bacteria, 
containing polycma-pl asmid recombinant molecules for the presence of 
infectious polyoma virus. 
2. Infection of tissue cultures with purified P-lambda or P-plasmid 
DNAs (since in tnese assays polyoma DNA rather than a prokaryotic 
DNA is the vector). 
3. V\ssay of £. col i lysates, demonstrated to be free of viable bacteria 
and polyoma lambda phage, for the presence of polyoma polypeptides. 
C. P2 Physical Containment 
1. In vitro ligation of restriction enzyme cleavage products. 
*2. Chemical characterization of purified recombinant DNA subsequent to 
cloning in bacteria and following phenol deproteini zati on. 
*3. Immunologic assays of polyoma HI antibody present in mouse serum 
following a 30-minute incubation of the serum at 56-60C. 
4. Following the treatment of bacterial lysates containing polyoma- 
lambda phage with phenol/SDS, the extracts can be handled at the P2 
containment level. 
V I . PROPOSED MEASURES FOR CONTAINMENT 
A. P4 
1. All safety practices will be carried out in accordance with the 
Safety and Operations Manual which is an appendix to this IMUA. 
2. The liquid disinfectant to be used at the P4, P3 and P2 levels of 
containment is a 1:10 dilution of a 4-6% solution of sodium hypochlorite 
or a 1:10 dilution of clorox or equivalent commercial preparation 
containing 5.25% sodium hypochlorite (Skulka, A. e£ a]_. , NARSM #69; 
Andrew Lewis, Jr., unpublished data). 
B. P3 f 
1. All safety practices described under P3 in the Recombinant DNA Research 
Guidelines (Federal Register 41(131 ) : 279 1 3 , July 7, 1976) will be 
adhered to. 
2. The choice of disinfectant is stated in 71. A. 2. 
*DNA preparations and sera will be tested for sterility prior to their 
removal from P4 containment. For procedures for testing, see IX. 
Transfer Of Materials Out Of Class III Cabinets. 
[Appendix C — 236] 
