1982 Summary Statement: Berg et al. 
Proc. Nat. Acad. Sci. USA 72 (1975) 
Containment of potentially biohazardous agents can be 
achieved in several ways. The most significant contribution to 
limiting the spread of the recombinant DNAs is the use of 
biological barriers. These barriers are of two types: (i) fas- 
tidious bacterial hosts unable to survive in natural environ- 
ments, and (it) nontransmissible and equally fastidious vec- 
tors (plasmids, bacteriophages, or other viruses) able to 
grow only in specified hosts. Physical containment, ex- 
emplified by the use of suitable hoods, or where applicable, 
limited access or negative pressure laboratories, provides an 
additional factor of safety. Particularly important is strict 
adherence to good microbiological practices which, to a large 
measure can limit the escape of organisms from the experi- 
mental situation, and thereby increase the safety of the 
operation. Consequently, education and training of all per- 
sonnel involved in the experiments is essential to the effec- 
tiveness of all containment measures. In practice, these 
different means of containment will complement one another 
and documented substantial improvements in the ability to 
restrict the growth of bacterial hosts and vectors could permit 
modifications of the complementary physical containment 
requirements. 
Stringent physical containment and rigorous laboratory 
procedures can reduce but not eliminate the possibility of 
spreading potentially hazardous agents. Therefore, investi- 
gators relying upon “disarmed” hosts and vectors for addi- 
tional safety must rigorously test the effectiveness of these 
agents before accepting their validity as biological barriers. 
lit. RECOMMENDATIONS FOR MATCHING TYPES OF 
CONTAINMENT WITH TYPES OF EXPERIMENTS 
No classification of experiments as to risk and no set of con- 
tainment procedures can anticipate all situations. Given our 
present uncertainties about the hazards, the parameters 
proposed here are broadly conceived and meant to provide 
provisional guidelines for investigators and agencies con- 
cerned with research on recombinant DNAs. However, each 
investigator bears a responsibility for determining whether, 
in his particular case, special circumstances warrant a higher 
level of containment than is suggested here. 
A. Types of containment 
1 . Minimal Risk. This type of containment is intended for 
experiments in which the biohazards may be accurately 
assessed and are expected to be minimal. Such containment 
can be achieved by following the operating procedures recom- 
mended for clinical microbiological laboratories. Essential 
features of such facilities are no drinking, eating, or smoking 
in the laboratory, wearing laboratory coats in the work area, 
the use of cotton-plugged pipettes or preferably mechanical 
pipetting devices, and prompt disinfection of contaminated 
materials. 
2. Low Risk. This level of containment is appropriate for 
experiments which generate novel biotypes but where the 
available information indicates that the recombinant DNA 
cannot alter appreciably the ecological behavior of the re- 
cipient species, increase significantly its pathogenicity, or 
prevent effective treatment of any resulting infections. The 
key features of this containment (in addition to the minimal 
procedures mentioned above) are a prohibition on mouth 
pipetting, access limited to laboratory personnel, and the 
use of biological safety cabinets for procedures likely to 
produce aerosols (e.g., blending and sonication). Though 
existing vectors may be used in conjunction with low risk 
procedures, safer vectors and hosts should be adopted as they 
become available. 
3. Moderate Risk. Such containment facilities are intended 
for experiments in which there is a probability of generating 
an agent with a significant potential for pathogenicity or 
ecological disruption. The principle features of this level of 
containment , in addition to those of the two preceding classes, 
are that transfer operations should be carried out in biological 
safety cabinets (e.g., laminar flow hoods), gloves should be 
worn during the handling of infectious materials, vacuum 
lines must be protected by filters, and negative pressure 
should be maintained in the limited access laboratories. 
Moreover, experiments posing a moderate risk must be done 
only with vectors and hosts that have an appreciably impaired 
capacity to multiply outside of the laboratory. 
4- High Risk. This level of containment is intended for 
experiments in which the potential for ecological disruption or 
pathogenicity of the modified organism could be severe and 
thereby pose a serious biohazard to laboratory personnel or 
the public. The main features of this type of facility, which 
was designed to contain highly infectious microbiological 
agents, are its isolation from other areas by air locks, a 
negative pressure environment, a requirement for clothing 
changes and showers for entering personnel, and laboratories 
fitted with treatment systems to inactivate or remove bio- 
logical agents that may be contaminants in exhaust air and 
liquid and solid wastes. All persons occupying these areas 
should wear protective laboratory clothing and shower at 
each exit from the containment facility. The handling of 
agents should be confined to biological safety cabinets in 
which the exhaust air is incinerated or passed through Hepa 
filters. High risk containment includes, in addition to the 
physical and procedural features described above, the use of 
rigorously tested vectors and hosts whose growth can be 
confined to the laboratory. 
B. Types of experiments 
Accurate estimates of the risks associated with different 
types of experiments are difficult to obtain because of 'our 
ignorance of the probability that the anticipated dangers 
will manifest themselves. Nevertheless, experiments involving 
the construction and propagation of recombinant DNA 
molecules using DNAs from ( i ) prokaryotes, bacteriophages, 
and other plasmids, (ii) animal viruses, and (in) eukaryotes 
have been characterized as minimal, low, moderate, and high 
risks to guide investigators in their choice of the appropriate 
containment. These designations should be viewed as interim 
assignments which will need to be revised upward or down- 
ward in the light of future experience. 
The recombinant DNA molecules themselves, as distinct 
from cells carrying them, may be infectious to bacteria or 
higher organisms. DNA preparations from these experiments, 
particularly in large quantities, should be chemically in- 
activated before disposal. 
1. Prokaryotes, Bacteriophages, and Bacterial Plasmids. 
Where the construction of recombinant DNA molecules and 
their propagation involves prokaryotic agents that are known 
to exchange genetic information naturally, the experiments 
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