7 
The organisms tested included Eh coli CS10 which is a wild-type strain, the 
EK-1 host system Eb_ coli HB101, and strain HB101 with the pBR beta T plasmid 
(HBlOl-pBR0T) . Plasmid pBRgT is a pBR-322 plasmid into which has been cloned, 
in the ampicillin resistance gene, DNA complementary to beta tubulin. The 
following subtilis strains were also tested: B^_ subtilis BR151, and BR151 
with the plasmid pHV14 (BR151-pHV14) . Plasmid pHV14 was constructed by ligating 
plasmid pBR322 with plasmid pC194. In B_^ subtilis hosts, the plasmid expresses 
chloramphenicol resistance. 
Ms. Hunt said preliminary laboratory characterization of the EL_ coli organisms 
shewed that HB101 and HBlOl-pBRBT require certain nutrients for replication on 
aqar medium. 
A second characteristic of HB101 and HBlOl-pBRBT was their decreased grewth rate 
compared to the wild-type CS10 when the organisms were grown in a shaking 
water bath at 37 degrees Centigrade. Strains HB101 and HBlOl-pBRBT grew at a 
reduced rate and reached logarithmic phase later than wild type strain CS10. 
HBlOl-pBRBT qrew at a reduced growth rate ccmpared to HB101. 
Ms. Hunt said Eh coli HB101 contains a recanbination-deficient mutation vhich 
results in increased sensitivity to ultraviolet light (UV). Strains HB101 and 
HBlOl-pBRBT demonstrated increased UV sensitivity ccmpared to the wild- type 
F. coli CS10 strain with HBlOl-pBRB T demonstrating an even greater sensitivity to 
UV lioht than HB101. 
Thus, results of preliminary tests indicated that HB101 and HBlOl-pBRBT are 
more debilitated in a laboratory environment than wild- type CS10. HBlOl-pBRBT 
demonstrated an even greater disadvantage than HB101 under these conditions. 
Ms. Hunt described the aerosol test system. She said aerosols of the test 
organisms were dispersed within a static chamber consisting of a modified Hot 
Pack incubator. The chamber volume was 842 liters. Air of the desired 
temperature and humidity flowed through a fiberglass filter (FG-50) into the 
aerosol chamber. The air was dispersed through the aerosol chamber by a 
dispersion fan. When the desired temperature and humidity were attained within 
the aerosol chamber, the fan was turned off and all of the dampers closed. 
A three- jet Collison nebulizer produced the test aerosol. The suspension 
fluid is drawn frem the bottom of the reservoir and impacted onto the side of 
the glass jar. Large particles reflux back dewn into the reservoir while 
smaller particles are released through the spray outlet. Pre-trial particle 
sizinq experiments revealed a mean particle size of 2.49 microns vhen organisms 
were aerosolized in their growth broths. This particle size is well within 
the respirable ranqe, and it has been estimated that 97 percent of particles 
of 2.6 microns or less will contain zero to one organism per droplet. The 
aerosal was sampled by all qlass impingers (AGI) at 5 time intervals within 
one hour. 
Ms. Hunt said organisms contained within the air stream are impinged into a 
liquid collection medium. The sampling liquid is diluted and plated on agar 
medium. 
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