Worst-case Challenge to Sterilization Efficacy
To characterize the microbial killing efficacy of the sterilants to be
evaluated under worst-case conditions, 10 uL of ATS containing - 108
cfu/ml of the test microorganisms is inoculated inside a surrogate lumen
carrier (3mm internal diameter, 2 cm long), and dried at room temperature
overnight. The inoculated test segment is then assembled into a lumen of
125cm total length and then processed through the sterilization process. The
test carriers consist of PVC plastic tubing cut into 2 cm lengths or PTFE
endoscopy biopsy channel tubing cut into 2 cm lengths.
For sterility testing, the central lumen segment is aseptically removed and
placed in a tube containing 2 mLs of sterile tryptic soy broth containing
10% fetal bovine serum. This segment is incubated at 35oC for 5 days for e.faecalis; 55oC for 5 days for G.stearothermophilus; and 30oC for 10 days for
M. chelonae. Because turbidity may be hard to detect for M.chelonae, blind
subcultures are performed after 10 days of incubation by sub-culturing the
broth to BA and incubating the plates at 30oC for 5 days.
To quantitate the level of residual viable test organisms post-treatment,
the entral segment of the duplicate set of lumen test carriers is
aseptically disassembled and placed into a sterile tube containing 2 mLs of
sterile tryptic soy broth containing 10% fetal bovine serum. The tube is sonicated in a Branson 1220 sonicator bath for 2 x 5 secs, and then mixed on
an S/P Multitube vortexer (American Dade, Miami, FL) on setting "2" for 10
minutes. Serial 1:10 dilutions are prepared in sterile tryptic soy broth
and 0.1 mLs plated on Tryptic Soy agar plates (BA plates for M.chelonae)
using the spread plate technique. The inoculated plates are incubated at
35oC for 24 - 48 hours for E.faecalis, 55oC for 24 - 48 hours for G.stearothermophilus and 30oC for 48 - 72 hours for M. chelonae. Colonies were
enumerated and the viable count per test carrier was calculated.
