Volume 26, Number 11—November 2020
Research
Validated Methods for Removing Select Agent Samples from Biosafety Level 3 Laboratories
Table 2
Preparation of select agents in different matrices*
| Agent | CFU/mL (matrix) | BAL fluid | Serum and plasma, µL | Culture | BAL cell pellet |
|---|---|---|---|---|---|
| Bacillus anthracis | 1010 (spore prep†) | 20 μL | 20§ | 20 µL | NT |
| Yersinia pestis | 109 (overnight culture) | Resuspend pellet¶ | 20 | Resuspend pellet¶ | NT |
| Burkholderia mallei | 109 (overnight culture) | Resuspend pellet# | 20** | Resuspend pellet†† | NT |
| Burkholderia pseudomallei | 109 (overnight culture) | 200 μL + 1.8 mL BAL | 20‡‡ | Resuspend pellet§§ | 2 × 106 CFU |
| Francisella tularensis | 109 (overnight culture‡) | 20 μL | 20¶¶ | Resuspend pellet## | NT |
*BAL, bronchoalveolar lavage; NT, not tested. †Spores for aerosol challenge were maintained in sterile water and diluted to the nebulizer-challenge concentration of »1 × 1010 CFU/mL. ‡All broth cultures will require a 2% supplement with Isovitalex (Becton Dickinson, https://www.bd.com) to obtain growth of F. tularensis. §Dilute spore prep 1:1000; transfer 20 μL to serum and plasma. ¶Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL BAL fluid or culture media. #Centrifuge 2 mL of overnight culture, resuspend in 2 mL BAL fluid. **Dilute overnight culture 1:100; transfer 20 μL to BAL fluid. ††Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media. ‡‡Dilute overnight culture 1:10 transfer 20 μL to serum or plasma. §§Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media. ¶¶Dilute overnight culture 1:10 transfer 20 μL to BAL fluid. ##Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media.