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Volume 26, Number 11—November 2020
Research

Validated Methods for Removing Select Agent Samples from Biosafety Level 3 Laboratories

Alexandria E. Kesterson, John E. Craig, Lara J. Chuvala, and Henry S. HeineComments to Author 
Author affiliation: Author affiliation: University of Florida, Orlando, Florida, USA

Main Article

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.

Main Article

Page created: August 05, 2020
Page updated: October 17, 2020 10:09 PM EDT
Page reviewed: October 17, 2020 10:09 PM EDT
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