Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues

Abstract

For safety, designated Select Agents in tissues must be inactivated and viability tested before the tissue undergoes further processing and analysis. In response to the shipping of samples of “inactivated” Bacillus anthracis that inadvertently contained live spores to nonregulated entities and partners worldwide, the Federal Register now mandates in-house validation of inactivation procedures and standardization of viability testing to detect live organisms in samples containing Select Agents that have undergone an inactivation process. We tested and validated formaldehyde and glutaraldehyde inactivation procedures for animal tissues infected with virulent B. anthracis , Burkholderia pseudomallei , Francisella tularensis , and Yersinia pestis . We confirmed that our fixation procedures for tissues containing these Tier 1 Select Agents resulted in complete inactivation and that our validated viability testing methods do not interfere with detection of live organisms. Institutions may use this work as a guide to develop and conduct their own testing to comply with the policy.

Effective methods for the inactivation of Francisella tularensis

“…Because F . tularensis does not form spores, inactivation is conducted with common methods, such as treatments with heat [23, 24], 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight [25], 4% PFA and 1% glutaraldehyde in 0.1M sodium cacodylate after formaldehyde [26], a combination of 10% sodium hypochlorite followed by 70% ethanol [27], and ultraviolet (UV) radiation [28]. F .…”

Strategies for Validation of Inactivation of Viruses with Trizol® LS and Formalin Solutions

“…Each of these studies demonstrated successful inactivation of a variety of viruses with either Trizol ® LS or formalin solutions, which when combined with previous literature and a recent publication of formalin efficacy against Tier 1 Select Agent bacteria demonstrates that these are both highly effective and reliable methods to inactivate Select Agents. 15,16 While sample cleanup and detection methods for viruses were optimized through these procedures, the studies presented do not demonstrate any new evidence of efficacy of formalin or Trizol-based solutions against viruses. In addition, our research institute had to absorb costs both in terms of supplies and personnel hours to reach these conclusions, adding up to over $200 000 for each of the assays.…”

“…All inactivation testing demonstrates that both formalin and Trizol ® LS solutions successfully inactivate virus infected cell lines and tissues, which is consistent with previously published literature. [5][6][7][8][9][10][11][12][13][14][15][16] Our objective in these studies was to demonstrate a series of methods that can be used to remove cytotoxic chemicals to allow research institutes working with Select Agents to comply with the latest regulations while minimizing the amount of resources needed to develop cleanup methods from scratch. In our methods, we pursued a series of strategies, the use of concentrating columns or desalting columns for removal of cytotoxic chemicals and retention of inactivated virus or HYPERFlasks that dilute any cytotoxic chemicals sufficiently to avoid degradation of the cell lines.…”

Biosafety during a pandemic: shared resource laboratories rise to the challenge

“…Fixation is often performed with the primary goal of stabilizing samples for downstream assays (e.g., intracellular staining). However, fixation protocols designed for stabilization may not necessarily result in pathogen inactivation and special care is needed in the assessment and development of fixation protocols (14, 26, 34–45). Commercial products, both within and across companies, often contain varying concentrations of fixative.…”