# Scanning electron microscopy and extended viability testing as a tool to evaluate the safety of MALDI-TOF extracts for risk group 3 spore-forming bacteria

## TL;DR

This study evaluates the safety of a MALDI-TOF extraction method for handling risk group 3 spore-forming bacteria using SEM imaging and viability testing.

## Contribution

The study introduces a method combining SEM and extended viability testing to assess the safety of inactivating RG3 bacteria for MALDI-TOF analysis.

## Key findings

- PVDF filtration effectively inactivated all tested bacterial preparations.
- SEM imaging revealed that cellulose filters were ineffective for exclusion.
- Microspheres smaller than the PVDF filter's pore size passed through, indicating potential contamination risks.

## Abstract

Introduction. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS for rapid identification of risk group 3 (RG3) bacteria is impeded by the following two main limitations: (a) equipment and maintenance costs for instruments placed within containment and (b) lack of a validated inactivation protocol to move RG3 material to a lower containment level. A validated inactivation method would improve operations of public health laboratories by allowing safe triage of potential RG3 agents. Albeit a validated, zero-risk inactivation protocol is unlikely, scientific interrogation of methods to identify and mitigate procedural biosafety risks is vital for institutional risk assessment.

Gap. To investigate the effect of a standard MALDI-TOF chemical extraction, hypothesized to alter cells, allowing passage through a filter and maintaining ability to replicate, this study paired visualization using a scanning electron microscope (SEM) with extended viability testing.

Aim. This work is intended to support risk assessments for the removal of material from a containment laboratory for MALDI-TOF MS.

Methodology. A standard set of Bacillus cereus and Bacillus anthracis vegetative and spore preparations was treated with a formic acid:acetonitrile extraction, with or without filtration, and plated on five types of media to monitor growth over 14 days. SEM images were taken of treated and untreated preparations, prior, during and after filtration across two filters. Reference beads provided accurate pore size measurements.

Results. SEM demonstrated no difference in treated and untreated cells but did indicate the ineffectiveness of cellulose filters compared to PVDF filters. Growth was observed in preparations that did not include PVDF filtration, whereas all preparations (n=60) that included PVDF filtration were 100% non-viable. Although non-viability was observed, an important finding was the passage of 0.262 and 0.173 µm microspheres through the 0.1 µm PVDF filter. Growth of unfiltered preparations was detected between 1 and 7 days.

Conclusions. This investigation demonstrates the value of interrogating materials used for bacterial inactivation, highlighting significant issues in the application of filters for exclusion purposes. Visual examination via SEM was key to providing evidence towards a low-risk inactivation method. These findings, with an understanding of limitations identified herein, can be used to inform risk assessments for the removal of RG3 bacteria from containment.