A flow cytometry method for safe detection of bacterial viability.
bacteria
flow cytometry - viability dye
health and safety
prevention
Journal
Cytometry. Part A : the journal of the International Society for Analytical Cytology
ISSN: 1552-4930
Titre abrégé: Cytometry A
Pays: United States
ID NLM: 101235694
Informations de publication
Date de publication:
03 Oct 2023
03 Oct 2023
Historique:
revised:
18
08
2023
received:
19
06
2023
accepted:
04
09
2023
medline:
3
10
2023
pubmed:
3
10
2023
entrez:
3
10
2023
Statut:
aheadofprint
Résumé
Flow cytometry is a relevant tool to meet the requirements of academic and industrial research projects aimed at estimating the features of a bacterial population (e.g., quantity, viability, activity). One of the remaining challenges is now the safe assessment of bacterial viability while minimizing the risks inherent to existing protocols. In our core facility at the Paris-Saclay University, we have addressed this issue with two objectives: measuring bacterial viability in biological samples and preventing bacterial contamination and chemical exposure of the staff and cytometers used on the platform. Here, we report the development of a protocol achieving these two objectives, including a viability labeling step before bacteria fixation, which removes the risk of biological exposure, and the decrease of the use of reagents such as propidium iodide (PI), which are dangerous for health (CMR: carcinogenic, mutagenic, and reprotoxic). For this purpose, we looked for a non-CMR viability dye that can irreversibly label dead bacteria before fixation procedures and maintain intense fluorescence after further staining. We decided to test on the bacteria, eFluor Fixable Viability dyes, which are usually used on eukaryotic cells. Since the bacteria had size and granularity characteristics very similar to those associated with flow cytometry background signals, a step of bacterial DNA labeling with SYTO or DRAQ5 was necessarily added to differentiate them from the background. Three marker combinations (viability-DNA) were tested on LSR Fortessa and validated on pure bacterial populations (Gram
Identifiants
pubmed: 37786349
doi: 10.1002/cyto.a.24794
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
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