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by claude@2026-07, 2026-07-03
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This study evaluated six fluorescent markers for their compatibility with halophilic archaea, focusing on cell envelope stability and activity in high-salt, high-membrane-potential conditions using Halobacterium salinarum and Haloferax volcanii. Using both bulk microplate-reader assays and cell-specific microscopy, the authors tested redox probes (alamarBlue/resazurin), membrane potential dyes (MitoTracker Orange-CMTMRos, Rhodamine 123), and nucleic-acid–binding dyes from a LIVE/DEAD kit (SYTO 9/propidium iodide), identifying how probe performance depends on physiology, growth phase, medium composition, and exposure time, including longer times needed to model haloarchaeal environmental states. A key limitation was that propidium iodide behaved unreliably, causing double-labeling and false interpretation of cells as dead. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.
Abstract
Prokaryotes, particularly those in extreme environments, are capable of diverse metabolic states resulting in altered cell envelope structure and function. However, these changes are difficult to assess as standard fluorescent probes are often incompatible with extreme conditions and/or extremophile cell physiology. Halophilic archaea present the challenge of near-saturated intra-/extra-cellular salts, high membrane potential, and extended survival in altered metabolic states including entrapped within salt crystal fluid inclusions. We evaluated the compatibility of six fluorescent markers of cell envelope stability and activity with two model species, Halobacterium salinarum and Haloferax volcanii . Redox activity markers alamarBlue and pure resazurin solutions, membrane potential probes MitoTracker™ Orange-CMTMRos and Rhodamine 123, and SYTO 9 and propidium iodide (LIVE/DEAD™ kit) to assess cell membrane integrity were evaluated for use in bulk (microplate reader) and cell-specific (microscopy) applications. Limitations of each probe were identified, clarifying the utilization of each based on cell physiology, growth phase, medium composition, and probe exposure time including extended timescales needed to simulate the environmental conditions of haloarchaea. Of particular note, propidium iodide behavior was unreliable leading to double-labeling of cells and false interpretation of cells as dead. These data provide important insights into the study of prokaryotes in non-standard conditions.
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Abstract
Prokaryotes, particularly those in extreme environments, are capable of diverse metabolic states resulting in altered cell envelope structure and function. However, these changes are difficult to assess as standard fluorescent probes are often incompatible with extreme conditions and/or extremophile cell physiology. Halophilic archaea present the challenge of near-saturated intra-/extra-cellular salts, high membrane potential, and extended survival in altered metabolic states including entrapped within salt crystal fluid inclusions. We evaluated the compatibility of six fluorescent markers of cell envelope stability and activity with two model species, Halobacterium salinarum and Haloferax volcanii. Redox activity markers alamarBlue and pure resazurin solutions, membrane potential probes MitoTracker™ Orange-CMTMRos and Rhodamine 123, and SYTO 9 and propidium iodide (LIVE/DEAD™ kit) to assess cell membrane integrity were evaluated for use in bulk (microplate reader) and cell-specific (microscopy) applications. Limitations of each probe were identified, clarifying the utilization of each based on cell physiology, growth phase, medium composition, and probe exposure time including extended timescales needed to simulate the environmental conditions of haloarchaea. Of particular note, propidium iodide behavior was unreliable leading to double-labeling of cells and false interpretation of cells as dead. These data provide important insights into the study of prokaryotes in non-standard conditions.
Competing Interest Statement
The authors have declared no competing interest.
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