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Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry
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Development of fungal spore staining methods for flow cytometric quantification and their application in

Gang Wen1, Ruihua Cao1, Qiqi Wan1

  • 1Key Laboratory of Northwest Water Resource, Environment and Ecology, MOE, Xi'an University of Architecture and Technology, Xi'an, 710055, PR China; Shaanxi Key Laboratory of Environmental Engineering, Xi'an University of Architecture and Technology, Xi'an, 710055, PR China.

Chemosphere
|January 31, 2020
PubMed

Insights

A new flow cytometry (FCM) method accurately quantifies fungal spores in water, assessing viability and disinfectant inactivation. This rapid technique offers an alternative to traditional, time-consuming methods for monitoring drinking water quality.

Area of Science:

  • Environmental microbiology
  • Analytical chemistry
  • Water quality assessment

Background:

  • Fungal contamination in drinking water is a growing concern.
  • Traditional methods like heterotrophic plate counts (HPC) are slow, labor-intensive, and cannot detect viable but non-culturable fungi.
  • Accurate enumeration and viability assessment of fungal spores are crucial for water safety.

Purpose of the Study:

  • To develop a rapid, simple, and accurate method for quantifying fungal spores in water.
  • To discriminate the viability of fungal spores using flow cytometry (FCM).
  • To evaluate the inactivation of fungal spores by chlorine-based disinfectants.

Main Methods:

  • Established an optimal staining protocol using SYBR Green I and Ethylene diamine tetraacetic acid (EDTA) for FCM analysis.
  • Pretreated spore suspensions with sonication.
  • Validated the FCM method against heterotrophic plate counts (HPC) and microscope observations.
  • Assessed fungal spore inactivation kinetics using FCM and plating after exposure to chlorine-based disinfectants.

Main Results:

  • The FCM method showed high correlation with HPC (R=0.996) and microscopy (R=0.988).
  • Optimal staining involved sonication, SYBR Green I, EDTA, incubation at 35°C for 20 min.
  • Fungal spores lost culturability before membrane integrity decreased during disinfection.
  • Inactivation rates followed the order: chlorine dioxide > chlorine > chloramine.

Conclusions:

  • Flow cytometry (FCM) provides an accurate and efficient alternative for enumerating fungal spores and assessing their viability in water.
  • The developed FCM method is suitable for evaluating the efficacy of disinfectants against fungal spores.
  • This technique aids in ensuring the safety of drinking water by monitoring fungal contamination and disinfection effectiveness.
Keywords:
Chlorine-based disinfectionFlow cytometryFungal sporesStaining methodViability

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