Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry

PLoS One. 2018 Feb 23;13(2):e0192836. doi: 10.1371/journal.pone.0192836. eCollection 2018.


Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Bacillus coagulans / physiology*
  • Bacteriological Techniques*
  • Bifidobacterium longum subspecies infantis / physiology
  • Citrus sinensis
  • Flow Cytometry*
  • Fruit and Vegetable Juices / microbiology
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Lactobacillus acidophilus / physiology
  • Lactobacillus casei / physiology
  • Lactobacillus rhamnosus / physiology
  • Microbial Viability*
  • Probiotics / analysis*

Grant support

This work was sponsored by Sami Labs Limited. There was no external funding involved. The funder provided support in the form of salaries for authors of both Sami Labs Limited and Sabinsa Corporation but did not have any additional role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. All the authors are affiliated to Sami Labs limited or Sabinsa Corporation.