Application of Flow Cytometry to Segregated Kinetic Modeling Based on the Physiological States of Microorganisms

Abstract

Flow cytometry (FC) has been introduced to characterize and to assess the physiological states of microorganisms in conjunction with the classical plate-counting method. To show the applicability of the technique, in particular for the development of kinetic models, pure culture fermentation experiments were followed over time, using both prokaryotic (Lactobacillus hilgardii) and eukaryotic (Saccharomyces cerevisiae) microorganisms growing in standard culture media (MRS and YPD). The differences observed between the active and viable cells determined by FC and CFU, respectively, allowed us to determine that a large number of cells were in a viable but nonculturable (VBNC) state, which resulted in a subpopulation much larger than the damaged-cell (double-stained) subpopulation. Finally, the determination of the evolution of viable, the VBNC, and the dead cells allowed us to develop a segregated kinetic model to describe the yeast and the bacteria population dynamics and glucose consumption in batch cultures. This model, more complete than that which is traditionally used, based only on viable cell measurements, describes better the behavior and the functionality of the cultures, giving a deeper knowledge in real time about the status and the course of the bioprocesses.

Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-Activated Cell Sorting Assay

“…In this research, we pursued an innovative approach to detecting infectious viruses in water using fluorescence-activated cell sorting (FACS). This method is rapid and sensitive, with an established record in microbiological research (29,34,39).…”

“…FACS is a specialized type of flow cytometry which provides a method for counting and sorting a heterogeneous mixture of biological cells into two or more kinds, one cell at a time, based upon the specific light-scattering and fluorescent characteristics of each cell (4,25,34,38). It is a useful method since it provides fast and quantitative recording of fluorescent signals from individual cells (14,16,34,47).…”

Responses of Lactic Acid Bacteria to Stress – VBNC State

“…Badanie tego rodzaju odpowiedzi uzasadnia stosowanie np. fluorescencyjnych technik, które pozwalają dokładnie ocenić stan populacji, z podziałem na subpopulacje [24,29]. Zastosowanie fluorescencyjnych technik umożliwia badanie fizjologii komórek bakterii pod względem różnych cech strukturalnych i funkcjonalnych [12].…”

“…Quirós i wsp. [24], stosując metody łączonego barwienia ChemChromeV6 ® z PI oraz CFU (ang. colony forming unit), przedstawili wiarygodny podział populacji na trzy podpopulacje, podkreślając znaczny udział komórek w VBNC podczas hodowli Lactobacillus hilgardii Lc2.…”

Flow cytometry: is this a novelty in microbiological diagnostics?

“…A kationos festékek a polarizált, az anionos festékek pedig a depolarizált felszíneken akkumulálódnak. A kationos rodamin-123 és a DiOC 6 (3) festékeket például gyakran használják baktériumok membránpotenciáljának mérésére [34]. Használhatók az anionos oxonol festékek is erre a célra, de mivel ezek bejutása a sejtekbe permeabilizálás nélkül nagyobb mér-tékben függ a membránintegritástól, mint a membrándepolarizáltságtól, ezért az eredmények értékelésekor az élő:elpusztult sejtarányt fi gyelembe kell venni.…”