Application of microplate scale fluorochrome staining assay for assessment of viability and stability of probiotic Bifidobacterium sp.

Hanna-Leena Alakomi, Jaana Mättö, Anu Vaari, Ilkka Virkajärvi, Maria Saarela

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

Abstract

Probiotic cultures encounter harsh conditions during production and in the GI-tract. There is a need for rapid and reliable methods predicting survival and activity of the probiotic cultures in various applications. In this study we describe a fluorescence staining assay for the determination of viability of Bifidobacterium cells in microplate scale. LIVE/DEAD BacLight Bacterial Viability Kit (SYTO9 and propidium iodide) was utilized for viability testing of fresh and freeze-dried Bifidobacterium cells and the fluorescence intensity was detected by a microplate fluorometer. Validation of the microplate scale assay was performed by comparing results to colony forming units, fluorescence microscopy and spectroscopy. Fresh and freeze-dried B. animalis cultures treated in acidic conditions (pH 2.5 and pH 3.0 alone and in combination with pepsin) or stored at different temperatures were used to study the applicability of the microplate assay for viability assessment of stressed cells. To reveal changes in membrane functions during acid treatment, DIBAC4 (a potentiometric fluorochrome) was additionally used for the analysis of the acid treated cells. In general, the results obtained with the microplate assay were comparable with plate count analysis and fluorescence microscopy. Microplate assay with viability stains gave an estimate of the viability of the probiotic preparations (detection level 106 cfu and the assay was applicable also for acid-treated cells. In the acid tolerance test, B. animalis was sensitive to pH 2.5, although pepsin had clearly protective effect in acidic conditions. Potentiometric measurements with DiBAC4 fluorochrome indicate that acidic conditions caused hyperpolarization of the cell membrane in B. animalis cells. Benefits from the fluorochromic viability assays are that changes in cell state in probiotic preparations can be estimated earlier compared to the results obtained with traditional cultivation methods.
Original languageEnglish
Title of host publication The Food, GI-tract Functionality and Human Health Cluster: 3rd PROEUHEALTH Workshop
Subtitle of host publicationAbstracts and posters
Place of PublicationEspoo
PublisherVTT Technical Research Centre of Finland
Pages55-55
ISBN (Electronic)951-38-6290-9
ISBN (Print)951-38-6289-5
Publication statusPublished - 2004
EventThe Food, GI-tract Functionality and Human Health Cluster: 3rd PROEUHEALTH Workshop - Sitges, Spain
Duration: 15 Mar 200417 Mar 2004

Publication series

NameVTT Symposium
PublisherVTT
Number232
ISSN (Print)0357-9387
ISSN (Electronic)1455-0873

Workshop

WorkshopThe Food, GI-tract Functionality and Human Health Cluster
CountrySpain
CitySitges
Period15/03/0417/03/04

Fingerprint

fluorescent dyes
Bifidobacterium
probiotics
viability
assays
cells
pepsin
fluorescence microscopy
fluorescence emission spectroscopy
fluorescence
acid tolerance
acids
staining
acid treatment
plate count
gastrointestinal system
protective effect
cell membranes
dyes
testing

Cite this

Alakomi, H-L., Mättö, J., Vaari, A., Virkajärvi, I., & Saarela, M. (2004). Application of microplate scale fluorochrome staining assay for assessment of viability and stability of probiotic Bifidobacterium sp. In The Food, GI-tract Functionality and Human Health Cluster: 3rd PROEUHEALTH Workshop : Abstracts and posters (pp. 55-55). Espoo: VTT Technical Research Centre of Finland. VTT Symposium, No. 232
Alakomi, Hanna-Leena ; Mättö, Jaana ; Vaari, Anu ; Virkajärvi, Ilkka ; Saarela, Maria. / Application of microplate scale fluorochrome staining assay for assessment of viability and stability of probiotic Bifidobacterium sp. The Food, GI-tract Functionality and Human Health Cluster: 3rd PROEUHEALTH Workshop : Abstracts and posters. Espoo : VTT Technical Research Centre of Finland, 2004. pp. 55-55 (VTT Symposium; No. 232).
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Alakomi, H-L, Mättö, J, Vaari, A, Virkajärvi, I & Saarela, M 2004, Application of microplate scale fluorochrome staining assay for assessment of viability and stability of probiotic Bifidobacterium sp. in The Food, GI-tract Functionality and Human Health Cluster: 3rd PROEUHEALTH Workshop : Abstracts and posters. VTT Technical Research Centre of Finland, Espoo, VTT Symposium, no. 232, pp. 55-55, The Food, GI-tract Functionality and Human Health Cluster, Sitges, Spain, 15/03/04.

Application of microplate scale fluorochrome staining assay for assessment of viability and stability of probiotic Bifidobacterium sp. / Alakomi, Hanna-Leena; Mättö, Jaana; Vaari, Anu; Virkajärvi, Ilkka; Saarela, Maria.

The Food, GI-tract Functionality and Human Health Cluster: 3rd PROEUHEALTH Workshop : Abstracts and posters. Espoo : VTT Technical Research Centre of Finland, 2004. p. 55-55 (VTT Symposium; No. 232).

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

TY - CHAP

T1 - Application of microplate scale fluorochrome staining assay for assessment of viability and stability of probiotic Bifidobacterium sp.

AU - Alakomi, Hanna-Leena

AU - Mättö, Jaana

AU - Vaari, Anu

AU - Virkajärvi, Ilkka

AU - Saarela, Maria

PY - 2004

Y1 - 2004

N2 - Probiotic cultures encounter harsh conditions during production and in the GI-tract. There is a need for rapid and reliable methods predicting survival and activity of the probiotic cultures in various applications. In this study we describe a fluorescence staining assay for the determination of viability of Bifidobacterium cells in microplate scale. LIVE/DEAD BacLight Bacterial Viability Kit (SYTO9 and propidium iodide) was utilized for viability testing of fresh and freeze-dried Bifidobacterium cells and the fluorescence intensity was detected by a microplate fluorometer. Validation of the microplate scale assay was performed by comparing results to colony forming units, fluorescence microscopy and spectroscopy. Fresh and freeze-dried B. animalis cultures treated in acidic conditions (pH 2.5 and pH 3.0 alone and in combination with pepsin) or stored at different temperatures were used to study the applicability of the microplate assay for viability assessment of stressed cells. To reveal changes in membrane functions during acid treatment, DIBAC4 (a potentiometric fluorochrome) was additionally used for the analysis of the acid treated cells. In general, the results obtained with the microplate assay were comparable with plate count analysis and fluorescence microscopy. Microplate assay with viability stains gave an estimate of the viability of the probiotic preparations (detection level 106 cfu and the assay was applicable also for acid-treated cells. In the acid tolerance test, B. animalis was sensitive to pH 2.5, although pepsin had clearly protective effect in acidic conditions. Potentiometric measurements with DiBAC4 fluorochrome indicate that acidic conditions caused hyperpolarization of the cell membrane in B. animalis cells. Benefits from the fluorochromic viability assays are that changes in cell state in probiotic preparations can be estimated earlier compared to the results obtained with traditional cultivation methods.

AB - Probiotic cultures encounter harsh conditions during production and in the GI-tract. There is a need for rapid and reliable methods predicting survival and activity of the probiotic cultures in various applications. In this study we describe a fluorescence staining assay for the determination of viability of Bifidobacterium cells in microplate scale. LIVE/DEAD BacLight Bacterial Viability Kit (SYTO9 and propidium iodide) was utilized for viability testing of fresh and freeze-dried Bifidobacterium cells and the fluorescence intensity was detected by a microplate fluorometer. Validation of the microplate scale assay was performed by comparing results to colony forming units, fluorescence microscopy and spectroscopy. Fresh and freeze-dried B. animalis cultures treated in acidic conditions (pH 2.5 and pH 3.0 alone and in combination with pepsin) or stored at different temperatures were used to study the applicability of the microplate assay for viability assessment of stressed cells. To reveal changes in membrane functions during acid treatment, DIBAC4 (a potentiometric fluorochrome) was additionally used for the analysis of the acid treated cells. In general, the results obtained with the microplate assay were comparable with plate count analysis and fluorescence microscopy. Microplate assay with viability stains gave an estimate of the viability of the probiotic preparations (detection level 106 cfu and the assay was applicable also for acid-treated cells. In the acid tolerance test, B. animalis was sensitive to pH 2.5, although pepsin had clearly protective effect in acidic conditions. Potentiometric measurements with DiBAC4 fluorochrome indicate that acidic conditions caused hyperpolarization of the cell membrane in B. animalis cells. Benefits from the fluorochromic viability assays are that changes in cell state in probiotic preparations can be estimated earlier compared to the results obtained with traditional cultivation methods.

M3 - Conference abstract in proceedings

SN - 951-38-6289-5

T3 - VTT Symposium

SP - 55

EP - 55

BT - The Food, GI-tract Functionality and Human Health Cluster: 3rd PROEUHEALTH Workshop

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Alakomi H-L, Mättö J, Vaari A, Virkajärvi I, Saarela M. Application of microplate scale fluorochrome staining assay for assessment of viability and stability of probiotic Bifidobacterium sp. In The Food, GI-tract Functionality and Human Health Cluster: 3rd PROEUHEALTH Workshop : Abstracts and posters. Espoo: VTT Technical Research Centre of Finland. 2004. p. 55-55. (VTT Symposium; No. 232).