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
T2 - The Food, GI-tract Functionality and Human Health Cluster
Y2 - 15 March 2004 through 17 March 2004
ER -