[Identification and characterization of non-cultivated forms of enterobacteria Erwinia carotovora in continuously incubated cultures].
OBJECTIVE
To determine overall number as well as number of viable cells in continuously incubated cultures of E. carotovora by methods of confocal microscopy and quantitative PCR-analysis.


METHODS
Strain E. carotovora atroseptica SCRI1043 was grown on LB medium to density 2x10(9) CFU/ml. Cells were aggregated by centrifugation and transferred on fresh LB medium, containing alkyloxybenzol, or on the AB medium, which was deficient on phosphorus and carbon. BacLight LIVE/ DEAD kit in combination with confocal laser microscopy as well as quantitative PCR were used for the determination of the number of viable cells.


RESULTS
Total number and number of viable cells in cultures on AB medium was high (10() - 10(9) and 10(7) - 10(8) cells/ml respectively) up to 3 - 5 months of cultivation. Though, number of cultivated cells significantly decreased in all variants of the experiment. Number of viable cells in such cultures was several orders greater than genomic copies detected by PCR. Efficacy of DNA amplification increased after dialysis and deproteinization of samples.


CONCLUSIONS
Loss of cultivation ability when number of viable bacteria is high points to possible switch of E. carotovora cells in non-cultivated state under unfavourable conditions. We assume that it is accompanied by formation of low-molecular components and DNA-bound proteins in cells, which inhibit PCR.
Gogolev
Iu V
IuV
Gorshkov
V Iu
VIu
Petrova
O E
OE
Mukhametshina
N E
NE
Ageeva
M V
MV
D015169:Colony Count, Microbial; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D050296:Microbial Viability; D016973:Pectobacterium carotovorum / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D016133:Polymerase Chain Reaction; D013997:Time Factors
D003470:Culture Media; D004269:DNA, Bacterial