Comparison of EMA-PCR with commonly used viable/dead methodsa

MethodViable/dead criteriaDetection methodAbsolute detection limit (log10 cells/g)Viable/dead differentiation ratio (log10)Time (h)SpecificityAnalyses of mixed culturesFlexiblebConsiderations
EMA-PCRMembrane integrityPCR24∼3Very highYesYesSensitive to high concentrations of material that quench light such as particle contamination
RT-PCRRNA stabilityRT-PCR3cNot quantitative∼5HighNoNoRNA is very heterogeneous and degradation is dependent on the environment and conditions of the cells
GrowthAbility to dividePlate counts, limiting dilution, conductance>1Does not detect dead cells>24Relatively lowYesdNoRecovery of viable cells is dependent on the medium used; growth may not be linked to viability
BacLightMembrane integrityFluorescence microscopy and flow cytometry34∼2NonspecificNoeNoLow sensitivity and sensitive to particle contamination
CTCMetabolic activityFluorescence microscopy and flow cytometry52∼5NonspecificNoNoLack of metabolic activity is not a good indicator of death
  • a Approximate values are extracted from references 5, 4, 6, 24, and 29. RT, reverse transcription; CTC, 5-cyano-2,3-ditotyltetrazolium chloride.

  • b Flexible means that the approach is easily adaptable to different organisms.

  • c Based on our own experience.

  • d If reliable selective media are present for the organisms of interest.

  • e Can potentially be adapted to analyses of mixed cultures by combining flow cytometry and cell sorting.