Table 4.

Comparison of methods to enumerate viable but non-culturable (VBNC) bacteria–suitability for sediment.

Technique	Direct or indirect	Method aim and calculation required	Advantages	Disadvantages	Suitable for sediment?	References
Nalidixic acid	Indirect	Nalidixic acid is bacteriostatic and inhibits cell division at low concentrations.	Allows differentiation of dividing viable cells from non-dividing VBNC cells.	“Resuscitated” cells would subsequently be inhibited by Nalidixic acid as they start to grow.	No	Ohtomo and Saito, 2001
		Need to run in conjunction with direct counts to ensure total counts do not change.		Some bacteria are Nalidixic acid-resistant.
		VBNC = (live-un-elongated cells under Nalidixic acid treatment)		“Injured” cells may only grow on non-selective media, difficult on sediment samples.
BacLight™	Indirect	Uses two dyes (SYTO9 and Propidium iodide) to stain live cells green and dead cells red. Cells are counted under the microscope.	Membrane integrity is one of the most conservative estimators of viability. Can result in overestimation of viability and VBNC fraction.	Step for disaggregation from sediment required.	No	Hassard et al., 2014
				Assumes that dead cells have disrupted membranes.
				Cells may form clusters and be difficult to count.
		VBNC = (live – culturable)
Fluorescent in-situ hybridisation (FISH) and peptide nucleic acid FISH (PNA-FISH).	Indirect	Oligonucleotide probes hybridise to target DNA/RNA and fluoresce under the microscope.	Relatively straight-forward technique. PNA probes have a superior binding capability than traditional FISH probes.	Need to find a species/strain-specific nucleotide probe.	No	Halpern et al., 2007; Malic et al., 2009
				Reliant on microscope quantification
		VBNC = (FISH positive – culturable).		Cells may form clusters and be difficult to count.
Immunomagnetic separation	Indirect	Antibodies for a specific species or strain are coated onto magnetic beads.	Not a standalone method for VBNC detection, Immunomagnetic separation can be used to isolate the organism of choice from environmental samples in conjunction with a quantification method.	Not 100% specific	Yes	Gwyther et al., 2013
		A magnet is used to pull the bacteria-linked beads from an environmental sample.		Step for disaggregation from sediment is required.
				Adds an extra step into the analysis time.
			Relatively straight-forward technique.	Quantification method required.
Flow cytometry (FCM)	Indirect	Cells are labeled with nucleic acid stains e.g., BacLight™. The flow cytometer sorts each cell individually, based on fluorescence backscatter which is used to determine abundance of live and dead cells.	FCM can distinguish between reproductively viable, metabolically active, intact and permeabilized cells.	Requires pure cultures or the quantification of entire populations.	Yes	Wallner et al., 1995; Khan et al., 2010
				Assumes that dead cells have disrupted membranes.
				Rapid in-situ analysis of single cells.
		VBNC = (live – culturable)		Difficult to distinguish between bacteria and phages if using environmental samples due to overlap of distributions and signal noise.
				FCM-FISH has been applied with limited success.
Propidium monoazide—quantitative PCR (PMA-qPCR)	Indirect	PMA binds to DNA in membrane-compromised cells, preventing DNA replication during PCR.	Can target specific species/strain of bacteria.	PMA-qPCR does not in itself distinguish VBNC cells, but enumerates the number gene equivalents from the bacteria with intact membranes.	Yes	Nocker et al., 2007; Gin and Goh, 2013
		ΔCTrefers to the difference in qPCR threshold cycles CT between total bacteria and live bacteria	qPCR is quantitative.	Detachment from sediments and particulate matter required as a pretreatment.
		VBNC = (PMA negative gene equivalents – culturable).
Ethidium monoazide—loop mediated isothermal amplification (EMA-LAMP).	Indirect	EMA binds to DNA in membrane-compromised cells, preventing DNA replication.	Can target specific species/strain of bacteria.	Similar limitations as PMA-qPCR	Yes	Wang et al., 2012,
			LAMP is quicker than PCR.
		VBNC = (EMA negative gene equivalents – culturable).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR).	Indirect	Quantitative PCR method used to detect expression levels of RNA e.g. rpoS gene mRNA.	Targets RNA expression, which is a proxy for activity in bacteria.	Environmental matrices, particularly sediment, may contain PCR inhibitors which restrict applicability.	Yes	Yaron and Matthews, 2002; Quilliam et al., 2011b; Wingender and Flemming, 2011
			Can detect target genes active in VBNC bacteria and compare to levels in culturable bacteria referenced against housekeeping genes.	Difficult to extract RNA. Requires suitable sampling regime and storage.
Autoinducers (AI) /resuscitation promotion factors (RPF).	Direct	Synthetic or biologically produced autoinducer 2 (AI2) or RPF to measure culturability of exposed and unexposed population.	Quantifies VBNC bacteria using the same quantification methodology as ‘culturable’ bacteria therefore a representative comparison.	Difficult to distinguish VBNC from additional growth due to autoinducer/RPF.	Yes	Bari et al., 2013; Ayrapetyan et al., 2014a
				Additional nutrients/cofactors may be required.
		VBNC = (AI or RPF culturable)- (normal culturable).	Species specific or broad spectrum RPF can be used depending on requirements.
				Often species specific.
				Theoretical distinction between resuscitation and growth based on growth rates.
Pre-rRNA analysis molecular viability testing.	Indirect	Detects innate synthetic activity of rRNA precursors.	Greater fraction of RNA pool than mRNA so easier to detect.	Possibility for false negatives lack of detection = VBNC cells.	Yes	Cangelosi et al., 2010,
				Requires a measure of abundance of species of interest.
		Direct indicator of growth as pre-rRNAs only formed in growing cells. In dormant cells pre-rRNA levels decline.	Species specific or constitutive precursors can be selected based on required resolution.
		After nutrient stimulation there is a ratiometric increase in abundance of pre-rRNA from viable cells but not non-viable cells.	Can be used to separate resuscitation from growth, as response time is quicker than that of growth rate of bacteria.	Requires nutrients to stimulate response.
				Reliant on RT-qPCR for detection so similar limitations at quantification step.
Dilution to extinction—resuscitation potential	Direct	VBNC bacteria can be distinguished from growth by a serial dilution method. Cells are subjected to a log dilution series below 1 CFU/ml of culturable cells. These diluted cells are cultured, if growth is determined then bacteria have resuscitated from VBNC.	Quantitative, utilises the same methodology to determine VBNC as culturable counts therefore directly comparable.	Dilution could inhibit quorum sensing based resuscitation.	Yes	Zhang et al., 2015
		VBNC = Resuscitated counts – culturable counts

Bold indicates the factors used to calculate VBNC.