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Table 1 Comparison of commonly used techniques to identify living and/or dead cells, particularly those applicable or potentially applicable to microbial communities

From: Schrödinger’s microbes: Tools for distinguishing the living from the dead in microbial ecosystems

Method Approach Compatible with next-generation sequencing? Compatible techniques Compatible sample types Applicable to low-biomass samples? Compatible biological entities Pros Cons References
Cultivation Plating and/or liquid culture to visualize actively multiplying cells Y Many Many (nearly all environments) Y Many (some representatives across broad phylogenetic groups of bacteria, archaea, fungi, spores, and viruses have been cultured) Unambiguous detection of viable microbes when cultivable Many microbes are not (yet) cultivable, therefore not practical for characterizing the viable portion of most microbial communities [33, 34]
Propidium iodide (PI) Dye binding to DNA in membrane-compromised cells and extracellular DNA; sometimes used in combination with total nucleic acid stains Na Many, e.g., epifluorescence microscopy, confocal laser scanning microscopy, flow cytometry, fluorometry Many (e.g., marine, freshwater, air, and soil samples), but samples must be in aqueous solution N Many (e.g., demonstrated for some psychrophilic, halophilic, and methanogenic archaea and some yeast, fungi, Gram + and Gram − bacteria) Absolute live/dead abundance quantification is possible when combined with dyes that can permeate intact membranes; readily available in commercial kits Known to stain viable cells of some species, and some organisms may not stain properly [52, 47]
Propidium monoazide (PMA) Dye binding to DNA in membrane-compromised cells and extracellular DNA Y Many, e.g., PCR, qPCR, MDA metagenomics, FISH, LAMP, microarrays, DGGE Many (e.g., marine, clean room, sediment, soil, biofilm, and wastewater treatment samples), but samples must be in aqueous solution Y Many (e.g., demonstrated for some methanogenic archaea, some Gram + and Gram − bacteria, some viruses, and some spores) Easy to perform and relatively fast; compared to EMA, more selective and less cytotoxic; several options for protocol trouble-shooting (see text) Optimization of the method might be necessary; known to stain viable cells of some species and not stain dead cells of other species (but generally more selective in this regard than EMA) [81, 73, 74, 75, 66, 69, 71, 70, 20]
Ethidium monoazide (EMA) Dye binding to DNA in membrane-compromised cells and extracellular DNA Y Flow cytometry and PCR Many (e.g., pure cultures from marine and food samples; likely similar to PMA, but not widely tested), but samples must be in aqueous solution Na Less well studied, but likely similar to PMA above Several options for protocol troubleshooting (see text) Known to stain viable cells of some species; less selective and more cytotoxic than PMA [60, 57, 58, 59]
Alexa Fluor Hydrazide (AFH) Dye binding to aldehydes and ketones in polysaccharides, glycoproteins, and/or in irreversibly damaged proteins (penetrates membrane-compromised cells) N Cultivation, flow cytometry, microscopy Unknown Unknown Only tested on eukaryotic cells and a few bacteria (e.g., E. coli) so far Low false-positive rate; does not require the presence of nucleic acids for staining; the ability to stain dead cells increases with cell age (as opposed to some nucleic acid stains) Has not be applied at the community scale [182]
RNA analyses (e.g., metatranscriptomics) Quantifying or sequencing mRNA and/or rRNA Y MVT (for pre-rRNA), qPCR, PCR, RNA sequencing Any, given sufficient RNA yield and quality Y (rRNA), N (mRNA) Many (e.g., archaea, Gram + bacteria, Gram − bacteria, fungi, spores if RNA can be extracted, actively replicating viruses, and RNA viruses) Can reveal phylogeny and metabolic potential (mRNA) of likely viable and/or recently active microbes mRNA has short half-life; rRNA is present in dormant cells; the extraction of high-quality RNA can be challenging [103, 105, 116, 115]
Cellular energy measurements Measuring ATP concentration N Flow cytometry, epifluorescence microscopy, CCD camera Many (e.g., marine, built environment, food, bioaerosols, and clean room samples) Y Many (e.g., archaea, Gram + bacteria, Gram − bacteria, and fungi) ATP concentration has high correlation with number of metabolically active cells; rapid and affordable assay Can overestimate ATP concentrations because of extracellular ATP; metabolically dormant spores will not be detected; lack of specificity [22, 140]
Bioorthogonal noncanonical amino acid tagging (BONCAT) with click chemistry Measuring translational activity via synthetic amino acid incorporation into proteins Y Many (e.g., FISH, AFH, flow cytometry, FACS, MDA, 16S rRNA gene sequencing, presumably, other DNA amplification and sequencing techniques and protein-based techniques) Presumably many; thus far, deep-sea methane seep sediments Unknown Presumably many; thus far, some archaea and Gram − bacteria, including slow growing Can reveal actively translating microbes in consortia and, in combination with downstream approaches, their phylogeny; insights into micron-scale interactions Application to microbial ecology is relatively new; broad applicability is presumed but not yet proven [176, 175]
Isothermal microcalorimetry (IMC) Measuring heat flow Y Many (the method is nondestructive) Many, including lakes, marine sediments, and soils Y Many (any actively metabolizing organisms generating heat) Will measure any sufficient metabolic activity Can only be applied to slow processes because of assay ramp-up time; possible false positive signatures (e.g. degradation of media) [183]
Stable-isotope probing (SIP) Tracing isotopically labeled substrates through an active microbial community Y PCR, FACS Many N Many (e.g., archaea, Gram + bacteria, Gram − bacteria, fungi, spores if actively incorporating substrates, and replicating viruses) Can determine metabolic activity and phylogeny in the same sample; can help to identify community members involved in the metabolism of specific labeled compounds of interest Long incubation times may be necessary; labeled substrates can be expensive; relatively large amount of biomass needed; the label can move through trophic networks during the incubation, so careful interpretations are necessary [153]
Proteomics/metaproteomics Identifying proteins via mass spectrometry N N/A, unless initial sample is split for multiple purposes Any, given sufficient protein yield and quality N Many (e.g., archaea, Gram + bacteria, Gram − bacteria, fungi, replicating viruses; can also measure viral structural proteins, which do not necessarily indicate infectivity) Can identify actively expressed proteins and metabolic pathways Requires exact protein sequence to be present in database for identification; often lower throughput than nucleic acid sequencing approaches [161]
  1. “Many” means that most of the possibilities for this category have been shown to be, or are likely to be, compatible; where practical, we have added examples from the literature. For abbreviations, see the list of abbreviations at the end of the main text
  2. aWe did not find evidence for attempts of this application for this technique