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 | |
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 | |
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) | |
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 | |
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 | |
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 | |
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 | |
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] |