Environmental Microbiology Reports (2021) 13(3), 248–252
doi:10.1111/1758-2229.12953
Correspondence
What do we mean by viability in terms of ‘viable but
non-culturable’ cells?
Da-Shuai Mu, 1* Zong-Jun Du,1* Jixiang Chen,2,3
Brian Austin4 and Xiao-Hua Zhang 2*
1
State Key Laboratory of Microbial Technology, Institute
of Microbial Technology, Shandong University, Qingdao,
266237, China.
2
College of Marine Life Sciences, and Frontiers Science
Center for Deep Ocean Multispheres and Earth System,
Ocean University of China, Qingdao, 266003, China.
3
School of Petrochemical Engineering, Lanzhou
University of Technology, Lanzhou, 730050, China.
4
Institute of Aquaculture, University of Stirling, Stirling
FK9 4LA, Scotland, UK.
In a recent Opinion article (‘Viable But Non-Culturable
Cells’ are Dead, https://doi.org/10.1111/1462-2920.15463),
Song and Wood (2021) argued the cells that can rejuvenate and reconstitute infections are not part of a dormancy
continuum. Instead, only persister cells are dormant and
capable of re-growth, whereas viable but non-culturable
(VBNC) cells are dead. We argue against this conclusion
in light of multiple previous publications. Thus, we discuss
the relations of death/non-viability, dormancy and the
culturability of Viable But Non-Culturable (VBNC) cells. In
essence, we address the issue about what is meant by
‘viability’?
suitability of the media and incubation conditions, and the
definition of viability which in this case reflected the
development of visible growth, i.e. colony formation
(Austin, 2017). Since its discovery, the term ‘VBNC’ was
a nidus of controversy that largely stemmed from the perspective of viability. Song and Wood assumed that if cells
lost their ability to grow or form visible colonies on media,
they were not viable, i.e. dead (Baquero and Levin, 2021;
Song and Wood, 2021). This begs the question about
cells that produce only limited growth, i.e. microcolony
formation. With the help of molecular techniques, it is
now understood that loss of culturability to produce visible growth does not equal death. Why should individual
bacteria multiply to produce millions of daughter cells that
form turbidity in liquid media or colonies on solid media?
Colony formation, such as occurs on agar media, is
highly artificial and does not represent the normal state of
bacteria. The loss of culturability of a living bacterium is
not uncommon; this is a challenge for culturing the
uncultured. Many microbial species in the biosphere that
would otherwise be ‘culturable’ may fail to grow because
of their growth state in nature, such as dormancy
(Mu et al., 2018) or specific requirements, such as for
nutrients, osmotic support or temperature.
Low density of cytosol of VBNC cells does not
mean death
Loss of culturability does not equal bacterial death
The VBNC state was first discovered and reported by Xu
et al. (1982) when these workers demonstrated that
some bacteria under conditions of stress maintained cell
functional viability but were not culturable on the media
upon which they were usually capable of growth. Specifically, the work emphasized the presence of intact cells of
Escherichia coli and Vibrio cholerae in marine and estuarine samples that failed to grow on conventional microbiological growth media. This begs the question about the
Received 9 April, 2021; accepted 11 April, 2021. For correspondence. *E-mail dashuai.mu@sdu.edu.cn; Tel. +86-0631-5688303.
**E-mail duzongjun@sdu.edu.cn; Tel. +86-0631-5688303. ***E-mail
xhzhang@ouc.edu.cn; Tel. +86-0532-82032767.
© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd
Song and Wood (2021) suggested that transmission electron microscopy (TEM) based methods should be used
along with colony counts to determine if cells are viable or
non-viable/dead (Kim et al., 2018). We believe that the
TEM method is a good approach for checking the morphology/state of cells, but we do not believe that VBNC
cells are necessarily dead/non-viable based on the current
data. In the previous study by Kim et al. (2018), it was
suggested that the cells were dead if the TEM analysis of
cells revealed a lack of normal cytosolic materials. Also,
they found that the old persisters had or tended to have
‘empty’ cytosol, which resembled cells of VBNC cultures.
We support this observation because it is believed that the
changes in the cell state represent a continuum between
The viability of VBNC cells
actively growing and dead cells (Ayrapetyan et al., 2018)
with VBNC cells being in a deeper state of dormancy than
persister cells (Fig. 1). However, based on the current
data, we do not believe that TEM analysis alone could
confirm that ’empty’ cells with intact membranes are dead.
The TEM images were generated using fixed, stained
and sectioned cells. This technique is known to affect or
obscure the delicate ultrastructure of cells. To avoid this,
electron cryo-tomography combined proteomic analysis
was used and the data showed the physiology of VBNC
cells and the drastically altered presence of their metabolic and structural proteins (Brenzinger et al., 2019). A
characteristic morphological feature of VBNC cells is
a smaller size and a round, coccoid shape with an
increased gap between the cytoplasmic and outer membrane (OM).
It is important to note that the observed low density of
cytosol in the cell by TEM analysis is not equal to the real
empty cytosol as the electron micrograph only showed
the 70 nm thickness parts of a cell (Kim et al., 2018). This
accounted for 10% of the total bacterial cytosol. The
low density of cytosol may be enough to maintain
the basic (low) metabolic activities of the cell and to
249
maintain selective permeation of the membrane (intact
membranes). Meanwhile, the membranes’ intact stability
should not be determined only by TEM. This is because
the observed data are not representative of the whole-cell
membranes and cannot determine whether the membrane is selectively permeable. However, TEM analysis
may be used as a method to determine the state of the
cell during the formation of persisters or VBNC cells.
There is another issue that not all the cells in a population may be capable of replication. This needs to be carefully considered in any discussion of viability versus nonviability.
Methods for determining VBNC cell viability
Song and Wood (2021) suggested the membrane or
DNA staining technique incorrectly viewed cell shells as
viable and were not suitable for determining cell viability.
However, many methods based on membrane or DNA
staining were effective to test the viability of VBNC cells.
The direct viable counting method was published by
Kogure et al. (1979). In this method, samples were incubated in yeast extract (final concentration, 0.025%) and
Fig. 1. Schematic diagram of the transition between culturable and unculturable cells based on the dormancy continuum hypothesis. Environmental stress induces cellular processes that lead to the degradation of intracellular cytoplasm. This affects cellular metabolism and culturability.
Persister cells are produced in the early stages of dormancy. When these persister cells are transferred to the media, their metabolic competence
allows them to go into the lag phase and exponential phase on media. However, if the stressful conditions continue or become more intense,
these persister cells may continue to degrade the intracellular cytoplasm (low electron density in TEM), and go deeper into dormancy then lose
their culturability (i.e. become VBNC). When VBNC cells are transferred to media, their metabolic competence does not allow them to form colonies. This is because they need adequate time and conditions to resuscitate to adapt to the culture conditions. Resuscitated cells will regain their
ability to grow on media. Attempts at culturing these cells any time prior to the completion of the resuscitation process will result in lack of visible
growth.
© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 13, 248–252
250 D.-S. Mu et al.
nalidixic acid (final concentration, 0.002%) at 26 C for
16 h before acridine orange staining. Cells, which were
elongated to at least twice the length of control cells (acridine orange direct counting), were scored as viable. In
this method, nalidixic acid is a specific inhibitor of DNA
synthesis and prevents cell division of Gram-negative
bacteria resulting in the formation of elongated filamentous cells. This elongation makes it easier to count cell
numbers by microscopy. Other methods based on the
permeability of live cell membrane, such as the LIVE/
DEAD staining assay (Orman and Brynildsen, 2013),
fluorescent indicator (mCherry) of cell lysis assay (Orman
and Brynildsen, 2013) and flow cytometry analysis could
also distinguish between VBNC cells and dead cells.
Moreover, a study using single-cell imaging and microfluidics differentiated VBNC and persister cells under
antibiotic exposure and identified differentially expressed
promoters to distinguish VBNC and persister cells
(Bamford et al., 2017). Also, these workers provided
strong evidence that VBNC cells were not dead and
shared molecular characteristics with persisters.
Besides the success of staining-based methods,
other approaches such as the metabolic assay
(Orman and Brynildsen, 2013), transcriptional analysis
(Asakura et al., 2007) and quantitative proteomics
(Mali et al., 2017) demonstrated the viability of VBNC
cells in different levels.
Remove stress
Log CFU/ml
Total viable cells
VBNC cells
Persister cells
Incubation time
Persister cells
resuscitation
VBNC cells
resuscitation
Fig. 2. Resuscitation dynamics of persistence and the VBNC state.
Both persister cells and VBNC cells are unable to generate visible
growth, i.e. colonies on media under a certain stress. When the
stress is removed and adequate conditions are met (vertical dotted
grey arrow), cells begin to alter their physiology toward resuscitation
(dotted orange line). Persister cells typically have a short lag phase
and produce the normal growth curve to form colonies on media
(solid orange line). A large portion of the population of VBNC cells
(dotted green line) needs a long time of resuscitation (dependent on
the stress and bacterial species) to proceed into the growth curve
(solid green line). If the large portion of VBNC cells regain the ability
to grow on nutrient media, they typically form a sharp exponential
phase to form colonies.
Resuscitation of VBNC cells
Song and Wood (2021) suggested that the resuscitation
of VBNC cells is possible from persister cells, but the yet
to be resuscitated VBNC cells are dead. We argue that
resuscitation from dormancy is the molecular process by
which cells repair oxidative damage, regain metabolic
competence and normalize their toxin–antitoxin ratios.
After resuscitation has occurred, cells are once again
able to produce visible growth, i.e. colonies. When
VBNC/dormant bacteria were re-inoculated onto media,
the growth conditions were a new type of stress for the
VBNC cells, i.e. they needed to resuscitate before adaption and proceeding into the normal growth curve (Fig. 1).
In fact, the VBNC cells resuscitated poorly because of
the oxidative stress imposed by nutrient media (Kong
et al., 2004). If VBNC cells are unable to go into the lag
phase in an appropriate time period (Fig. 1), they may
well maintain dormancy or even lose viability on the
media (Mu et al., 2021; Zhang et al., 2021). This is a
major difference between VBNC and persister cells,
which pertains to resuscitation dynamics. Although persisters are typically able to form colonies on nutrient
media following a short lag phase after removing stress
(Aldridge et al., 2012), VBNC cells are unable to do so
even after removal of the inducing stress (Ayrapetyan
et al., 2015). VBNC cells require a much longer resuscitation period away from nutrient media (Li et al., 2014).
Once the cells are resuscitated, they divide at the same
rate, independent of whether they may be regarded as
VBNC or persisters (Bruhn-Olszewska et al., 2018).
Thus, the lag in regrowth is not due to slower-growing
cells but rather the time necessary for VBNC cells to
resuscitate (Fig. 2). Normally, stress treated wild-type cultures contain significantly more VBNCs than persisters
(Du et al., 2007b; Orman and Brynildsen, 2013), which
also reflect on the resuscitation curve (Du et al., 2007a).
When small numbers of persister cells are resuscitated
after removal of stress, they proceed into a normal
growth curve. Moreover, when a mass of VBNC cells are
resuscitated after a long period of resuscitation, they form
a sharp growth curve in a short time, which is different
from the exponential phase (Fig. 2).
It should be recognized that the VBNC state was not a
laboratory phenomenon, as such cells may be readily
detected in the natural environment and in infections
(Oliver, 2010). For example, increased cases of vibriosis
occurred in areas of the world where the disease was typically nonexistent, because of the ability of Vibrio to resuscitate from the VBNC state based on temperature changes
(Oliver et al., 1995). Therefore, for detecting pathogenic
bacteria, traditional culture-dependent methods are not
fully reliable for finding VBNC cells or those which do not
develop visible growth, such as exemplified by micro-
© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 13, 248–252
The viability of VBNC cells
colony formation. Thus, molecular detection methods
should be used to seek for pathogenic bacteria (Guo
et al., 2021).
Perspectives
By arguing the meaning of viability in terms of VBNC
cells, we do not wish to draw a line between VBNC and
persisters but hope to shed light on the importance of this
relationship. It is vital that we consider all of the viable
cells that are in a culture. There is an interesting angle
that if a population of cells enters a VBNC state, it does
not necessarily mean that all the cells remain viable and
could be resuscitated - this could be restricted to a
smaller number of cells. This small subpopulation of cells
could permit the survival of the culture. Clearly, we need
to establish methods to eradicate any dormant cells from
animals subjected to anti-infective therapy in order to
reduce recurrence and the possibility for the emergence
of antibiotic-resistant bacteria. Thus, we need to target
the cells encompassing the entire continuum between
those that are actively growing and inactive/dead in order
to attempt to determine the critical point between deep
dormancy and death.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (41876166).
References
Aldridge, B.B., Fernandez-Suarez, M., Heller, D.,
Ambravaneswaran, V., Irimia, D., Toner, M., and Fortune, S.M.
(2012) Asymmetry and aging of mycobacterial cells lead
to variable growth and antibiotic susceptibility. Science
335: 100–104.
Asakura, H., Ishiwa, A., Arakawa, E., Makino, S., Okada, Y.,
Yamamoto, S., and Igimi, S. (2007) Gene expression profile of Vibrio cholerae in the cold stress-induced viable but
non-culturable state. Environ Microbiol 9: 869–879.
Austin, B. (2017) The value of cultures to modern microbiology. Anton Leeuw Int 110: 1247–1256.
Ayrapetyan, M., Williams, T., and Oliver, J.D. (2018) Relationship between the viable but nonculturable state and
antibiotic persister cells. J Bacteriol 200: e00249–e00218.
Ayrapetyan, M., Williams, T.C., Baxter, R., and Oliver, J.D.
(2015) Viable but nonculturable and persister cells coexist
stochastically and are induced by human serum. Infect
Immun 83: 4194–4203.
Bamford, R.A., Smith, A., Metz, J., Glover, G., Titball, R.W.,
and Pagliara, S. (2017) Investigating the physiology of viable but non-culturable bacteria by microfluidics and timelapse microscopy. BMC Biol 15: 121.
Baquero, F., and Levin, B.R. (2021) Proximate and ultimate
causes of the bactericidal action of antibiotics. Nat Rev
Microbiol 19: 123–132.
251
Brenzinger, S., van der Aart, L.T., van Wezel, G.P.,
Lacroix, J.M., Glatter, T., and Briegel, A. (2019) Structural
and proteomic changes in viable but non-culturable Vibrio
cholerae. Front Microbiol 10: 793.
Bruhn-Olszewska, B., Szczepaniak, P., Matuszewska, E.,
Kuczynska-Wisnik, D., Stojowska-Swedrzynska, K.,
Moruno Algara, M., and Laskowska, E. (2018) Physiologically distinct subpopulations formed in Escherichia coli
cultures in response to heat shock. Microbiol Res 209:
33–42.
Du, M., Chen, J., Zhang, X., Li, A., and Li, Y. (2007a) Characterization and resuscitation of viable but nonculturable
Vibrio alginolyticus VIB283. Arch Microbiol 188: 283–288.
Du, M., Chen, J., Zhang, X., Li, A., Li, Y., and Wang, Y.
(2007b) Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate. Appl Environ
Microbiol 73: 1349–1354.
Guo, L., Wan, K., Zhu, J., Ye, C., Chabi, K., and Yu, X.
(2021) Detection and distribution of vbnc/viable pathogenic bacteria in full-scale drinking water treatment plants.
J Hazard Mater 406: 124335.
Kim, J.S., Chowdhury, N., Yamasaki, R., and Wood, T.K.
(2018) Viable but non-culturable and persistence describe
the same bacterial stress state. Environ Microbiol 20:
2038–2048.
Kogure, K., Simidu, U., and Taga, N. (1979) A tentative
direct microscopic method for counting living marine bacteria. Can J Microbiol 25: 415–420.
Kong, I.S., Bates, T.C., Hulsmann, A., Hassan, H., Smith, B.
E., and Oliver, J.D. (2004) Role of catalase and oxyR in
the viable but nonculturable state of Vibrio vulnificus.
FEMS Microbiol Ecol 50: 133–142.
Li, L., Mendis, N., Trigui, H., Oliver, J.D., and Faucher, S.P.
(2014) The importance of the viable but non-culturable
state in human bacterial pathogens. Front Microbiol
5: 258.
Mali, S., Mitchell, M., Havis, S., Bodunrin, A., Rangel, J.,
Olson, G., et al. (2017) A proteomic signature of dormancy
in the Actinobacterium Micrococcus luteus. J Bacteriol
199: e00206-17.
Mu, D.S., Liang, Q.Y., Wang, X.M., Lu, D.C., Shi, M.J.,
Chen, G.J., and Du, Z.J. (2018) Metatranscriptomic and
comparative genomic insights into resuscitation mechanisms during enrichment culturing. Microbiome 6: 230.
Mu, D.S., Ouyang, Y., Chen, G.J., and Du, Z.J. (2021) Strategies for culturing active/dormant marine microbes. Mar
Life Sci Technol 3: 120–130.
Oliver, J.D. (2010) Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol
Rev 34: 415–425.
Oliver, J.D., Hite, F., Mcdougald, D., Andon, N.L., and
Simpson, L.M. (1995) Entry into, and resuscitation from,
the viable but nonculturable state by Vibrio-vulnificus in an
estuarine environment. Appl Environ Microbiol 61: 2624–
2630.
Orman, M.A., and Brynildsen, M.P. (2013) Establishment of
a method to rapidly assay bacterial persister metabolism.
Antimicrob Agents Chemother 57: 4398–4409.
Song, S., and Wood, T.K. (2021) ‘Viable but non-culturable
cells’ are dead. Environ Microbiol . https://doi.org/10.
1111/1462-2920.15463.
© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 13, 248–252
252 D.-S. Mu et al.
Xu, H.S., Roberts, N., Singleton, F.L., Attwell, R.W.,
Grimes, D.J., and Colwell, R.R. (1982) Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in
the estuarine and marine-environment. Microb Ecol 8:
313–323.
Zhang, X.-H., Ahmad, W., Zhu, X.-Y., Chen, J., and
Austin, B. (2021) Viable but nonculturable bacteria and
their resuscitation: implications for cultivating uncultured
marine microorganisms. Mar Life Sci Technol 3:
188–202.
© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 13, 248–252