MINIREVIEW

Strategies for culture of ‘unculturable’ bacteria

Sonia R. Vartoukian, Richard M. Palmer & William G. Wade
King’s College London Dental Institute, Infection Research Group, London, UK

Correspondence: William G. Wade, Abstract

Department of Microbiology, King’s College
Molecular ecology methods are now well established for the culture-independent

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London, Floor 28, Tower Wing, Guy’s
Campus, London SE1 9RT, UK. Tel.: 144 20 characterization of complex bacterial communities associated with various envir-
7188 3872; fax: 144 20 7188 3871; onmental and animal habitats and are revealing the extent of their diversity. By
e-mail: william.wade@kcl.ac.uk comparison, it has become clear that only a small minority of microorganisms are
readily cultivated in vitro, with the majority of all bacteria remaining ‘unculturable’
Received 30 November 2009; revised 11 March using standard methods. Yet, it is only through the isolation of bacterial species in
2010; accepted 9 April 2010. pure culture that they may be fully characterized, both for their physiological and
Final version published online 17 May 2010.
pathological properties. Hence, the endeavour to devise novel cultivation methods
for microorganisms that appear to be inherently resistant to artificial culture is a
DOI:10.1111/j.1574-6968.2010.02000.x
most important one. This minireview discusses the possible reasons for ‘uncultur-
Editor: Ian Henderson
ability’ and evaluates advances in the cultivation of previously unculturable
bacteria from complex bacterial communities. Methods include the use of dilute
Keywords nutrient media particularly suited for the growth of bacteria adapted to oligo-
microbiome; uncultivable bacteria; 16S rRNA trophic conditions, and the provision of simulated natural environmental condi-
gene; bacterial culture. tions for bacterial culture. This has led to the recovery of ‘unculturables’ from soil
and aquatic environments, likely to be due to the inclusion of essential nutrients
and/or signalling molecules from the native environment.
MICROBIOLOGY LETTERS

the community and the entire ecosystem in the environment

Introduction or pathogens of plants and animals may be overlooked if
For the purpose of this minireview, the terms ‘unculturable’ they are unculturable. Consequently, with the development
and ‘as yet uncultivated’ are used to describe organisms that of molecular culture-independent techniques, there has
have yet to be grown on artificial media in vitro. been a move towards the characterization of mixed bacterial
It is well established that only approximately 1% of populations within biomass from the environment and in
bacteria on Earth can be readily cultivated in vitro – the so- samples from animals (including humans) using PCR
called ‘great plate count anomaly’, based on the observation amplification of housekeeping genes particularly that en-
that microscopic counts are considerably larger than the coding 16S rRNA gene, cloning for purification and sequen-
equivalent total viable counts (Staley & Konopka, 1985; cing for identification (Giovannoni et al., 1990; Pace, 1997).
Amann et al., 1995; Hugenholtz et al., 1998). There are As a result, numerous novel phylotypes have been identified
currently estimated to be 61 distinct bacterial phyla, of among bacterial communities from a wide range of habitats:
which 31 have no cultivable representatives (Hugenholtz from seawater and soil to the health- and disease-associated
et al., 2009). The topology of the archaeal phylogenetic tree microbiota of humans (Munson et al., 2002; Rappe &
remains uncertain, but it is clear that the 54 species of Giovannoni, 2003; Zhou et al., 2004; Aas et al., 2005).
Archaea cultured to date represent only a fraction of the total Despite the availability of varied molecular methods for
diversity, with 49 lineages mostly uncultured (Auguet et al., the evaluation of bacterial communities, cultural analyses
2010). are far from redundant. It is only through the isolation of
Because the majority of bacteria and archaea remain individual bacterial species in pure culture that a compre-
unculturable, the diversity of complex bacterial commu- hensive characterization of physiological properties and a
nities is inevitably underestimated using standard cultiva- full assessment of virulence potential may be undertaken.
tion methods. Furthermore, organisms of key importance to Hence, there has been a significant focus in recent years on

FEMS Microbiol Lett 309 (2010) 1–7

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
2 S.R. Vartoukian et al.

developing methods for the in vitro culture of those species Some bacterial growth factors have been considered
hitherto refractory to cultivation. analagous to mammalian cytokines – circulating regulatory
molecules that mediate cellular communication. The term
‘bacterial cytokine’ was coined by Mukamolova et al. (1998)
Reasons for ‘unculturability’ for the resuscitation-promoting factor (Rpf), a protein that
The finding that certain bacterial species have never been revived dormant Micrococcus luteus cells and increased the
identified by culture may be a simple matter of coincidence: growth rate of vegetative cells. Rpf also stimulated the
an organism that has a low prevalence or is particularly growth of other members of the Actinobacteria including
slow-growing may have been overlooked in cultural ana- Mycobacterium tuberculosis, and a family of related growth
lyses. Additionally, many genetically distinct phylotypes factors was identified (Kell & Young, 2000). A family of
are phenotypically indistinguishable and are lumped proteins with a similar function in the Firmicutes was
together if conventional biochemical methods for identifica- subsequently discovered (Ravagnani et al., 2005). Rpf was

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tion are used. later demonstrated to have a lysozyme-like structure and
Conversely, some bacteria are genuinely resistant to muralytic activity (Cohen-Gonsaud et al., 2005). How Rpf
culture in isolation on conventional media. Certain bacteria stimulates the growth of dormant cells remains to be
have fastidious growth requirements including the need for determined, but it is possible that remodelling of the
specific nutrients, pH conditions, incubation temperatures peptidoglycan in the cell walls of dormant cells is required
or levels of oxygen in the atmosphere. Kopke et al. (2005) before growth can resume. Interestingly, it has been demon-
investigated the effect of different substrates and culture strated recently that peptidoglycan fragments bind to PrkC,
conditions on the growth of bacteria from comparable a serine/threonine protein kinase, in Bacillus subtilis to
samples of coastal sediments, and found that the various stimulate spore germination (Shah et al., 2008). Muropep-
cultivation approaches resulted in the isolation of different tides generated by Rpf degradation of peptidoglycan may
groups of bacteria specific to each method, confirming the interact with PknB, a homologue of PrkC in M. tuberculosis,
impact of cultivation conditions on the yield of culture. and thereby initiate resuscitation and stimulate growth
Thus, if the specific requirements for the growth of a (Kana & Mizrahi, 2009).
bacterium are not met by the artificial medium and incuba- Signalling molecules present only within the natural
tion conditions, or if there is competition for nutrients habitat are thought to be essential for the growth of many
among mixtures of organisms cultured together, some bacteria (Lewis, 2007; Nichols et al., 2008).
bacteria may not grow. Growth may also be inhibited by In the absence of these beneficial interactions and signals,
bacteriocins released from other bacteria in a mixed culture some bacteria may struggle to grow in monoculture.
or by antibacterial substances present within the medium Furthermore, faced with an unfamiliar environment devoid
(Tamaki et al., 2005). In order to make the best estimate of of essential factors, bacteria may, as a survival strategy, enter
the true diversity of the community present, multiple into a temporary state of low metabolic activity accompa-
methods of cultivation should be used. nied by the inability to proliferate or to form colonies on
The formation of biofilms appears to be an inevitable result culture media (Barcina et al., 1990; Colwell, 2000; Lewis,
of bacterial colonization of surfaces and has been identified in 2007; Nichols et al., 2008), which may be mistaken for a
the earliest fossil records (Hall-Stoodley et al., 2004). Bacterial constitutional resistance to culture.
biofilms have many of the features of multicellular organisms
and individual species within biofilms cooperate to resist
Techniques used to culture the
external stresses (Stoodley et al., 2002). Such interactions
enable the biofilm to function as a complex unit (Stoodley
‘unculturables’
et al., 2002; Marsh, 2005; ten Cate, 2006). There may be cross- Significant efforts have been made in recent years to devise
feeding or metabolic cooperation between species for the culturing methods for as-yet-uncultivated species. Develop-
provision of nutrients (Belenguer et al., 2006), such as the ments in the last decade, particularly in the field of environ-
production of lactic acid (through fermentation of carbohy- mental microbiology, have led to the recovery of
drates) by Streptococcus mutans, which is utilized as a source of unculturables from diversely populated habitats including
carbon by Veillonella spp. (Mikx & Van der Hoeven, 1975). soil and aquatic (marine and freshwater) environments.
Another key feature of biofilm communities is bacterial The majority of culture media used to date have been
communication through networks of signals (Davey, 2008). nutrient-rich. It is now thought that these conditions may
These include quorum-sensing mechanisms that are involved favour the growth of faster-growing bacteria at the expense
in the regulation of the bacterial community structure, of slow-growing species, some of which thrive in nutrient-
properties and survival (De Kievit et al., 2001; Konaklieva & poor environments (Koch, 1997; Connon & Giovannoni,
Plotkin, 2006; ten Cate, 2006). 2002), and may be inhibited by substrate-rich conventional

c 2010 Federation of European Microbiological Societies FEMS Microbiol Lett 309 (2010) 1–7
Published by Blackwell Publishing Ltd. All rights reserved
Culturing the unculturable 3

media. Consequently, the use of dilute nutrient media has bacteria under laboratory conditions have sometimes been
led to the successful cultivation of previously unculturable met with success only when these bacteria are cocultivated
bacteria from various aquatic and terrestrial habitats (Watve with helper strains (Ohno et al., 1999, 2000; Nichols et al.,
et al., 2000; Connon & Giovannoni, 2002; Rappe et al., 2002; 2008).
Zengler et al., 2002). Factors released from helper bacteria into the environ-
Various methods have been used to physically reduce the ment are often growth-stimulatory for otherwise uncultur-
number and diversity of bacteria within mixed samples able bacteria even in the absence of the actual helper strain.
before cultivation. These include filtration methods (Hahn Thus, the conditioning of media with spent culture super-
et al., 2004), density-gradient centrifugation or elutriation natants or cell-free extracts derived from helper strains has
and extinction-dilution whereby samples are diluted, ideally been used for the growth stimulation of species such as
down to single cells, before their culture in isolation (Watve Catellibacterium spp., Psychrobacter spp., Sphingomonas spp.
et al., 2000; Connon & Giovannoni, 2002; Ben-Dov et al., and Symbiobacterium spp. (Tanaka et al., 2004; Bae et al.,

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2009; Song et al., 2009; Wang et al., 2009). 2005; Kim et al., 2008a, b; Nichols et al., 2008).
Many bacteria, particularly those that are oligotrophic in Signalling molecules may be responsible for such growth
the environment, are very slow-growing. Extended incuba- promotion. Empirical testing of known signal molecules,
tion times are a prerequisite for the cultivation of such cyclic AMP (cAMP) and acyl homoserine lactones was
bacteria, with the added benefit that faster-growing mem- shown to significantly increase the cultivation efficiency of
bers within the mixed populations progressively die off over marine bacteria (Bruns et al., 2002) – the addition to liquid
time, reducing the bacterial competition. The culture of soil media of 10 mM cAMP led to cultivation efficiencies of up to
bacteria for up to 12 weeks has revealed increasing colony 100%. This remarkable result has not, however, been corro-
counts and an increased recovery of rarely isolated strains borated by other studies investigating the effect of cAMP on
with time (Davis et al., 2005). Similarly, long-term incuba- the growth of individual species. Coppola et al. (1976)
tion for up to 24 weeks has been successful for the isolation observed a growth inhibition of Escherichia coli in media
of strains from the SAR11 clade (Song et al., 2009). Even supplemented with 5 mM cAMP, and in a study by Chen &
members of the TM7 Division, which have yet to be Brown (1985), the addition of cAMP at levels ranging from
cultivated in isolation, were able to form colonies visible to 0.01 to 100 mM showed no consistent influence on the
the naked eye when incubation times of 50 days were growth rates of Legionella pneumophila. A cAMP concentra-
used [unpublished observation reported in a review by tion-dependent effect on growth may explain the differences
Hugenholtz (2002)]. in the results of the various studies. It is also possible that
Many bacteria have specific nutrient or chemical require- use of the most-probable-number method in the study by
ments for growth (Graber & Breznak, 2005; Tripp et al., Bruns et al. (2002) led to an overestimation of cell numbers.
2008). For example, members of the genera Abiotrophia and Another study (Nichols et al., 2008), in this case investigat-
Granulicatella, previously known as the nutritionally variant ing the growth stimulation of a Psychrobacter strain, success-
streptococci, require pyridoxal or L-cysteine for growth fully characterized the growth-promoting factor responsible
(Ruoff, 1991), while Tannerella forsythia requires an exogen- and identified this as a 5-amino-acid peptide.
ous source of N-acetyl muramic acid (Wyss, 1989). The An alternative approach for the culture of as-yet-unculti-
characterization of phylogenetically related species may vated organisms is to simulate their natural environment in
provide clues to the metabolic requirements of organisms vitro. Kaeberlein et al. (2002) constructed a diffusion
that are so far resistant to culture. Cultivation media may be chamber that allowed the passage of substances from the
modified or enriched with this in mind, resulting in the natural environment (intertidal marine sediment) across a
isolation of previously ‘unculturable’ organisms (Sait et al., membrane and successfully grew bacteria from marine
2002; Davis et al., 2005). However, simply adding the sediment that were previously uncultivated. These bacteria
required substrate to cultivation media may not, in all cases, were subsequently cultured on solid media, but grew only in
enable culture of the target organism. For example, slow- the presence of other bacteria, implying codependency.
growing acetotrophs of the genus Methanosaeta are often Similar diffusion chambers have been constructed since, to
outcompeted by faster-growing Methanosarcina spp. in culture ‘uncultivable’ or rarely cultivated bacteria from
mixed culture. On the other hand, Janssen (2003) found marine (Nichols et al., 2008) and freshwater environments
that the incorporation of acetone and isopropanol as (Bollmann et al., 2007). The latter study reported a signifi-
enrichments led to the production (by species that ferment cantly greater diversity of recovered isolates using the
these substrates) of a slow and steady source of acetate that diffusion chamber than on conventional agar plates.
allowed Methanosaeta spp. to flourish. Also mimicking the natural environment, sterile fresh-
Because of a reliance on beneficial bacterial interactions (Stingl et al., 2008; Wang et al., 2009) and marine- (Rappe
within the source environment, attempts to cultivate certain et al., 2002; Song et al., 2009) waters have been used to

FEMS Microbiol Lett 309 (2010) 1–7

c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
4 S.R. Vartoukian et al.

culture previously uncultivated bacteria. Ben-Dov et al. permeabilization and fixation procedures, and may not
(2009) encapsulated individual bacteria (following dilution therefore be subsequently cultured in isolation.
of samples) in agar spheres encased in a polysulphonic The colony hybridization method, on the other hand, is
polymeric membrane before incubation in a simulated or a undertaken on membrane transfers from plate cultures that
natural environment, and was successful in growing several remain viable (Salama et al., 1993). Consequently, hybridi-
novel organisms. zation detections on membranes may be used to locate
Another innovative technique mimicking natural condi- matched microcolonies within mixed cultures, from where
tions, this time used for the microcolony cultivation of they may be isolated. This method has been used in recent
uncultivated soil bacteria, is the soil substrate membrane work (Vartoukian et al., 2010), leading to the successful
system (Ferrari et al., 2005, 2008), which includes a poly- isolation from dental plaque of a previously uncultivated
carbonate membrane support and soil extract as a substrate. member of the Synergistetes phylum. Enrichment of the
Although this system allowed the microcultivation of novel subcultured microcolonies with candidate feeder organisms

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bacterial strains, the bacteria remained part of a mixed from the original mixed cultures was found to facilitate the
community on the membrane. A recent development of the growth of the microcolony-forming bacteria.
method has enabled the detection of live microcolonies on Flow cytometry and cell sorting (FACS) is a method with
the membrane using viability staining, and the subsequent numerous applications in microbiology (Alvarez-Barrientos
micromanipulation of such colonies for their isolation et al., 2000). In an effort to cultivate as-yet-uncultivated
(Ferrari & Gillings, 2009). taxa, Zengler et al. (2002) used gel microdroplets to en-
The study of bacteria with an obligate intracellular life- capsulate single bacterial cells (from dilutions of mixed
style presents a particular challenge and it can be difficult to environmental samples), which then formed microcolonies
determine and reproduce the environmental conditions in situ. Based on characteristic light-scattering properties,
required for metabolic activity. For example, initial work any microdroplets that contained microcolonies (as op-
investigating the metabolism of Coxiella burnetii used posed to single or no cells) were detected and sorted by
neutral pH buffers and concluded that there was negligible FACS, and subsequently analysed phylogenetically. When
activity (Ormsbee & Peacock, 1964). When acidic buffers the intention is to detect and sort specific bacterial species,
were used, metabolism was markedly enhanced (Hackstadt however, target-specific fluorochrome-labelled antibody or
& Williams, 1981). Further refinements of this approach oligonucleotide probes are usually required. Whereas anti-
including the use of a citrate buffer, provision of complex body-conjugated probes may preserve cellular viability,
nutrients and high (140 mM) chloride have enabled meta- oligonucleotide probes do not, preventing the subsequent
bolic activity to be maintained for over 24 h (Omsland et al., cultivation of sorted cells. Although FACS of ‘unculturable’
2008), enabling the investigation of the physiology of this bacterial cells may not therefore directly lead to their
important species. cultivation, FACS in conjunction with whole-genome am-
Many of the methods described above use an open-ended plification has been used to obtain a partial genome
approach with the aim of cultivating all bacteria present in a sequence for a member of the TM7 phylum (Podar et al.,
sample. As a result, they have led to the cultivation of 2007). Knowledge of the genomes of as-yet-uncultivated
numerous fastidious bacteria. However, the phylogenetic organisms will help characterize these species and provide
targeting of specific bacterial strains of interest requires clues that will aid their in vitro cultivation in the future. For
alternative approaches. example, genomic analysis of ‘Candidatus Pelagibacter ubi-
que’ has revealed a deficiency of the genes that are necessary
for assimilatory sulphate reduction in the production of
Isolation of targeted ‘unculturables’ sulphur, which is essential for biosynthesis in aerobic marine
Advances in molecular biology have enabled the detection bacteria (Tripp et al., 2008).
and sorting of specific target bacteria with a view to their The micromanipulation of single bacterial cells for their
selective enrichment or physical isolation. Oligonucleotide isolation in pure culture has potential applications for the
probes can be designed to target phylotypes with no known isolation of ‘unculturable’ bacteria (Frohlich & Konig,
cultivable representatives. Using methods such as FISH or 2000). Optical tweezers, in the form of an infrared laser, are
catalysed reporter deposition (CARD)-FISH for added used to trap and isolate single cells within a cell separation
sensitivity, target-specific probes can detect cells of pre- unit from where they are ultimately transferred to growth
viously ‘unculturable’ taxa among mixed populations media for cultivation. This method was used successfully by
(Amann et al., 1995, 2001; Ferrari et al., 2006; Vartoukian Huber et al. (1995) to isolate a previously uncultivated
et al., 2009), enabling the visualization of their cellular archaeal strain following visual recognition of its cellular
morphology. A limitation of these methods is that the cells morphology from targeted whole-cell hybridization. Raman
detected within a sample are no longer viable after cell tweezers, as used by Huang et al. (2009), involve a similar

c 2010 Federation of European Microbiological Societies FEMS Microbiol Lett 309 (2010) 1–7
Published by Blackwell Publishing Ltd. All rights reserved
Culturing the unculturable 5

technique of optical trapping differing only in the method of Bollmann A, Lewis K & Epstein SS (2007) Incubation of
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