it is a common misconception that microorganisms isolated in pure culture from an environment represent the numerically dominant and/or functionally significant species in that environment . in fact , microorganisms isolated using standard cultivation methods are rarely numerically dominant in the communities from which they were obtained : instead , they are isolated by virtue of their ability to grow rapidly into colonies on high - nutrient artificial growth media , typically under aerobic conditions , at moderate temperatures . easily isolated organisms are the ' weeds ' of the microbial world and are estimated to constitute less than 1% of all microbial species ( this figure was estimated by comparing plate counts with direct microscopic counts of microorganisms in environmental samples ; it has been called the " great plate - count anomaly " ) . given that the study of a microorganism is simpler if you have it in pure culture on an agar plate , it is not surprising that most of what we know about microbiology comes from the study of microbial weeds . for example , approximately 65% of published microbiological research from 1991 to 1997 was dedicated to only eight bacterial genera , escherichia ( 18% ) , helicobacter ( 8% ) , pseudomonas ( 7% ) , bacillus ( 7% ) , streptococcus ( 6% ) , mycobacterium ( 6% ) , staphylococcus ( 6% ) and salmonella ( 5% ) , all of which are relatively simple to grow on agar plates . intuitively , it seems unlikely that this handful of organisms can be representative of the approximately 5,000 validly described prokaryotic species , but exactly how unrepresentative are they ? and if more than 99% of microorganisms in the environment are unculturable using standard techniques , how representative are cultivated microorganisms of prokaryotic diversity as a whole ? to answer these questions , we need a framework for placing prokaryotic species and genera in a broader evolutionary context . the pioneering work of carl woese and colleagues   on comparative analysis of small - subunit ribosomal rnas ( 16s and 18s rrnas ) provided an objective framework for determining evolutionary relationships between organisms and thereby ' quantifying ' diversity as sequence divergence on a phylogenetic tree . woese found that cellular life can be divided into three primary lineages ( domains ) , one eukaryotic ( eucarya , also called eukaryota ) and two prokaryotic ( bacteria and archaea ) , and he also defined 11 major lineages ( phyla or divisions ) within the bacterial domain on the basis of 16s rrna sequences obtained from cultivated organisms . this analysis revealed distant relationships not suspected from phenotypic characterization , such as the association between the genera bacteroides and flavobacterium . the leading reference source in prokaryotic taxonomy , bergey 's manual of systematic bacteriology , has adopted a 16s rrna framework to classify prokaryotes , replacing the previous ad hoc scheme that was based on traditional phenotypic characterization . the manual proposes a standardized prokaryote nomenclature that has mostly been fitted to a classical taxonomic hierarchy ( species , genus , family , order , class , phylum ) ; i will adhere to this system as far as possible in this article ( see the taxonomic outline available at ) . the phylum is the highest - level grouping in the bacterial and archaeal domains   and , therefore , is a useful rank for overviewing prokaryotic diversity . the eight most intensively studied prokaryotic genera listed in the introduction are members of only three bacterial phyla : proteobacteria ( escherichia , helicobacter , pseudomonas , salmonella ) , firmicutes ( bacillus , streptococcus , staphylococcus ) and actinobacteria ( mycobacterium ) . moreover , the top 25 most - studied genera are all members of these three phyla , with the exceptions of chlamydia and borrelia ( clinically important genera of the bacterial phyla chlamydiae and spirochaetes , respectively ) . in a recent study , 177 environmental , veterinary and clinical isolates that were not identifiable by traditional phenotypic characterization were evaluated by comparative 16s rrna analysis . the isolates included a large number of different genera and species , but at the phylum level all except one of the 177 were members of only four bacterial phyla : proteobacteria ( 82 isolates ) , firmicutes ( 61 ) , actinobacteria ( 29 ) and bacteroidetes ( 4 ) . this cultivation bias towards four bacterial phyla ( the ' big four ' ) is also reflected in microbial culture collections ; for example , 97% of prokaryotes deposited in the australian collection of microorganisms   are members of the big four ( figure 1a ) . in fact , it is a challenge to obtain isolates that do not belong to the big four , and these four phyla therefore dominate our present understanding of microbiology . a logical question to ask is how many prokaryotic phyla there are altogether , in order to estimate how biased a sampling of four may be . in the mid 1980s , norman pace and colleagues outlined a molecular approach that bypassed the need to cultivate a microorganism in order to determine the sequence of its 16s rrna gene ( 16s rdna ) . essentially , bulk nucleic acids are extracted directly from environmental samples , 16s rdna sequences are isolated from the bulk dna , typically via pcr ( using primers broadly targeting 16s rdnas ) and cloning , and these sequences are compared with known sequences ( figure 2 ) . gene sequences obtained in this manner ( ' environmental clone sequences ' ) can then be assigned a location in a phylogenetic tree and can thus act as a marker for the organism from which they were obtained . the approach can be brought full circle by applying 16s rrna - targeted nucleic - acid probes specific for the organisms of interest to visualize and quantify the target group in the environmental sample using techniques such as whole - cell fluorescence in situ hybridization ( fish ) and membrane hybridization   ( figure 2 ) . many researchers have applied the rrna approach to a wide variety of environmental samples over the past decade and , perhaps not surprisingly given the great plate - count anomaly , the number of recognized bacterial phyla has exploded from the original estimate of 11 in 1987   to 36 in 1998 . this increase is due not only to environmental sequences that have filled out the tree , but also to a steady trickle of sequences from ' exotic ' cultured organisms , particularly thermophiles , that highlight new lineages . figure 3a presents a recent conservative estimate of bacterial diversity at the phylum level ; it is conservative because it includes only phyla for which at least four near - full - length 16s rdna sequences ( over 1,300 nucleotides ) are known . the total number of phylum - level lineages in this tree is 35 , 22 ( 63% ) of which have one or more cultivated representatives and 13 ( 37% ) of which are known only from environmental sequences . there are at least another ten phylum - level lineages , however , that are present in the bacterial domain but are not shown in figure 3a because they are represented by too few and/or only partial sequences . these lineages include cultivated bacteria such as chrysiogenes and dictyoglomus , which are recognized as representing independent phyla in the taxonomic outline of bergey 's manual of systematic bacteriology . as more 16s rdna sequences accumulate from both cultured and uncultured prokaryotes , the boundaries of existing phyla are being challenged and need to be re - evaluated . for example , the bacterial phylum firmicutes , as currently defined , may not be monophyletic and may comprise at least four distinct phylum - level lineages that include the haloanaerobiales , thermoanerobacteriales , and sulfobacillus groups . higher - level associations between bacterial phyla have not been resolved in 16s rdna trees , with the exceptions of the sister - group affiliations of the bacteroidetes and chlorobi , and of the chlamydiae and verrucomicrobia . this is presumably because such relationships are beyond the resolution that can be obtained from the 16s rrna molecule and/or the current inference methods . recently , trees based on concatenated ribosomal proteins obtained from complete genome sequences have suggested higher - order associations between chlamydiae and spirochaetes , between thermotogae and aquificae , and between actinobacteria , deinococcus - thermus and cyanobacteria . the phylum verrucomicrobia is also likely to be a member of the same group as chlamydiae and spirochaetes , given that it is a sister group to chlamydiae ; this prediction can be tested when a completed genome sequence becomes available for the verrucomicrobia . several ' candidate ' phyla , comprising only environmental clone sequences , have developed into large groups with sequence divergences similar to or greater than those within the big four phyla ( examples include op11   and ws6 ) , and yet we know nothing about these lineages beyond a crude outline of their environmental distribution . most have not even been ( knowingly ) observed under the microscope . in a preliminary investigation of one candidate phylum , tm7 , we determined that representatives of the group had typical gram - positive cell envelopes and that they may have archaea - like streptomycin resistance . detailed study of lineages like this one may yield insights into the evolutionary history of gram - positive bacteria ( including , perhaps , a radical proposal that gram - positive bacteria are related to archaea ) , which so far appear to have a restricted phylum - level distribution within the bacterial domain ( actinobacteria and firmicutes ) . tm7 bacteria have also been implicated in human subgingival ( gum ) disease , which might promote their study . the archaea are formally divided into two phyla , crenarchaeota and euryarchaeota , from 16s rrna phylogeny , but these groupings may be artifacts because analysis of concatenated ribosomal protein sequences suggests that euryarchaeota , at least , is not a monophyletic group . figure 3b presents a current estimate of the major lineages in the archaeal 16s rdna tree below the level of the crenarchaeota and euryarchaeota ( indicated to the right of the tree ) , using the same criteria and annotation used for the bacterial tree ( figure 3a ) . the total number of phylum - level lineages in the archaeal tree is 18 , of which 8 ( 44% ) have cultivated representatives and 10 ( 56% ) have none . a higher tally of 23 phyla is arrived at if lineages not meeting the selection criteria are included in the estimate . these include methanopyri , currently represented by a single sequence , and environmental group c3 , which has no full - length representatives . most archaeal research has concentrated on the cultivated methanogenic ( such as methanococci ) and thermophilic ( such as thermoprotei and thermococci ) lineages ( figure 3b ) . as is the case with the bacteria , most candidate archeal phyla are completely uncharacterized at this point . a notable exception is candidate phylum c1 ( figure 3b ) , which contains cenarchaeum symbiosum , an uncultured archaeon that has been amenable to detailed study , including partial genome sequencing , because it exists as a near monoculture in a marine sponge . the advent of large - scale dna sequencing has provided unprecedented access to molecular data for inferring the tree of life . currently , complete genome sequences of prokaryotes have been obtained only from pure cultures and hence , at the phylum level , microbial genomics reflects the bias towards the big four phyla ( figures 1b,3 ) . this bias ( 71% from the big four ) is not as extreme as in culture collections ( 97% ; figure 1 ) because phyla containing human pathogens , such as chlamydiae and spirochaetes , are better represented by genome sequences ( figure 3a ) , as are archaea ( figure 3b ) . increasing efforts are being made to select phylogenetically diverse prokaryotes ( archaea for example ) for genome sequencing , using the 16s rrna phylogeny as a guide . but is selection solely on the basis of an exotic location in a 16s rrna tree justified ? the implicit assumption is that the evolutionary history of 16s rrna represents the evolutionary history of the whole organism ( the whole genome ) , but the concept of a unified organismal phylogeny has been significantly compromised by the finding of widespread lateral gene transfer ( lgt ) between organisms . lgt appears to affect the informational genes ( those involved in transcription and translation ) to a lesser extent than metabolic and other operational genes , leading to the hypothesis that a core set of vertically transmitted informational genes define organismal phylogeny . recent evidence suggests that this may not be the case for the euryarchaeota , however ; here , informational genes are apparently no less subject to lgt than operational genes . reliable detection of lgt by comparison of gene trees is complicated by gene duplication and loss , and different methods for detecting lgt are not particularly consistent . the extent to which lgt blurs organismal phylogenies is therefore unclear at this point . at one extreme , if genomes are largely chimeric assemblages of genes with different histories , then any random sampling of organisms should provide a representative ' window ' into genome space . on the other hand , if a core of vertically transmitted genes ( which includes 16s rdna ) defines the organism , then striving to obtain genome sequences from all major lineages in the 16s rrna tree   seems justified . either way , a more complete sampling of phyla defined using 16s rrna should help to resolve the issue . the number of prokaryote genome - sequencing projects completed or in progress as of 20 august 2001   is shown for each phylum - level lineage in the bacterial ( figure 3a ) and archaeal ( figure 3b ) domains . phylum - level lineages comprising only environmental clone sequences ( figure 3 ) also need to be sampled for genome sequences ; this could best be achieved by obtaining one or more representatives of each phylum in pure culture . the classical approach to cultivating microorganisms is to prepare a solid or liquid growth medium containing an appropriate carbon source , energy source and electron acceptor depending on the physiological type of organism being isolated . the medium is then inoculated with a suitable source of microorganisms and left to incubate at a desired growth temperature until organisms multiply to the point at which we become aware of their presence by colony formation or increased turbidity . this approach is not phylogenetically directed , however , and , as discussed above , typically ends up collecting fast - multiplying microbial weeds . to isolate representatives of novel environmental lineages , a directed form of cultivation is required . in one such approach , the first step is to select a target group and design group - specific oligonucleotide probes   to detect or visualize the target organisms in environmental samples ( figure 2 ) . the probes can be used to screen a range of samples and , hopefully , to identify a habitat that is a rich source of the target group . the target organisms then need to be either selectively enriched on the basis of their phenotype or physically isolated from other non - target organisms present in the sample . as it is likely that we know nothing about the physiology of the target environmental group , physical isolation is the preferred route . several methods have been used successfully to physically isolate microorganisms , including sample dilution , filtration , micromanipulators and optical tweezers , density - gradient centrifugation , and cell sorting using flow cytometry ( for an excellent review , see ) . sample dilution may work when the target organism is numerically dominant in a microbial community . the sample is simply diluted until only the target organism remains , albeit at a much lower cell density than in the starting material . sample filtration separates cells according to size , so if the target group is particularly large or small , this might be useful for initial sorting away from the primary inoculum . micromanipulators and optical tweezers are instruments for physically moving single cells or tight clusters of cells from a mixture of cells to fresh growth medium , where the cell(s ) can grow in isolation . these methods are most suitable for isolation of large , morphologically conspicuous microorganisms , such as filaments . density - gradient centrifugation separates cells according to buoyant density and may be useful for initial sorting of communities to enrich for the target organisms . cell sorting by flow cytometry is a high - throughput method for quickly isolating target cells from a mixed culture ; it is most suitable for singly - occurring cells because cell aggregates can interfere with the hydrodynamic focusing in the apparatus . when individual cells are being isolated ( by micromanipulators or optical tweezers ) , the isolation procedure can not be directly monitored by fish because cells are killed ( by fixation with paraformaldehyde ) and isolated cells must be viable for the next step in culturing ; procedures in which subsamples can be sacrificed ( such as filtration or density - gradient centrifugation ) can be monitored by fish . once individual target cells have been physically isolated , a range of growth conditions can be tested to try to promote growth without the complication of overgrowth by non - target cells . strategies include using habitat - simulating growth media , diffusion - gradient enrichments and longer incubation times ( reviewed in ) . common growth media , such as tryptic soy agar , poorly simulate most natural habitats because they are overly substrate - rich ; media that more closely resemble the inoculum habitat will therefore have a greater chance of supporting target - organism growth . the use of cell - free filtrates of the inoculum habitat as the basis for the growth medium is one way of achieving this . diffusion - gradient enrichments facilitate rapid determination of the optimal growth conditions for two parameters at a time , such as ph and nutrient concentrations , usually applied as gradients over a solid or semisolid medium at right angles to each other . finally , simply allowing inoculated growth media to incubate for longer periods than the standard overnight to two - week period may increase the chance of successful isolation of target organisms ( see below ) . throughout the process a phylogenetically directed isolation approach has been successfully demonstrated for an archaeal clone sequence , psl91 , obtained from a hot spring . sequence - specific fish probes were designed and applied to an enrichment from the hot spring , and grape - like cell clusters were highlighted by fish . clusters demonstrating this morphotype were then physically isolated using optical tweezers , grown in pure culture in a liquid medium and confirmed as the target archaeon by fish . the psl91 sequence represents a member of the thermoprotei , however   ( figure 3b ) , and this phylum contains other cultivated representatives , including one genus , desulfurococcus , relatively closely related to psl91 ( 96% 16s rdna sequence identity ) . this may have provided physiological clues as to how to grow the target organism , given that close phylogenetic relatives often ( but not always ) have similar phenotypes . in some instances , for example , we discovered that micromanipulated filaments belonging to candidate phylum tm7   ( figure 3a ) could form colonies visible to the naked eye on low - nutrient solid media ( r2a ) under aerobic conditions ; the only catch was that they took 50 days to do so ( p.h . this may be the case for a wide range of uncultivated organisms , with simple removal of the target organism from the weeds in the inoculum , and a little patience , being all that is required for success . there are likely to be many prokaryotes that will never be brought into pure culture , however , such as organisms that live in obligately interdependent relationships , because the conditions for their growth are too exacting ( and thus can not be reproduced in the laboratory ) . for such organisms , genomes of uncultured prokaryotes can be accessed by a relatively straightforward adaptation of the rrna approach ( figure 2 ) . high - molecular - weight dna extracted from environmental samples can be cloned directly into large - insert cloning vectors , such as cosmids or bacterial artificial chromosomes ( bacs ) . with careful handling of the environmental dna , this results in access to large contiguous portions of microbial genomes - 35 - 40 kilobases ( kb ) for cosmids and up to 200 kb for bacs - without the need for cultivation . bacs have the additional advantage that heterologous expression of some of the insert genes may be possible in the escherichia coli host harboring the vector . clones can be sequenced using shotgun or chromosome - walking methods and comparatively analyzed ( figure 2 ) . if a 16s rrna gene or another conserved gene is identified in a clone then the phylogenetic identity of the genome segment can be determined . perhaps the most impressive application of this approach to date is the discovery of proteorhodopsin in an uncultured lineage of marine bacterioplankton belonging to the gammaproteobacteria . an open reading frame encoding proteorhodopsin was found on a 130 kb genomic fragment together with a 16s rdna sequence identifying its owner as a member of the ' sar86 ' group in the gammaproteobacteria . members of the sar86 group had been detected on numerous occasions in culture - independent surveys of marine habitats , but no function could be inferred for them because there are no close cultivated representatives for the group . the discovery of proteorhodopsin , which is phylogenetically related to the light - driven proton pump bacteriorhodopsins , suggests that the sar86 lineage lives phototrophically in the marine environment . ideally , we would like to reconstruct entire genomes from uncultured prokaryotes using large - insert cloning - vector approaches . this is a daunting task given the species complexity of most microbial communities and the genomic microheterogeneity within prokaryotic populations . it will probably be an impossible task for habitats such as soil , containing thousands of individual genomes . it remains to be seen , however , whether it is possible to reconstruct complete genomes from a low - diversity microbial community . in conclusion , several major lineages of bacteria ( but not archaea ) containing isolated representatives lack even a single sequenced genome . over a third of phylum - level prokaryotic lineages are represented exclusively by sequences of uncultured prokaryotes that have been repeatedly detected in culture - independent habitat surveys over the past decade . the mere existence of such large phylogenetically conspicuous groups , about which we know virtually nothing , should be reason enough to study them . yet there remains a reluctance amongst many microbiologists to accept these ' virtual bacteria '   as bona fide members of the microbial world . by analogy , imagine that we were unaware of the metazoa until a few years ago , when we began detecting them in environmental surveys using phylogenetic markers . imagine that metazoa - specific probes were designed to allow us to see this new group under the ' macroscope ' . what do these creatures do for a living ? what other organisms remain to be discovered in this group ? this is approximately the stage we are at in the description of candidate prokaryotic phyla . at the very least , uncharacterized prokaryotic phyla will probably contain members with impressive physiological repertoires and interesting evolutionary histories , worthy of study and of genome sequencing . i thank mark ragan , norman pace and george garrity for providing comments on the manuscript . pie charts showing the phylum - level distribution of prokaryotic isolates ( a ) in the australian collection of microorganisms   and ( b ) in the prokaryote genome sequences completed or in progress as of 20 august 2001 . ' full - cycle ' rrna approach to characterizing microorganisms in their natural settings without the need for cultivation . access to whole genomes of uncultivated organisms is also possible using the same basic approach but with large - insert cloning vectors , such as bacs , which remove the need for pcr . evolutionary distance dendrograms of ( a ) bacterial and ( b ) archaeal diversity derived from comparative analysis of 16s rrna gene sequences . the trees were constructed using the arbsoftware package and a sequence database modified from the march 1997 arb database release   using 50% consensus sequence filters for each domain and the olsen correction and neighbor - joining options . this modified database will be available from the ribosomal database project   user - submitted alignments download site . major lineages ( phyla ) are shown as wedges with horizontal dimensions reflecting the known degree of divergence within that lineage . phyla with cultivated representatives are in gray and , where possible , named according to the taxonomic outline of bergey 's manual . phyla known only from environmental sequences are in white ; because they are not formally recognized as taxonomic groups , they are usually named after the first clones found from within the group . note that environmental groups e2 and e3 defined in   are part of the thermoplasmata phylum in the archaeal tree in ( b ) . the number of genome sequences completed or in progress for each phylum is given in brackets after the phylum name , with the exception of methanopyrus kandleri , which is not included in the tree because it is represented by a single sequence . bacterial phyla with cultured representatives but without representative sequenced genomes   where possible , the nomenclature of the taxonomic outline for bergey 's manual of systematic bacteriology   has been used .