so the cells that are metabolically or physiologically active but can not be cultured on specific media are the viable but nonculturable cells ( vbnc ) .
in fact , less than 1% of the microorganisms in natural water and soil samples are cultured in viable count procedures . in 1982
, xu and coworkers introduced the term  viable but nonculturable bacterial cells ( vbnc )  to distinguish particular cells that could not form colonies on solid media but retained metabolic activity and the ability to elongate after the administration of nutrients . according to oliver , 
vbnc can be defined as a metabolically active bacterial cell that crossed a threshold in this way , for known or unknown reasons , and became unable to multiply in or on a medium normally supporting its growth . 
most of the bacteria that enter vbnc state are gram - negative species belonging to the gamma subclass of the proteobacteria branch , except for rhizobium , agrobacterium , and helicobacter - campylobacter species .
bashford and colleagues   announced that they had recovered vibrio cholerae from streams and drainage ditches , including sites with negligible chance of sewage contamination . around the same time , colwell et al .   also found vibrio cholerae in maryland , usa .
she and her coworkers showed that , in artificial sea water , vibrio cholera and e. coli remained viable though they lost the capacity to form colonies on culture media . soon
salmonella enteritidis , shigella sonnei , and legionella pneumophila joined the list of organisms known to be capable of entering a state in which they failed to show up on nutrient agar yet took up substrates and signaled in other ways that they were certainly not dead .
the use of laboratory media to recover and enumerate bacteria and to link them with or absolve them from pathological and other activities became obsolete by the new discoveries , and the term vbnc ( viable but nonculturable ) was introduced .
microorganisms that do not grow in culture media , but are still metabolically active and capable of causing infections in animals and plants , are said to be in a vbnc state .
traditional laboratory culture conditions and methods can not meet the requirements of vbnc organisms to resume growth .
vbnc cells exhibit active metabolism in the form of respiration or fermentation , incorporate radioactive substrates , and have active protein synthesis but can not be cultured or grown on conventional laboratory media .
they have been detected by observing discrepancies between plate count enumeration of bacterial population and direct staining and microscopic counts .
for example , viable but nonculturable cells of vibrio cholerae , enteropathogenic e. coli , legionella pneumophila , and various other bacteria have been shown to regain culturability after they have entered the intestinal tracts of animals .
the vbnc state is defined as a state of dormancy triggered by harsh environmental conditions , such as nutrient starvation , extreme temperatures , and sharp changes in ph or salinity ; osmotic stress , oxygen availability [ 20 , 21 ] , and damage to or lack of an essential cellular component including dna ; exposure to food preservatives   and heavy metals [ 23 , 24 ] ; exposure to white light ; activation of lysogenic phages or suicide genes such as sok / hak or autolysins ; and decontaminating processes such as pasteurization of milk   and chlorination of wastewater .
the characteristics of bacteria in the vbnc state can be summarized as follows : maintaining apparent cell integrity ; possessing some form of measurable cellular activity ; possessing apparent capacity to regain culturability in vivo ; responding to external stimulus as shown by specific gene expression ; having low metabolic activity ; exhibiting dwarfing ; having reduced nutrient transport ; containing a high atp level and exhibit high membrane potential ; having extensive modifications to the fatty acid composition in cytoplasmic membranes ; within the cell wall peptidoglycan , increased crosslinking , increased muropeptides bearing covalently bound lipoprotein , and shortening of the average length of glycan strands ; higher autolytic capability than exponentially growing cells ; retained plasmids ; increased antibiotic resistance due to lower metabolic activity ; changes in outer - membrane protein profile ; continuous gene expression .the vbnc state continues to be disputed due to the difficulties of differentiating vbnc cells and dormant cells through resuscitation and phenotypic studies .
however , recent molecular studies have generated data to support the existence of the vbnc state .
maintaining apparent cell integrity ;   possessing some form of measurable cellular activity ; possessing apparent capacity to regain culturability in vivo ;   responding to external stimulus as shown by specific gene expression ;   having low metabolic activity ;   exhibiting dwarfing ;   having reduced nutrient transport ;   containing a high atp level and exhibit high membrane potential ;   having extensive modifications to the fatty acid composition in cytoplasmic membranes ;   within the cell wall peptidoglycan , increased crosslinking , increased muropeptides bearing covalently bound lipoprotein , and shortening of the average length of glycan strands ;   higher autolytic capability than exponentially growing cells ;   increased antibiotic resistance due to lower metabolic activity ;   changes in outer - membrane protein profile ;   continuous gene expression .
the vbnc is also considered an important reservoir of many human pathogens in the environment .
the following list includes but is not limited to the pathogenic bacteria that can enter the vbnc state : aeromonas hydrophila , agrobacterium tumefaciens , burkholderia cepacia , campylobacter jejuni , enterobacter aerogenes , enterobacter cloacae , enterococcus faecalis , escherichia coli ( including ehec ) , helicobacter pylori , klebsiella pneumoniae , legionella pneumophila , listeria monocytogenes , mycobacterium tuberculosis , pseudomonas aeruginosa , salmonella typhi , salmonella typhimurium , shigella dysenteriae , shigella flexneri , shigella sonnei , streptococcus faecalis , vibrio alginolyticus , vibrio cholerae , vibrio harveyi , vibrio parahaemolyticus , and vibrio vulnificus ( types 1 and 2 ) .
many believe that pathogens in the vbnc state are unable to induce infection / disease despite retaining their virulent properties .
however , when vbnc pathogens pass through a host animal , resuscitation and resumption of metabolic activity have led to infections and diseases [ 40 , 41 ] .
the first evidence of pathogenicity of nonculturable cells was the demonstration of fluid accumulation in the rabbit ileal loop assay ( rica ) by vbnc vibrio cholerae o1 , followed by human volunteer experiments .
vbnc e. coli nonculturable cells were also reisolated after passaging through rabbit ileal loops 4 days after inoculation , and chick embryos died when injected with nonculturable cells of legionella pneumophila , leading to the conclusion that vbnc pathogens remain potentially pathogenic .
many indicator bacteria and pathogenic bacteria which exist in aquatic habitats have been shown to have a vbnc state .
water is routinely tested for such indicators and pathogens , and if they are not detected or enumerated at a concentration below guidelines , waters are deemed to be safe for public consumption . therefore , where circumstances indicate possible presence of vbnc pathogens , additional molecular methodology needs to be used to reduce the risk of infective disease outbreaks .
thus , food and environmental and clinical samples no longer can be considered free from pathogens if culturing yields negative results . for the general public
, the presence of vbnc in water and food may be related to low - grade infections or the so - called aseptic infections .
in many cases , the infections are incorrectly attributed to viruses since no bacteria were detected .
for example , vibrio cholerae o1 in the surface water have been shown to remain in nonculturable state .
these water sources are used for domestic purpose regularly and pose a risk of infection .
when conditions are not favorable for growth , then it transforms to the nonculturable state in association with crustacean copepods .
persistence of vibrio cholerae in water in the vbnc state is an important public health factor , since detection will not be successful if only conventional cultural methods are used .
similarly , shigella can undergo vbnc state in water but become a threat when they enter the human body .
additional studies have indicated that a good number of pathogenic bacteria can survive food and water treatment processes and persist as well as retain virulence in processed food , pasteurized milk , potable water , and the environment .
one study demonstrated that recurrent urinary tract infections in many individuals were caused by uropathogenic e. coli cells which remained in vbnc state .
other studies showed that uropathogenic vbnc e. coli retain enteropathogenicity as shown by continual production of enterotoxin .
nilsson et al .   showed that vbnc helicobacter pylori cells can express virulence factors such as caga , vaca , and vrea .
food and its surrounding environment are a complex system , in which physiochemical characteristics ( ph , aw , and chemical composition ) and environmental factors ( storage temperature and time , decontamination treatments , and packaging under modified atmosphere ) act simultaneously on contaminating bacteria leading to the vnbc state .
for example , it has been demonstrated that refrigerated pasteurized grapefruit juice induced the vbnc state in e. coli o157  :  h7 and s. typhimurium within 24 hours of incubation .
reported that in pasteurized milk which has undergone thermal treatment , contaminating bacteria such as e. coli and pseudomonas putida enter into the vbnc state but retain transcription and translation functions .
several foodborne outbreaks have been reported in japan , where pathogens such as salmonella enterica subsp .
enterica   and e. coli o157   in the vbnc state were responsible for outbreaks .
the vnbc is also critical in determining shelf life and microbial stability of food and beverages .
for example , acetic acid and lactic acid bacteria entered the vbnc state in wine as a consequence of lack of oxygen and presence of sulphites .
nalidixic acid ( 2040  mg / l ) is used to stop cell division . after exposure to nalidixic acid , viable cells continue to grow and will appear elongated , whereas the nonviable metabolically inactive cells will retain their original shape and size .
viable cells will be seen as elongated , whereas vbnc / dormant cells will be seen as oval and large .
frequently used stains are acridine orange , 4,6-diamino-2-phenyl indole ( dapi ) , fluorescein isothiocyanate ( fitc ) , indophenyl - nitrophenyl - phenyltetrazolium chloride ( int ) , and 5-cyano-2,3-ditolyl tetrazolium chloride ( ctc ) .
the mode of action of these dyes and the reactions observed are summarised in table 1 . in recent years
, a new differential staining assay , the baclight live / dead assay , has been developed .
the assay allows simultaneous counting total and viable ( metabolically active ) cells , by using two nucleic acid stains , that is , green - fluorescent syto 9 stain and red - fluorescent propidium iodide stain .
syto 9 stains both live and dead bacteria , whereas propidium iodide penetrates only bacteria having damaged membranes . when used together , propidium iodide reduces syto 9 fluorescence in dead bacteria with damaged membranes resulting in red fluorescent cells , whereas the live bacteria will fluoresce green .  
are nucleic acids ( dna / rna ) which have been chemically or radioactively labeled and are used to detect complementary target dna / rna . specific amplification of dna targets in bulk dna extracts from environmental and clinical samples permits detection of specific organisms or groups of related organisms without the need to cultivate them , provided the appropriate unique primers are used .
these procedures do not discriminate between culturable and nonculturable forms of the target organisms . due to the failure of distinguishing between dead or live cells by dna - based methods ,
rna - based methods are a more valuable estimate of gene expression and/or cell viability under different conditions .
this technique is more able to discriminate between culturable and nonculturable forms of an organism .
furthermore , reverse transcriptase pcr ( rt - pcr ) can distinguish between live and dead cells .
this is possible because it is an mrna - based method and mrna is short - lived ( half - life less than 1 minute ) .
messenger rna is only present in metabolically active cells and not found in nature after cell death . rt pcr can detect nonculturable but active or live cells .
even though traditional culture methods fail to detect the presence of specific vbnc in a sample , the presence of these microbes can be demonstrated using some of the molecular techniques described .
more specifically , oligonucleotide probes of l820 nucleotides are proving most useful because they hybridize rapidly to specific dna sequences of target organisms .
the detection of vbnc cells directly from the environmental samples can also be achieved using different types of blotting such as colony blot , slot blot , dot blot , and southern blot .
the principle of blotting is the use of radio- or nonradioactive or fluorescence labeled probe .
fluorescent in situ hybridization ( fish ) is an alternative format for hybridization probes in which fluorescence labeled dna or rna probes are hybridized with target nucleic acids in whole , permeabilized cells . the application of this method to the detection of single microbial cells by using
this is done through selective targeting of regions of rrna , which consist of conserved and variable nucleotide regions . by choosing the appropriate rrna probe sequence ,
fish can be used to detect all bacterial cells ( a universal probe ) or a single population of cells ( a strain - specific probe ) of vbnc .
from the above discussions , it is evident that a number of nonspore - forming human pathogenic bacteria can enter the vbnc state with maintained cellular structure and biology and persistent gene expression but remain nonculturable by traditional cultural techniques .
they can survive and revert to culturable conditions when provided with appropriate conditions , hence , being a significant threat to public health and food safety .
further research is needed to elucidate mechanisms leading to the vbnc and the development of methodologies to confirm their existence .
 the viable but nonculturable ( vbnc ) state is a unique survival strategy of many bacteria in the environment in response to adverse environmental conditions . 
 vbnc bacteria can not be cultured on routine microbiological media , but they remain viable and retain virulence . 
 the vbnc bacteria can be resuscitated when provided with appropriate conditions . 
 a good number of bacteria including many human pathogens have been reported to enter the vbnc state . though there have been disputes on the existence of vbnc in the past , 
 extensive molecular studies have resolved most of them , and vbnc has been accepted as a distinct survival state . 
 vbnc pathogenic bacteria are considered a threat to public health and food safety due to their nondetectability through conventional food and water testing methods . 
 a number of disease outbreaks have been reported where vbnc bacteria have been implicated as the causative agent . 
 further molecular and combinatorial research is needed to tackle the threat posed by vbnc bacteria with regard to public health and food safety . 
1. Introduction
2. History
3. VBNC
4. VBNC Pathogens
5. Public Health Significance of VBNC
6. VBNC State of Foodborne Bacteria: A Challenge in Food Safety
7. Methods of Detection of VBNC Bacteria
8. Conclusion