Introduction

Growing bacteria on nutrient agar is one of the most familiar techniques in microbiology. When a sample is spread onto a suitable culture medium and incubated under appropriate conditions, some bacterial cells multiply and form visible colonies. These colonies allow microbiologists to isolate bacteria, study their characteristics, and estimate the number of colony-forming units in a sample. However, the absence of visible colonies does not necessarily mean that all bacteria in the sample are dead. Some bacterial cells can remain viable while losing their ability to grow under laboratory conditions that would normally support them. This condition is known as the viable but nonculturable (VBNC) state.

The VBNC state has been investigated in several bacterial species, including Escherichia coli, Vibrio cholerae, Listeria monocytogenes, and Salmonella. Under stressful environmental conditions, some bacteria may undergo physiological changes that reduce their metabolic activity and prevent them from forming colonies on routine culture media. Nevertheless, they may retain measurable characteristics associated with living cells, and some can regain culturability when conditions become favourable. This phenomenon is particularly relevant to food microbiology, environmental monitoring, and bacterial physiology because conventional cultivation does not always detect every viable bacterial population.

Figure 1: The conceptual image was created by AI for Microbes World.

What Is the Viable but Nonculturable State?

The VBNC state describes a condition in which bacterial cells retain evidence of viability but cannot be cultivated under conditions that normally support their growth. Under favourable circumstances, bacteria obtain nutrients, maintain cellular functions, and reproduce. When environmental conditions become stressful, however, some bacterial populations reduce their physiological activity and eventually lose culturability. Although these cells no longer produce detectable growth on routine culture media, some may retain intact membranes, measurable metabolic activity, or other characteristics associated with living organisms.

To understand this phenomenon, it is important to distinguish between viability and culturability. Viability concerns whether a bacterial cell remains alive, although determining this experimentally requires appropriate biological evidence. Culturability refers to the ability of a cell to multiply under specified laboratory conditions and produce detectable growth. These properties are closely related but are not identical. A bacterium may be viable without being culturable under the conditions tested. Conversely, detecting an intact membrane or bacterial DNA does not independently prove that a cell is alive.

Not every bacterium that fails to grow on agar is necessarily in the VBNC state. Some microorganisms require specific nutrients, temperatures, oxygen concentrations, or other growth conditions that are not provided by routine culture media. Others may experience temporary cellular injury that prevents immediate growth. Researchers must therefore distinguish genuinely nonculturable populations from dead cells, injured bacteria, and organisms that simply require alternative cultivation conditions. This distinction remains an important methodological challenge in VBNC research.

Discovery of the VBNC State

The modern concept of the VBNC state developed from environmental microbiology research during the early 1980s. In a study published in 1982, Xu and colleagues investigated the survival of E. coli and V. cholerae in estuarine and marine environments. They compared conventional culture-based measurements with direct microscopic and physiological methods for assessing bacterial survival. During exposure to saltwater conditions, the number of bacteria recovered by cultivation declined considerably, while other measurements indicated that some cells remained viable. These observations showed that the loss of culturability did not necessarily correspond to the death of the entire bacterial population.

The findings encouraged further research into bacterial survival under environmental stress. Subsequent studies reported similar behaviour in other bacterial species and examined the physiological changes associated with nonculturability. Researchers also investigated whether nonculturable bacteria could recover their ability to grow when environmental conditions improved. Although the VBNC state is now widely recognized, aspects of its biological definition and the methods used to identify it remain under discussion, particularly when distinguishing it from dormancy and cellular injury.

Why Do Bacteria Enter the VBNC State?

Bacteria encounter changing environmental conditions throughout their lives, and these changes can influence their growth and survival. Temperature, nutrient availability, salinity, acidity, oxidative stress, and exposure to antimicrobial substances are among the factors associated with reduced bacterial culturability. In some species, prolonged exposure to unfavourable conditions may lead to a physiological state in which cellular activity decreases while certain functions remain detectable. However, the response varies between bacterial species, strains, and environmental conditions. Not every stress produces a VBNC population, and not every bacterium responds to stress in the same way.

Low temperature is one factor associated with the development of nonculturable populations in some bacterial species. Cold conditions can reduce metabolic activity and affect cellular processes required for growth. Prolonged exposure may eventually lead to a loss of culturability, even when some cells retain measurable signs of viability. Nutrient limitation can produce related effects. When essential nutrients become scarce, bacteria may reduce their metabolic activity and alter their physiological processes. In certain organisms, prolonged nutrient deprivation contributes to nonculturability. These responses are particularly relevant in natural environments, where nutrient availability and temperature often fluctuate.

Changes in salinity, pH, and oxidative conditions may also influence bacterial culturability. For example, bacteria entering natural aquatic environments can encounter conditions very different from those in nutrient-rich laboratory media. Some cells may die, while others remain culturable or develop physiological characteristics associated with the VBNC state. Exposure to disinfectants can produce similarly varied outcomes. Depending on the treatment conditions, disinfectants may kill bacterial cells, cause sublethal injury, or leave populations that are no longer recoverable by conventional culture methods. Experimental studies have reported VBNC-associated populations following certain disinfectant treatments, but this does not mean that disinfection generally causes bacteria to enter the VBNC state.

The biological mechanisms underlying nonculturability are complex and remain incompletely understood. Some researchers consider the VBNC state a form of physiological adaptation that may help certain bacteria persist during environmental stress. Others have emphasized that severe cellular injury can produce similar observations. The VBNC state should therefore not automatically be described as a deliberate or universally beneficial survival strategy.

How Is the VBNC State Different from Bacterial Dormancy and Antibiotic Resistance?

The VBNC state is closely related to bacterial dormancy, but the two terms are not interchangeable. Dormancy broadly describes a condition of reduced growth or metabolic activity, whereas the VBNC state specifically involves the loss of culturability under conditions that would normally support bacterial growth. Some dormant bacteria remain culturable, while VBNC populations do not produce detectable growth using the relevant culture methods. The distinction becomes more complicated when considering bacterial persister cells, which can survive certain antibiotic treatments without necessarily carrying inherited resistance mechanisms. Persisters and VBNC cells may share physiological characteristics, and their relationships continue to be investigated.

Antibiotic resistance represents a different phenomenon. Resistant bacteria can survive or grow at antimicrobial concentrations that inhibit susceptible organisms, often because of inherited or acquired genetic mechanisms. Bacteria in a VBNC state may exhibit altered susceptibility or increased tolerance to certain stresses, but this does not automatically establish genetic antibiotic resistance. These distinctions matter because bacterial survival following antimicrobial exposure can result from several biological processes, and colony formation alone cannot identify the underlying mechanism.

How Can Scientists Detect VBNC Bacteria?

Detecting VBNC bacteria requires more than observing whether colonies appear on an agar plate. Researchers must first establish that a bacterial population has lost culturability under normally supportive conditions and then investigate whether some cells retain convincing evidence of viability. Different laboratory techniques provide information about different cellular properties, so combining complementary methods is generally more informative than relying on a single test.

Conventional Culture Methods

Conventional cultivation remains one of the most important techniques in microbiology because it demonstrates that bacteria can reproduce under specified conditions. When colonies develop on an appropriate medium, researchers can isolate the organisms and investigate their characteristics. However, when no colonies are detected, the result does not necessarily establish that the sample contains no viable bacteria. Cells may be injured, nonculturable, or unable to grow under the selected incubation conditions. Culture results therefore describe the population recoverable using a particular procedure rather than providing a complete measurement of every viable cell. When investigating possible VBNC populations, researchers may compare routine cultivation with alternative recovery conditions and additional viability measurements.

PCR and Quantitative PCR

Polymerase chain reaction (PCR) and quantitative PCR (qPCR) detect selected DNA sequences without requiring bacteria to grow on culture media. These methods are valuable for identifying bacterial genetic material in clinical, environmental, and food samples, including samples containing organisms that are difficult to cultivate. However, DNA may remain detectable after bacterial cells have died. Consequently, a positive PCR result does not automatically demonstrate the presence of living bacteria. This limitation is also important in microbiome research, where DNA-based sequencing can identify microbial genetic material without directly establishing whether the detected organisms are viable or metabolically active.

Viability PCR

Viability PCR is designed to reduce the amplification of DNA associated with certain dead or membrane-damaged cells. One commonly used reagent is propidium monoazide (PMA), while related reagents such as PMAxx are also available. These compounds are intended to enter cells with compromised membranes and interact with their DNA following light activation, reducing subsequent PCR amplification. When combined with qPCR, this approach can help distinguish DNA associated with relatively intact cells from DNA associated with some membrane-damaged cells. However, membrane integrity is an indirect indicator of viability rather than definitive proof that a bacterium is alive. Some dead cells may retain intact membranes, while living but injured cells may temporarily show membrane damage. Sample composition and experimental conditions can also influence assay performance. PMA-based methods should therefore be interpreted cautiously and, where possible, supported by other measurements.

Microscopy and Metabolic Activity Measurements

Fluorescence microscopy, flow cytometry, and metabolic assays provide additional ways to investigate bacterial populations that are difficult to cultivate. Depending on the technique, researchers may examine membrane integrity, enzyme activity, respiration, or other physiological characteristics. These measurements can help identify cells that retain biological activity despite reduced culturability. Nevertheless, each method has limitations because it measures a particular cellular property rather than every aspect of viability. A population that no longer forms colonies but retains membrane integrity and measurable metabolic activity may be investigated as a possible VBNC population, although additional evidence is needed to establish its physiological condition.

Can VBNC Bacteria Become Culturable Again?

Some VBNC bacteria can regain their ability to grow when environmental conditions become favourable. This process is commonly called resuscitation. Depending on the bacterial species and experimental conditions, recovery of culturability has been associated with changes in temperature, nutrient availability, or the removal of environmental stress. However, resuscitation is not universal, and the conditions required for recovery differ among organisms. Some nonculturable populations may remain unable to grow even after the original stress has been removed.

Demonstrating genuine resuscitation is experimentally challenging. Suppose researchers expose a bacterial population to stressful conditions until no colonies are detected and then observe bacterial growth after transferring the sample to a more favourable environment. The result could indicate that VBNC cells regained culturability. However, it could also arise from a small number of culturable cells that remained below the detection limit of the original assay and subsequently multiplied. Researchers must account for this possibility before concluding that resuscitation has occurred. This distinction is important because the reappearance of colonies alone does not necessarily demonstrate that previously nonculturable cells recovered.

What Have Recent Studies Revealed About VBNC Bacteria?

Recent investigations have provided additional evidence about the behaviour and detection of VBNC-associated bacterial populations, particularly in food microbiology. These studies are relevant because conventional culture methods remain central to food-safety monitoring, while environmental stresses and sanitation procedures can influence bacterial recovery.

VBNC Listeria monocytogenes in Food-Processing Environments

A study published by Brauge and colleagues in 2026 investigated whether VBNC-associated Listeria monocytogenes populations originating from biofilms on stainless-steel surfaces could be transferred to a ready-to-eat seafood product and subsequently regain culturability. Biofilms are surface-associated microbial communities embedded in extracellular material, and they can present challenges for cleaning and sanitation in food-processing environments. The researchers exposed bacterial biofilms to a quaternary ammonium-based disinfectant and examined the resulting populations using microbiological and viability-associated detection methods.

The researchers then investigated bacterial transfer to vacuum-packed smoked herring. Under the experimental conditions, populations associated with the VBNC state were transferred from treated surfaces to the food, where recovery of culturability was subsequently observed during refrigerated storage. These findings suggest that culture-negative or poorly culturable bacterial populations may require additional attention when evaluating microbial survival following sanitation. However, the study was conducted using a defined experimental system and does not establish how frequently this phenomenon occurs in commercial food production. It also does not demonstrate that every VBNC L. monocytogenes population can recover under refrigerated conditions.

Detection of Salmonella in Meat Samples

In another study published in 2026, Pham and colleagues evaluated an optimized PMAxx real-time PCR method for detecting viable and VBNC-associated Salmonella populations in meat samples. The researchers examined experimentally prepared bacterial populations, including cells exposed to prolonged cold conditions that reduced culturability. They also evaluated the assay using retail meat samples and compared its performance with conventional culture methods. The study demonstrated that the molecular approach could retain detectable signals from experimentally induced nonculturable populations, illustrating its potential usefulness for investigating bacteria that may not be adequately represented by routine cultivation.

The researchers also tested 33 retail chicken and pork samples, of which 23 were positive, with agreement between the optimized molecular method and conventional culture in that sample set. These findings support the potential application of viability-associated PCR as a complementary method, but they do not establish that it detects every viable cell or consistently outperforms conventional cultivation. In particular, a positive PMAxx-qPCR result does not independently prove that the detected bacteria can resuscitate, multiply, or cause infection. The study therefore provides evidence about assay performance under the investigated conditions rather than a universal solution to VBNC detection.

Why Does the VBNC State Matter in Microbiology?

The VBNC state has implications for food microbiology, environmental monitoring, and laboratory investigations of bacterial survival. In food-processing environments, microbiologists use cultivation to detect microorganisms that may affect food quality or safety. In environmental microbiology, culture-based methods help investigate bacterial contamination and survival in water, sediments, and other habitats. When some bacterial populations lose culturability while retaining viability, conventional cultivation may underestimate the number of viable cells. This possibility is especially relevant when interpreting samples exposed to changing environmental conditions or antimicrobial treatments.

However, the VBNC concept does not make conventional culture methods unnecessary or unreliable. Cultivation remains essential for isolating bacteria, studying their physiological characteristics, and conducting many antimicrobial susceptibility investigations. Instead, VBNC research demonstrates that different methods answer different biological questions. Colony counts measure recoverable colony-forming units under specified conditions, PCR detects selected genetic targets, and membrane-based assays examine particular cellular properties. None of these approaches independently describes every aspect of bacterial survival.

The presence of VBNC-associated bacteria also does not automatically establish a health risk. Whether a microorganism can recover, multiply, produce harmful substances, or cause infection depends on the organism and the circumstances involved. Understanding these limitations allows researchers to interpret laboratory results more accurately and avoid assuming that the absence of detectable growth necessarily means the absence of viable microorganisms.

Conclusion

The viable but nonculturable state demonstrates that bacterial survival cannot always be assessed through colony formation alone. Under certain stressful conditions, some bacteria lose their ability to grow on normally supportive culture media while retaining measurable characteristics associated with living cells. This phenomenon has been investigated in several bacterial species and is particularly relevant to understanding microbial survival in food and environmental samples.

Although some VBNC populations can regain culturability, recovery depends on the organism and environmental conditions, and genuine resuscitation must be distinguished from the multiplication of previously undetected culturable cells. Recent studies involving Listeria monocytogenes and Salmonella have further illustrated the importance of combining cultivation with appropriate physiological and molecular measurements. The central lesson is that bacterial viability and culturability are related but distinct properties, and reliable interpretation of microbial survival requires an understanding of what each laboratory method measures.

References

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