• Journal of Microbiology
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• v.43 no.spc1
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• pp.93-100
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• 2005

It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the 'viable but nonculturable' (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

• Korean Journal of Soil Science and Fertilizer
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• v.38 no.5
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• pp.287-293
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• 2005

In this work, to develop soil inoculant which maintains stable viable cells and normalized quality, studies on micro-encapsulation with bacteria and yeast cells were performed by investigating materials and methods for micro-encapsulation as well as variation and stability of encapsulated cells. Preparation of capsule was conducted by application of extrusion system using micro-nozzle and peristaltic pump. K-carragenan and Na-alginate were selected as best carrier for gelation among K-carageenan, Na-alginate, locust bean gum, cellulose acetate phthalate (CAP), chitosan and gelatin tested. Comparing the gels prepared with Bacillus sp. KSIA-9 and carriers of 1.5% concentration, although viable cell of K-carragenan and Na-alginate was six times higher than those of other, Na-alginate was finally selected as carrier for gelation because it is seven times cheaper than K-carragenan. The gel of 1.5% Na-alginate was also observed to have the best morphology with circular hardness polymatrix and highest viable cell. When investigating the stability of encapsulated cells and the stabilizer effect, free cells were almost dead within 30 or 40 days whereas encapsulated cells decreased in 10% after 30 days and 15-30% even after 120 days. As stabilizer for maintaining viable cell, both 1% starch and zeolite appeared to possess the level of 70-80% cell for bacteria and yeast until after 120 days.

• KSBB Journal
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• v.18 no.1
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• pp.14-18
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• 2003

In this study of the simultaneous saccharification and fermentation (SSF) of paper sludge, fed-batch cultivation of Lactobacillus paracasei subsp. paracasei KLB58 was attempted to produce viable KLB58 cells and lactic acid. Optimal culture conditions, including the temperature and concentration of the supplemented enzyme, were examined in terms of lactic acid production and viable cell count. When the effects of culture temperature and $\beta$-glucosidase concentration were examined in fed-batch SSF, the highest viable cell counts and lactic acid production (i.e. 5$\times$$10^9$ CFU/ml and 45 g/L, respectively) were obtained at 37$^{\circ}C$ and 2 unit/ml of $\beta$-glucosidase.

• Journal of fish pathology
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• v.37 no.1
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• pp.49-60
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• 2024

A viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when faced with unfavorable environmental conditions, rendering them unable to grow on nutrient agar while maintaining low metabolic activity. This study explored the impact of temperature and nutrient availability on inducing VBNC state in Edwardsiella piscicida, the most important bacterial fish pathogen, and assessed its pathogenicity at VBNC state. E. piscicida was suspended in filtered sterile seawater and exposed to three different temperatures (4, 10, and 25℃) to induce the VBNC state. Subsequently, the induced VBNC cells were subjected to resuscitation by either raising the temperature to 28℃ or inoculating them in brain heart infusion broth supplemented with 1% NaCl. A propidium monoazide (PMA)-qPCR method was also developed to selectively quantify live (VBNC or culturable) E. piscicida cells. The results showed that the bacteria entered the VBNC state after approximately 1 month at 4℃ and 25℃, and 2 months at 10℃. The VBNC E. piscicida cells were successfully revived within 3 days in a nutrient-rich environment at 28℃, highlighting the significance of temperature and nutrition in inducing and resuscitating the VBNC state. In pathogenicity tests, resuscitated E. piscicida cells exhibited high pathogenicity in olive flounder comparable to cultured bacteria, while VBNC cells showed no signs of infection, suggesting they are unlikely to resuscitate in fish. In conclusion, this study contributes to our understanding of fish pathogen ecology by investigating the characteristics of the VBNC state under varying temperature and nutrition conditions.

• Korean Journal of Agricultural Science
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• v.49 no.4
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• The Korean Journal of Food And Nutrition
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• v.6 no.3
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• pp.208-210
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• 1993

In order to know the influences of heat treatment of yoghurt on pH, $\beta$-galactosidase and viable cells, yoghurt sample was made by general method with Lactobacillus bulgaricus and Streptococcus thermophilus, and the changes in pH, $\beta$-galactosidase-activity and viable cell-count were determined during heating at 55$^{\circ}C$ and 7$0^{\circ}C$. The pH of yoghurt was not changed when the yoghurt was heated at 7$0^{\circ}C$, but at 55$^{\circ}C$ it decreased slightly. The stability of $\beta$-galactosidase was not affected markedly by heat treatment at 55$^{\circ}C$, but was rapidly inactivated at 7$0^{\circ}C$. The heat treatment of yoghurt at 55$^{\circ}C$ had the halb of viable cell in 1 hour, but the heat treatment at 7$0^{\circ}C$ had considerable effect on viable cell in 5 minutes.

• International Journal of Oral Biology
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• v.35 no.1
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• pp.1-5
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• 2010

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells ($1.09\;{\times}\;10^5\;cells/ml$). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR:64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% ($3.0\;{\times}\;10^4\;cells/ml$) or 20% KSR ($4.8\;{\times}\;10^4\;cells/ml$) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.

• KSBB Journal
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• v.13 no.3
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• pp.251-258
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• 1998

A structured kinetic model is proposed to describe cell growth and secondary metabolite, flavone glycosides, synthesis in batch suspension culture of Scutellaria baicalensis G. The model has been developed by representing the physiological state of cell described as the activity and viability which can be estimated based on the culture fluorescence. In the model, three type of cells are considered; active-viable, nonactive-viable and dead cells. Viable cell weight could be determined based on the relative fluorescence intensity. The flavone glycosides could be produced by both active-viable and non-active viable cells with a different production rate. And the model includes the cell expansion due to glucose concentration and death phase which accounts for the release of intracellular secondary metabolite into medium. Dependent variables include substrate concentration(glucose), cell mass(dry cell weight and fresh cell weight), product concentration(flavone glycosides), activity and viability. Satisfactory agreement between the model and experimental data is obtained from shake flask culture of Scutellaria baicalensis G. The proposed model can predict the cell growth and flavone glycosides synthesis as well as intermediate materials.

• Restorative Dentistry and Endodontics
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• v.33 no.6
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• pp.537-544
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• 2008

Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treatment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.

• Microbiology and Biotechnology Letters
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• v.28 no.2
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• pp.117-123
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• 2000

The object of this work is to improve the maintenance of bioluminescence from stored Photobacterium phosphoreum in a view of developing continuous monitoring system for pollutants. The long-term experiments were performed to determine the effect of storage temperature and immobilization on the maintenance of bioluminescence and viability of P. phosphoreum. A naturally luminescent bacterium, P. phosphoreum was starved in 2.5% Nael solution at $20^{\circ}C$, $4^{\circ}C$, -$20^{\circ}C$ and -$70^{\circ}C$ for 30 days. In vivo luminescence was measured by luminometry, and total cell concentrations and concentrations of culturable and viable cells were determined by acridine orange staining, dilution plate counting, and direct viable counting, respectively. The bioluminescence emission from cells stored at 4De was maintained up to 10 days while those with starved cells at other temperature ranges decreased to background level within 3 days. In terms of viability of cells, concentrations of cells stored at $20^{\circ}C$ were rapidly decreased as a result of cell lysis, leading to a drop in culturable and viable counts while cells stored at $4^{\circ}C$ was shown viable but nonculturable state during starvation. With immobilized cells on strontium alginate, the bioluminescence showed higher maintenance than free cells and decreased with count number of nonculturable cells.