An assessment of the metabolic activity of starved and vegetative bacteria using two redox dyes

Walsh, Sarah K; Lappin‐Scott, Hilary M.; Stockdale, H.; Herbert, B. N.

doi:10.1016/0167-7012(95)00046-1

Cited by 26 publications

(15 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the present study, we worked with CTC in biofilm and planktonic cultures under strict anaerobic conditions, and the results obtained were similar to preliminary experiments in aerobic cultures (data not shown). These data suggest that the reduction of CTC by bacteria is independent of the availability of oxygen, thus confirming the results from previous studies showing CTC reduction by a variety of anaerobes during all phases of growth (Bhupathiraju et al, 1999;Fukui & Takii 1989;Walsh et al, 1995). Seemingly, intracellular enzymes (e.g.…”

Section: Discussionsupporting

confidence: 80%

Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation

2008

View full text Add to dashboard Cite

The ability of oral bacteria to enter a non-growing state is believed to be an important mechanism for survival in the starved micro-environments of the oral cavity. In this study, we examined the reactivation of nutrient-deprived cells of two oral bacteria in biofilms, Streptococcus anginosus and Lactobacillus salivarius. Non-growing cells were generated by incubation in 10 mM potassium phosphate buffer for 24 h and the results were compared to those of planktonic cultures. When both types of cells were shifted from a rich, peptone-yeast extract-glucose (PYG) medium to buffer for 24 h, dehydrogenase and esterase activity measured by the fluorescent dyes 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and fluorescein diacetate (FDA), respectively, was absent in both species. However, the membranes of the vast majority of nutrient-deprived cells remained intact as assessed by LIVE/DEAD staining. Metabolic reactivation of the nutrient-deprived biofilm cells was not observed for at least 48 h following addition of fresh PYG medium, whereas the nongrowing planktonic cultures of the same two strains were in rapid growth in less than 2 h. At 72 h, the S. anginosus biofilm cells had recovered 78 % of the dehydrogenase activity and 61 % of the esterase activity and the biomass mm "2 had increased by 30-35 %. With L. salivarius at 72 h, the biofilms had recovered 56 % and 75 % of dehydrogenase and esterase activity, respectively. Reactivation of both species in biofilms was enhanced by removal of glucose from PYG, and S. anginosus cells were particularly responsive to yeast extract (YE) medium. The data suggest that the low reactivity of non-growing biofilm cells to the introduction of fresh nutrients may be a survival strategy employed by micro-organisms in the oral cavity.

show abstract

Section: Discussionsupporting

confidence: 80%

Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation

2008

View full text Add to dashboard Cite

show abstract

“…The assay is based on the ability of metabolically active cells to reduce INT to insoluble red formazan. However, the feasibility of this method to characterize metabolic activity of lactobacilli has so far not been demonstrated, and previous investigators have used the microscope to determine the deposition of formazan crystals in individual cells (43,45). To develop a miniaturized assay for the rapid analysis of a large amount of samples, we evaluated whether the initial rate of formazan production by cell suspensions is a suitable means to estimate their metabolic activity.…”

Section: Resultsmentioning

confidence: 99%

“…The staining of cells with tetrazolium salts was proposed as a rapid method for determination of cell viability (9,43,45). Tetrazolium reduction to formazan by lactobacilli depends on the activity of NADH-dependent enzymes, such as NADH oxidase or peroxidase activities, or specific NADH dehydrogenases that are present in LAB (4,20,24,30,37,39).…”

Section: Discussionmentioning

confidence: 99%

Effects of High Pressure on Survival and Metabolic Activity of Lactobacillus plantarum TMW1.460

Ulmer

Gänzle

Vogel

2000

Appl Environ Microbiol

124

View full text Add to dashboard Cite

The application of high pressure (HP) for food preservation requires insight into mechanisms of HPmediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10 6 cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.

show abstract

“…INT and other tetrazolium salts, i.e., CTC, had been widely used in vivo to decipher the fraction of active cells in a sample, that is, those cells capable of reducing INT or CTC reagent to insoluble formazans (the nonfluorescent INT-F and the fluorescent CTC-F, respectively), providing qualitative data on the presence or absence of activity but failing to estimate rates of activity (Zimmermann et al 1978;Tabor and Neihof 1982;Walsh et al 1995;Griebe et al 1997;Gasol and Arístegui 2007). The careful assessment of the adequate INT concentration, together with the determination of the optimal incubation time, allowed us to obtain reliable microbial respiration rates measurements using the in vivo INT reduction method.…”

Section: Discussionmentioning

confidence: 99%

In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

Martínez‐García

Fernández

Aranguren-Gassis

et al. 2009

Limnology & Ocean Methods

108

View full text Add to dashboard Cite

Oxygen consumption during in vitro dark bottle incubations is the most common method to estimate planktonic respiration. This method is time consuming and labor instensive and, consequently, the database of planktonic respiration rates is scarce. Electron Transport System (ETS) activity measurement has gained acceptance as a routine technique to estimate respiration due to its high sensitivity. However, the in vitro ETS assay commonly used yields potential rates. Hence, the empirically determined ratio between in situ respiration and potential ETS activity is not constant, varying over two orders of magnitude, highly limiting the routine application of this technique to estimate microbial respiration. We hypothesized that in vivo ETS activity should be a reliable estimator of in situ respiration in marine plankton communities. The aim of this study was to develop a methodology to estimate in vivo ETS activity rates by using tetrazolium salt 2‐para (iodo‐phenyl)‐3(nitrophenyl)‐5(phenyl)tetrazolium chloride (INT) as electron acceptor. We established a procedure to apply the in vivo INT reduction method to natural marine microplankton samples. Optimum incubation times were found to be lower than 2–6 h, even for oligotrophic waters. The method was tested in natural waters, covering a very wide range of respiration rates and trophic conditions. A significant linear relationship was found between in situ respiration and in vivo INT reduction. Moreover, the ratio between oxygen consumption and INT reduction was constant across contrasting environments, which reveals the INT reduction method as a simple, quick, sensitive, and robust technique suitable to substantially improve the microbial plankton respiration database.

show abstract

An assessment of the metabolic activity of starved and vegetative bacteria using two redox dyes

Cited by 26 publications

References 18 publications

Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation

Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation

Effects of High Pressure on Survival and Metabolic Activity of Lactobacillus plantarum TMW1.460

In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

Contact Info

Product

Resources

About