A comparison of methods for assessing yeast viability

Summary Eight different methods were used to assess the cell viability of four strains of Saccharomyces. Staining with Mg-ANS, primuline yellow, FITC and methylene blue gave a good index of yeast cell viability. The standard plate count technique and microcolony formation also gave a good measure of cell viability. Fluorescent staining with acridine orange was the least useful of the methods tested. INT dye reduction gave a good index of respiring cells depending upon the yeast strain tested.     

Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli

The effects of substrates of primary aerobic dehydrogenases, and inorganic phosphate on aerobic INT and CTC reduction in Escherichia coli were examined. In general, INT produced less formazan than CTC, but INT (+) cell counts remained near values of CTC (+) cells. INT and CTC (+) cell numbers were higher than plate counts on R2A medium using succinate, formate, lactate, casamino acids, glucose, glycerol (INT only) and no substrate. Formate resulted in the greatest amount of INT and CTC formazan. Reduction of both INT and CTC was inhibited above 10 mmol 1^-1 phosphate, and this appeared to be related to decreased rates of O₂ consumption. Formation of fluorescent CTC (+), but not INT (+) cells was also inhibited in a concentration dependent manner by phosphate above 10 mmol 1^-1. From light microscopic observations it appeared CTC formed increasing amounts of poorly or non-fluorescent formazan with increasing phosphate. Therefore, use of phosphate buffer in excess of 10 mmol 1^-1 may not be appropriate in CTC and INT reduction assays.     

Assessment of bacterial endospore viability with fluorescent dyes

AIM: To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. METHODS AND RESULTS: Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0.02). CONCLUSION: It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.     

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.     

Detection of respiratory enzyme activity in Giardia cysts and Cryptosporidium oocysts using redox dyes and immunofluorescence techniques.

The fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), combined with fluorescein-labeled antibodies, was tested for the simultaneous detection of the respiratory electron transport system (ETS) activity and enumeration of Giardia cysts and Cryptosporidium oocysts by spectral microfluorometry and epifluorescence microscopy. The reduction of CTC and p-iodonitrotetrazolium violet (INT), a non-fluorescent redox dye, was compared with propidium iodide (PI) and fluorescein diacetate (FDA) for the measurements of Giardia cyst viability over time. According to the PI and FDA staining techniques, nearly 60% of the cysts tested viable at the beginning of the observations; after 21 days their viability decreased to 5%. The redox dyes indicated that approximately 4-10% of the cysts were metabolically active 48 h after they were shed, followed by a decline in enzyme activity to near undetectable levels after 4 days. Spectral analysis on individual cysts indicated that the fluorescence emission of the reduced CTC and the fluorescein-labeled antibodies is distinctive for each compound and suitable for their simultaneous determination by microphotometry, flow cytometry and epifluorescence microscopy. The fluorescence signal remained without alteration when the cysts were transferred onto microscope slides coated with an optical embedding medium and stored at -20 degrees C. The fluorescence intensity of the reduced CTC, when properly standardized, can provide quantitative measurements of ETS activity of the cysts. This is the first report of a method to determine enzyme redox activity on intact cysts applicable to water, laboratory and animal samples.     

Analysis of the microbial functional diversity within water-stressed soil communities by flow cytometric analysis and CTC+ cell sorting

Total and active cell counts within soil samples were determined by culture-independent methods using flow cytometry and preparative Nycodenz gradient centrifugation. Whole cells were purified from soil cores and total extractable cell counts assessed by SYBR Green II fluorescence, while active cell counts were determined by 5-cyano-2,3-ditolyl tetrazolium chloride reduction (CTC+ cells). Parallel microcosms, maintained at either field water capacity or subjected to drying, indicated that the total extractable cell count remained between 10(8) and 10(9) g(-1) (dry weight). In contrast, the CTC+ active count fell threefold in dried microcosms (6% of total cell count) when compared to wetted microcosms (18% of total cell count). Specifically, these data highlighted an overall deactivation of microbial biomass during water stress, with 16S rDNA analyses of flow-sorted CTC+ cells demonstrating shifts within the active diversity. Flow cytometry coupled with cell purification techniques represents a significant tool for operationally defining an active and redundant microbial component within soil communities and is demonstrated during water stress.     

Metabolism of Escherichia coli injured by copper

Escherichia coli injured by copper in carbonate buffer simulating the drinking water environment showed decreased oxygen utilization. Oxygraph measurements revealed that copper-injured bacteria had a rate of oxygen utilization that was less than 25% of that of control cells. Respirometry experiments measured rates over a longer period of time and showed similar trends. Nuclear magnetic resonance spectroscopy (13C nmr) and gas chromatography were used to identify differences in metabolism between healthy and injured populations of E. coli. The rate of glucose utilization by injured cells under anaerobic conditions was 64% of that of healthy cells. The rates of lactate and ethanol accumulation were 88 and 50% of the control, respectively. The 13C nmr studies of oxygenated cultures revealed differences in the accumulation of acetate and glutamine. Aerobic utilization of glucose and succinate by injured cells were 87 and 21% of the rate of the controls, respectively. Additional studies revealed injured cells had a decreased ability to reduce 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) with a variety of carbohydrate substrates. Injured cells reduced greater quantities of INT than healthy cells when NADH was used as a substrate. A comparison of metabolic end products suggested that injured cells also had considerable differences in carbon flow compared with healthy cells.     

Use of a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to high-intensity pulsed electric fields

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal in food-borne bacteria exposed to high-intensity pulsed electric fields (PEF). METHODS AND RESULTS: A rapid cellular staining method using the fluorescent redox probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4',6-diamidino-2-phylindole was used for enumerating actively respiring cells of Listeria mononcytogenes, Bacillus cereus and Escherichia coli. This respiratory staining (RS) approach provided good agreement with the conventional plate count agar method for enumerating untreated and high-intensity PEF-treated bacteria suspended in 0.1% (w/v) peptone water. However, test organisms subjected to similar levels of lethality by heating at 56 degrees C resulted in ca 3-log-unit difference in surviving cell numbers ml(-1) when enumerated by these different viability indicators. PEF-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: While PEF-treatment did not produce sublethally injured cells (P < 0.05), substantial subpopulations of test bacteria rendered incapable of forming colonies by heating may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: The fluorescent staining method offers interesting perspectives on assessing established and novel microbial inactivation methods. Use of this approach may also provide a better understanding of the mechanisms involved in microbial inactivation induced by PEF.     

Factors Affecting the Determination of Respiratory Activity on the Basis of Cyanoditolyl Tetrazolium Chloride Reduction with Membrane Filtration

Deficiencies in traditional bacterial enumeration techniques which rely on colony formation have led to the use of total direct counting methods, such as the acridine orange direct count technique for the enumeration of planktonic bacteria. As total direct counts provide no information on the viability or activity of the organisms, demonstration of respiratory activity with the fluorochrome cyanoditolyl tetrazolium chloride (CTC) has been employed. We have modified this technique by performing filtration prior to CTC incubation. Cells captured on a polycarbonate membrane were incubated on absorbent pads saturated with medium containing CTC. Following counterstaining with DAPI (4(prm1),6-diamidino-2-phenylindole) total and respiring cells were enumerated by epifluorescence microscopy. Factors affecting CTC reduction by Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli K-12 were investigated. With K. pneumoniae, nutrient additions to the CTC medium did not increase the number of respiring cells detected. CTC reduction by all three organisms decreased in response to an increase of the pH of the CTC medium above pH 6.5. Increasing phosphate concentrations contributed to this inhibitory effect. CTC-membrane filter counts of K. pneumoniae, S. typhimurium, and E. coli K-12 and of bacteria in well water corresponded closely with plate counts (r = 0.987). The results show that careful attention should be given to the composition of CTC-containing media which are used to enumerate respiring bacteria. With an appropriate medium, reliable enumeration of respiring bacteria can be achieved within a few hours.     

Direct estimate of active bacteria: CTC use and limitations

During the last 10 years, the dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been used to determine the in situ number of "active" bacteria in different ecosystems. A part of this success is due to a simple protocol, which does not require sophisticated equipment. However, it has not been established whether the method determines viable cells, e.g. those capable of growth and cell division, as opposed to cells that are active in the sense of having some detectable metabolic activity. In this study, the number of CTC-positive cells through the growth stages of Escherichia coli was estimated and compared to counts of the total number of bacteria, the culturability (CFU counts) and respiratory activity (CO(2) evolution). There was a good correlation between the number of CTC-positive cells and the CFU count, regardless of the growth phase. However, CTC could still be reduced by a large part of the population during the first hours of stationary phase even if the bacteria were no longer releasing CO(2). Thus, the reduction of CTC is a good estimator for cell viability, rather than cell activity. Additionally, a review of the literature showed that there is presently no standardized protocol for using CTC, which makes difficult at present the comparison of active bacterial numbers in different samples from different sites.     

Evaluation of the redox dye 5-cyano-2,3-tolyl-tetrazolium chloride for activity studies by simultaneous use of microautoradiography and fluorescence in situ hybridization

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.     

The effect of temperature on viability of carbon- and nitrogen-starved Escherichia coli

Escherichia coli was grown in a defined medium at optimum temperature and then transferred to each of five different starvation regimes at 5°C, 20°C, or 37°C, for 1000 hours. Cells were maintained with growth-limiting amounts of carbon or nitrogen, or without either or both nutrients. Bacterial cell viability was assessed by dilution plating, the reduction of 2(p-indophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT), direct viable counts (DVC), and microcolony development. The recoverability of cells on solid medium declined most rapidly, and to the greatest extent in most cases, in cultures maintained at 37°C. Only nitrogen-starved cells maintained at 5°C became completely nonculturable. The reduction of INT consistently indicated higher numbers of viable cells compared to the other methods in all cultures. The viabilities of carbon- and nitrogen-limited cells, assessed by all methods, were similar to one another at each of the temperatures. Viability was lowest at 37°C. Nutrient-downshifted cells also followed a temperature-dependent pattern of survival with viability lowest at 37°C. Morphological differences were noted at different temperatures but were most obvious for nitrogen-starved cells at 37°C, which increased in length. Correspondence to: R.W. Attwell     

Effects of starvation and osmotic stress on viability and heat resistance of Pseudomonas fluorescens AH9

This study addresses the responses to starvation and osmotic stress of Pseudomonas fluorescens isolated from spoiled fish. Culturability and viability of stressed cells were determined. Cells maintaining an active electron transport system were considered to be viable and this activity was assessed by the ability of the cells to reduce the 5-cyano-2,4-ditolyl tetrazolium chloride (CTC) to fluorescent CTC-formazan. Cells starved of carbon maintained high culturability and a high proportion of the cells were capable of reducing CTC during short-time (up to 5 d) experiments. ATP concentrations were lower in carbon-starved than in log-phase cells but the measured levels suggested that metabolic activity was retained. Carbon-starved cells developed an increased heat resistance and prolonged starvation resulted in further protection. Viable, but non-culturable cells were found during heat challenge implying that culture methods underestimate the recovery potential of these cells. Osmotically-stressed Ps. fluorescens maintained a high viability, whereas culturability was rapidly lost. In contrast to starved cells, no protection against a subsequent heat challenge was found in osmotically-stressed (4 or 18 h) cells, but an increased salinity of the heating menstruum alone resulted in elevated heat resistance.     

The modification and evaluation of some cytochemical techniques for the enumeration of metabolically active heterotrophic bacteria in the aquatic environment

Variants of the tetrazolium-reduction, nalidixic acid-inhibition and fluorescein diacetate-hydrolysis techniques for enumeration of metabolically active bacteria were compared, using samples of planktonic, benthic, and epiphytic freshwater bacteria. Results obtained by these methods generally showed statistically significant differences. However, an INT reduction technique, without added substrate, and a slightly modified nalidixic acid procedure gave values which did not differ at the 5% level. The results indicated that from 10 to 40% of total bacteria in the samples examined were metabolically active. These values were up to two orders of magnitude higher than those obtained by conventional plate count techniques.     

Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.     

Circulating tumor cells: highlight on practical implications

Circulating tumor cells (CTCs) are cells of presumed epithelial origin, whose prognostic and predictive value in metastatic cancer patients has recently been demonstrated. To date, the count of CTCs through the CellSearch? system represents a valid approach for monitoring disease status in patients with metastatic colorectal, breast, and prostate cancer; in these cancer types, a rise in the CTC count at any time during treatment predicts a poor outcome. Nevertheless, the clinical utility of monitoring CTC counts remains controversial, and what to do when CTC counts rise during therapy still remains an unanswered question. In this report, we suggest how to integrate CTC counts with their molecular characterization to better translate biologic information obtained on CTCs into daily clinical practice.     

Tetrazolium reduction in Saccharomyces cerevisiae

Summary The tetrazolium salt, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) was used to determine viable respiring cells in batch cultures of Saccharomyces cerevisiae. Respiring cells reduce INT to water insoluble iodonitrotetrazolium formazan (INT-formazan) which is deposited within the respiring cell. The INT-formazan granules can be observed by brightfield microscopy. This allows a rapid quantitative determination of the percentage of respiring cells and total cells within the same microscopic field.In actively growing batch cultures of S. cerevisiae, the respiring cell count was equal to the total cell count for the first 72 h of the growth cycle. After 144 h of incubation only 22.7% of the total cell numbers were actively respiring.     

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.     

A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water.

Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria. Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium. A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter. The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA. Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water. Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E. coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC. Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997). Differences in injury did not appear to affect CTC reduction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.     

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems

BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.