BacLight Bacterial Viability Kit Research Articles

Viable But Nonculturable State of Foodborne Pathogens in Grapefruit Juice: A Study of Laboratory

Several foodborne human pathogens, when exposed to harsh conditions, enter viable but nonculturable (VBNC) state; however, still open is the question whether VBNC pathogens could be a risk for public health, because, potentially, they can resuscitate. Moreover, cultural methods for food safety control were not able to detect VBNC forms of foodborne bacteria. Particularly, it has not been established whether food chemophysical characteristics can induce VBNC state in contaminating pathogen bacterial populations, especially in food, such as salads and fresh fruit juices, not subjected to any decontamination treatment. In this preliminary study, we intentionally contaminated grapefruit juice to determine whether pathogen bacteria could enter VNBC state. In fact, grapefruit juice contains natural antimicrobial compounds, has an average pH of about 3 and low content in carbohydrates. Such characteristics make grapefruit juice a harsh environment for microbial survival. For this purpose, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium ATCC 14028, Listeria monocytogenes ATCC 7644, and Shigella flexneri ATCC 12022, at two different inoculum sizes, have been used. Viability by the LIVE/DEAD BacLight Bacterial Viability kit and culturability by plate counts assay were monitored, whereas "resuscitation" of nonculturable populations was attempted by inoculation in nutrient-rich media. The data showed that L. monocytogenes lost both culturability and viability and did not resuscitate within 24 h independently on inoculum size, whereas E. coli O157:H7 was able to resuscitate after 24 h but did not after 48 h. Salmonella Typhimurium and S. flexneri, depending on inoculum size, lost culturability but maintained viability and were able to resuscitate; moreover, S. flexneri was still able to form colonies after 48 h at high inoculum size. In conclusion, entry into VBNC state differs on the species, depending, in turn, on inoculum size and time of incubation.

Rapid molecular methods for enumeration and taxonomical identification of acetic acid bacteria responsible for submerged vinegar production

The aim of the present study was to search for a rapid and reliable method to enumerate viable acetic acid bacteria (AAB) and to identify to genera and species level AAB isolates from vinegars in full acetic fermentation elaborated by the submerged method from cider, wine and spirit ethanol in industrial bioreactors. Results showed that the rapid epifluorescence staining method using the LIVE/DEAD BacLight bacterial viability kit and direct counts in Neubauer chamber rendered consistent and reliable data for viable cell counts of bacteria in all the studied vinegars. A linear correlation was shown between viable cell counts and fermentation rates. The highest fermentation rates and viable cell counts were found in cider vinegars, whereas spirit vinegars showed the lowest values for both parameters. Eighty-four AAB pure isolates were recovered from 41 different vinegar samples and were submitted to DNA extraction. PCR amplification of the 16S–23S intergenic spacer region of rDNA and subsequent sequencing were carried out to identify isolates to species level. Results showed that Gluconacetobacter europaeus was the predominant cultivable species, appearing in 79% of the total isolates. This was the unique species found in spirit vinegars, and this is the first time that AAB from spirit vinegars are taxonomically identified. Ga. europaeus was as well the predominant cultivable species in white wine vinegars. Cider vinegars presented the highest variability of species: Ga. europaeus (35.3% appearance among cultivable isolates), Ga. xylinus (35.3%), Acetobacter pasteurianus (17.6%) and Ga. hansenii (11.8%). Red wine vinegars showed cultivable isolates of the species Ga. xylinus (71.4%) and Ga. europaeus (28.6%). Summarising, both described methods for AAB enumeration and taxonomical identification proved to be fast and reliable methods, and results revealed Ga. europaeus as the cultivable major species in vinegars in full fermentation conducted by the submerged method, suggesting that Ga. europaeus strains can constitute excellent starter cultures for the elaboration of vinegars by the submerged method.

A Novel Antibacterial Silicone Implant Material with Short- and Long-Term Release of Copper Ions

Sir: Capsular contracture after silicone implants is still an unresolved and serious complication. The initial adhesion of specific skin bacteria to the implant surface is believed to be the major cause of early or late infection and may also be associated with capsular contracture.1 Studies of patients with breast implants have shown that many implant pockets are germ culture-positive. The predominant isolate germs are from the local skin flora (e.g., coagulase-negative staphylococci, Propionibacterium acnes, and Corynebacterium species). Preventing implant colonization by microorganisms and formation of bacterial biofilm could be effective in improving the outcome.1,2 Various investigations with different strategies with administration of systemic antibiotics and antiseptic irrigation of the implant pocket have been performed to reduce or prevent the bacterial contamination of silicone implants.2 An alternative approach consists of implant surface modification by antimicrobial agents. With all different methods, it is fundamental not to enforce antibacterial properties at the expense of biocompatibility. The toxic effect of metal ions on either tissue cells or bacteria is well known and has been reported in multiple studies.3 Silver (Ag+) has been widely used as an antimicrobial agent in different fields of medicine. The strong antibacterial effect of Ag+ is mainly enforced at the expense of cytocompatibility.3 Heidenau et al. demonstrated in growth inhibition tests with tissue cells and bacteria that, among Al3+, Co2+, Zn2+, Hg2+, Ag+, and Cu2+, the copper ions had by far the best compromise between antibacterial effectiveness and cytotoxicity.3 Consequently, we modified the surface of silicone sheets from implants with covalent linking of 20 chitosan layers dipped in a copper solution for 1 hour (Fig. 1). Chitosan is a linear biodegradable polysaccharide with high biocompatibility. The advantage of our modification technique with chitosan layers and copper ions is that it achieves a combination of short-term and long-term release of copper ions.4Fig. 1.: Scanning electron microscopic image of a silicone surface with chitosan coating and copper additives.For quantification of bacterial adherence and to determine the proportion of live and active bacteria and dead and inactive bacteria, the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Eugene, Ore.) was used, which provides a two-color fluorescence assay of bacterial viability.5 The silicone specimens were incubated in a Staphylococcus epidermidis suspension isolated from the human skin for 120 minutes at 4°C. Subsequently, the bacterial suspension was extracted and the silicone sheets were washed with saline to remove nonadhering bacteria. The live or dead stain was prepared in recommended proportions, so that green fluorescent staining was achieved of bacteria with intact cell membranes and red fluorescent staining was achieved of bacteria with damaged membranes. Consequently, the ratio of green to red fluorescence intensities was measured as a quantitative index of bacterial viability. In our in vitro tests, we found a significantly lower amount of adhering bacteria and a higher percentage of dead S. epidermidis on our silicone sheets with copper additive than on our pure silicone sheets (Fig. 2). Despite these preliminary results, an animal study would still be needed to confirm the antibacterial effect of our implants in vivo. Possibly we can also reduce the development and progress of capsular fibrosis.Fig. 2.: Fluorescence microscopic images by live and dead staining. Vital (green) and dead (red) staphylococci on (left) pure silicone material and (right) silicone material with copper additives (original magnification, × 200). Display window, 150 × 188 μm.Lukas Prantl, M.D., Ph.D Ralf Bürgers, D.D.S., Ph.D. Stephan Schreml, M.D. Johannes Zellner, M.D. Department of Plastic and Reconstructive Surgery Martin Gosau, M.D., D.M.D. Department of Craniomaxillofacial Surgery University Medical Center Regensburg Regensburg, Germany DISCLOSURE Dr. Prantl is one of the inventors of the new implant material and is one of the applicants for a patent. Silicone was supplied by the Mentor Corporation (Irving, Texas). ACKNOWLEDGMENTS The authors thank the Friedrich-Baur-Research Institute for Biomaterials, University of Bayreuth, Germany, for the excellent work to modify silicone sheets with copper ions for antibacterial effect. They also thank Mentor Corporation (Irving, Texas) for their generous supply of silicone material.

Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real‐time quantitative PCR

To optimize ethidium monoazide (EMA) coupled with real-time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems. EMA (0.9-45.5 microg ml(-1)) and propidium monoazide (PMA, 0.9 and 2.3 microg ml(-1)) combined with qPCR (i.e. EMA-qPCR and PMA-qPCR, respectively) were applied to unheated and heated (70 degrees C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight-EM). The effects of nontarget microflora and sample matrix on the performance of EMA-qPCR were also evaluated. In comparison with BacLight-EM results, qPCR with EMA at 2.3 microg ml(-1) was determined as the optimal EMA-qPCR assay, which performed equally well as PMA-qPCR for unheated Leg. pneumophila but better than PMA-qPCR for heated Leg. pneumophila (P < 0.05). Moreover, qPCR with EMA at 2.3 microg ml(-1) accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella-like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0.05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. The qPCR with EMA at 2.3 microg ml(-1) may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems. The EMA-qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.

Determination of Pseudomonas Putida Live Cells with Classic Cultivation and Staining with “Live/Dead Baclight Bacterial Viability Kit”

ABSTRACTPeriodic culture of Pseudomonas putida strain ATCC 12633=NBPMCC1090 was investigated in a 24 h experiment for sensitivity to zinc oxide thin films constructed under different conditions. The growth of bacteria was followed at every three hours and three methods were used to determine live cells. Optical density, cultivation and microscopic methods were applied and compared. To distinguish active and inactive cells LIVE/DEAD BacLight Bacterial Viability Kit staining was compared with classical cultivation methods. Bacterial quantity determinated on nutrient media, appear higher than those of live cells counted on epifluorescent microscope.

Gamma Radiation Used as Hygienization Technique for Foods Does Not Induce Viable but Non-Culturable State (VBNC) in Salmonella enterica subsp. enterica serovar Typhimurium

Gamma radiation has been widely used for hygienization of food products. Whether gamma radiation stress induces VBNC state in Salmonella is of great concern. Therefore, the study was carried out to determine whether gamma radiation exposure induces VBNC state in Salmonella enterica subsp. enterica serovar Typhimurium (S. typhimurium). The parameters tested were culturability on agar medium, transcriptional activity by RT-PCR, cytoplasmic membrane integrity, and direct viable count using LIVE/DEAD BacLight bacterial viability kit. The LIVE/DEAD BacLight counts for S. typhimurium cells treated with 0.5 and 1.0 kGy radiation dose were 0.8 and 0.1% of the control, respectively. Plate counts for S. typhimurium cells treated with 0.5 and 1.0 kGy radiation dose were 0.7 and 0.05% of the control, respectively. No viable cells of S. typhimurium were detected by both plate count and LIVE/DEAD BacLight after radiation treatment with 2 kGy. No transcriptional activity was detected in cells treated with 2 kGy radiation dose. If there were VBNC cells present, then significant differences in the counts between the LIVE/DEAD BacLight microscopic counts and plate agar counts must be observed. No significant difference (P > 0.05) in the counts were observed. Thus, it can be concluded that treatment with 2 kGy results in complete killing and does not induce VBNC state in S. typhimurium.

COMPARATIVE STUDY OF SHELL SWAB AND SHELL CRUSH METHODS FOR THE RECOVERY OF SALMONELLA FROM SHELL EGGS

ABSTRACT Swabbing is the standard methodology for the recovery of resident microorganism from shell eggs in Japan. A comparative study of shell swab (SW) and shell crush (CR) techniques was performed to recover the laboratory‐inoculated Salmonella from shell eggs. It was found that the recovery of Salmonella by CR methods was significantly higher (4.5–7.5 log cfu/egg) than that of SW methods (3.1–6.3 log cfu/egg). However, analyses with quantitative real‐time polymerase chain reaction (invA as a target gene), fluorescent microscopic and quantitative analyses with a Live/Dead BacLight bacterial viability kit revealed that not all of the inoculated Salmonella spp. populations were recovered as intact cells by either method. The chemiluminescent bacterial viability assay showed that chemiluminescence intensity (CI) began to increase after 30 min in CR samples; on the other hand, SW samples did not show any increase in CI for 2 h. These results suggest that SW might cause more damage and lethality to cells than CR. In addition, to determine the most appropriate method for recovering resident aerobic bacteria, coliforms and Salmonella spp from shell eggs, 4,000 commercial eggs were collected and sampled by shell rinse (SR) and CR techniques using phosphate‐buffered saline (PBS) warmed to different temperatures. PBS at 37C was found to be the best recovery solution and temperature, respectively, for recovering aerobic microorganisms from shell eggs by both methods and the CR methods recovered a higher population than did the SR methods (4.9 versus 5.8 log cfu/egg for SR and CR methods, respectively; n = 500 eggs/method). Therefore, the CR method along with recovery buffer (PBS) at 37C could be an effective technique for the recovery of microorganisms from post‐processed shell eggs. PRACTICAL APPLICATIONSThere is a need to develop a rapid and highly sensitive method for the recovery of microorganisms from shell eggs. Such recovery methods are also useful for evaluating the efficacy of newly developed shell egg disinfection techniques. Many methods involving rinsing, swabbing, and crushing of shell eggs have been reported; however, we performed a comparative study of the method used to recover the Salmonella from shell eggs. We found that the shell crush method (CR) was superior to the shell swab method (SW) for the recovery of Salmonella spp., and phosphate‐buffered saline (PBS) at 37C was found to be the best recovery solution and temperature, respectively, for recovering microorganisms from shell eggs by both methods. Therefore, the CR method along with recovery buffer (PBS) at 37C could be an effective technique for the recovery of microorganisms from post‐processed shells. Use of this method could be recommended for the microbial evaluation of post‐processed shell eggs in industries.

Long-Term Survival of Legionella pneumophila in the Viable But Nonculturable State After Monochloramine Treatment

Legionella pneumophila, a facultative intracellular human pathogen, can persist for long periods in natural and artificial aquatic environments. Eradication of this bacterium from plumbing systems is often difficult. We tested L. pneumophila survival after monochloramine treatment. Survival was monitored using the BacLight Bacterial Viability Kit (Molecular Probes), ChemChrome V6 Kit (Chemunex), quantitative polymerase chain reaction and culturability on buffered charcoal-yeast extract agar. In nonculturable samples, regain of culturability was obtained after addition of the amoeba Acanthamoeba castellanii, and esterase activity and membrane integrity were observed after >4 months after treatment. These results demonstrate for the first time that L. pneumophila could persist for long periods in biofilms into the viable but nonculturable (VBNC) state. Monitoring L. pneumophila in water networks is generally done by enumeration on standard solid medium. This method does not take into account VBNC bacteria. VBNC L. pneumophila could persist for long periods and should be resuscitated by amoeba. These cells constitute potential sources of contamination and should be taken into account in monitoring water networks.

Efficacy of common hospital biocides with biofilms of multi-drug resistant clinical isolates

The hospital environment is particularly susceptible to contamination by bacterial pathogens that grow on surfaces in biofilms. The effects of hospital biocides on two nosocomial pathogens, meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, growing as free-floating (planktonic) and adherent biofilm populations (sessile) were examined. Clinical isolates of MRSA and P. aeruginosa were grown as biofilms on discs of materials found in the hospital environment (stainless steel, glass, polyethylene and Teflon) and treated with three commonly used hospital biocides containing benzalkonium chloride (1 % w/v), chlorhexidine gluconate (4 % w/v) and triclosan (1 % w/v). Cell viability following biocide treatment was determined using an XTT assay and the LIVE/DEAD BacLight Bacterial Viability kit. The minimum bactericidal concentration (MBC) of all biocides for planktonic populations of both organisms was considerably less than the concentration recommended for use by the manufacturer. However, when isolates were grown as biofilms, the biocides were ineffective at killing bacteria at the concentrations recommended for use. Following biocide treatment, 0-11 % of cells in MRSA biofilms survived, and up to 80 % of cells in P. aeruginosa biofilms survived. This study suggests that although biocides may be effective against planktonic populations of bacteria, some biocides currently used in hospitals are ineffective against nosocomial pathogens growing as biofilms attached to surfaces and fail to control this reservoir for hospital-acquired infection.

Whey Starter for Grana Padano Cheese: Effect of Technological Parameters on Viability and Composition of the Microbial Community

This work aimed to investigate the effects of thermal treatments and yeast extract addition on the composition of the microbial community of natural whey starters for Grana Padano cheese. Different natural whey starter samples were held at 4°C for 24h (cooling treatment), or at −20°C for 24h (freezing treatment) to evaluate the possibility of conservation, or at 54°C for 1h (heat treatment) to evaluate the effect of the temperature commonly used during curd cooking. Separately, another set of samples was enriched with 0.3, 0.5, and 1.0% (wt/vol) of yeast extract to study its effect on the growth of lactic acid bacteria (LAB) in the starter. The new approach in this study is the use of 2 culture-independent methods: length heterogeneity (LH)-reverse transcription (RT)-PCR and fluorescence microscopy. These techniques allowed us to easily, quickly, and reproducibly assess metabolically active LAB in the control and treated samples. The LH-RT-PCR technique distinguished microorganisms based on natural variations in the length of 16S rRNA amplified by RT-PCR, as analyzed by using an automatic gene sequencer. Fluorescence microscopy counts were performed by using a Live/Dead BacLight bacterial viability kit. The repeatability of LH-RT-PCR showed that this technique has great potential to reveal changes in the microbial community of natural whey starters for Grana Padano cheese. All species showed low sensitivity to cold (4°C). However, after the freezing (−20°C) and heating (54°C) treatments, different behaviors of the species were reported, with significant changes in their viability and relative composition. Heating treatment during curd cooking profoundly affected the viability and composition of the community that remained in the cheese and that consequently modified the microbial population. At the same time, this treatment produced the selection of LAB in whey and could be considered as the first step in natural whey starter production. Addition of yeast extract stimulated the growth of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. lactis to the detriment of Lactobacillus helveticus species. Because the yeast extract altered the microflora balance, whey starter conservation at −20°C and yeast extract addition cannot be suggested as technological innovations.

In Vitro Assessment of the Gastrointestinal Transit Tolerance of Taxonomic Reference Strains from Human Origin and Probiotic Product Isolates of Bifidobacterium

Next to health promoting effects, the functional aspect of probiotic strains also involves their capacity to reach the colon as viable metabolically active cells. The present study aimed to assess the potential of 24 probiotic product isolates and 42 human reference strains of Bifidobacterium to survive gastrointestinal transit under in vitro conditions. The survival capacity of exponential and stationary phase cultures upon exposure to gastric and small intestinal juices was determined using a recently developed microplate-based assay in combination with the LIVE/DEAD BacLight Bacterial Viability kit. All 66 strains tested displayed a considerable loss in viability during exposure to an acidic pepsin containing solution (pH 2.0). Among the 10 taxa tested, cultures of B. animalis ssp. lactis appeared to be most capable to survive gastric transit. Although to a lesser extent, the presence of bile salts also affected the viability of most of the strains tested. Except for 3 strains, all 66 strains showed bile salt hydrolase activity using an agar-based assay. In contrast, the bifidobacterial strains used in this study appeared to possess a natural ability to survive the presence of pancreatin (pH 8.0). Although the effect was not significant, a slightly enhanced tolerance to gastrointestinal transit was observed when cells were in the stationary phase, especially when exposed to acid, compared with cells being in the exponential phase. Survival in the gastrointestinal tract appeared to be largely strain-dependent and hence implies that different strains will likely display a different behavior in functionality. The assay used in this study allows an initial assessment of strains for use as probiotic cultures prior to selecting potential candidate strains for further investigation in vivo.

Lichens Survive in Space: Results from the 2005 LICHENS Experiment

This experiment was aimed at establishing, for the first time, the survival capability of lichens exposed to space conditions. In particular, the damaging effect of various wavelengths of extraterrestrial solar UV radiation was studied. The lichens used were the bipolar species Rhizocarpon geographicum and Xanthoria elegans, which were collected above 2000 m in the mountains of central Spain and as endolithic communities inhabiting granites in the Antarctic Dry Valleys. Lichens were exposed to space in the BIOPAN-5 facility of the European Space Agency; BIOPAN-5 is located on the outer shell of the Earth-orbiting FOTON-M2 Russian satellite. The lichen samples were launched from Baikonur by a Soyuz rocket on May 31, 2005, and were returned to Earth after 16 days in space, at which time they were tested for survival. Chlorophyll fluorescence was used for the measurement of photosynthetic parameters. Scanning electron microscopy in back-scattered mode, low temperature scanning electron microscopy, and transmission electron microscopy were used to study the organization and composition of both symbionts. Confocal laser scanning microscopy, in combination with the use of specific fluorescent probes, allowed for the assessment of the physiological state of the cells. All exposed lichens, regardless of the optical filters used, showed nearly the same photosynthetic activity after the flight as measured before the flight. Likewise, the multimicroscopy approach revealed no detectable ultrastructural changes in most of the algal and fungal cells of the lichen thalli, though a greater proportion of cells in the flight samples had compromised membranes, as revealed by the LIVE/DEAD BacLight Bacterial Viability Kit. These findings indicate that most lichenized fungal and algal cells can survive in space after full exposure to massive UV and cosmic radiation, conditions proven to be lethal to bacteria and other microorganisms. The lichen upper cortex seems to provide adequate protection against solar radiation. Moreover, after extreme dehydration induced by high vacuum, the lichens proved to be able to recover, in full, their metabolic activity within 24 hours.

Response to alkaline stress by root canal bacteria in biofilms

To determine whether bacteria isolated from infected root canals survive alkaline shifts better in biofilms than in planktonic cultures. Clinical isolates of Enterococcus faecalis, Lactobacillus paracasei, Olsenella uli, Streptococcus anginosus, S. gordonii, S. oralis and Fusobacterium nucleatum in biofilm and planktonic cultures were stressed at pH 10.5 for 4 h, and cell viability determined using the fluorescent staining LIVE/DEAD BacLight bacterial viability kit. In addition, proteins released into extracellular culture fluids were identified by Western blotting. Enterococcus faecalis, L. paracasei, O. uli and S. gordonii survived in high numbers in both planktonic cultures and in biofilms after alkaline challenge. S. anginosus, S. oralis and F. nucleatum showed increased viability in biofilms compared with planktonic cultures. Alkaline exposure caused all planktonic cultures to aggregate into clusters and resulted in a greater extrusion of cellular proteins compared with cells in biofilms. Increased levels of DnaK, HPr and fructose-1,6-bisphosphate aldolase were observed in culture fluids, especially amongst streptococci. In general, bacteria isolated from infected roots canals resisted alkaline stress better in biofilms than in planktonic cultures, however, planktonic cells appeared to use aggregation and the extracellular transport of specific proteins as survival mechanisms.

Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains

To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed.

Cooperative biodegradation of geosmin by a consortium comprising three gram-negative bacteria isolated from the biofilm of a sand filter column

To isolate and identify bacteria from a sand filter column capable of degrading the taste and odour compound, geosmin. In doing so, to investigate if these organisms degrade geosmin either individually or if an alternative mechanism is utilized. Geosmin-degrading bacteria from a biologically active sand filter column were enriched by their growth in a minimal medium supplemented with geosmin as the sole carbon source. By day 51, 21.7 mg l(-1) of geosmin had been degraded as determined by solid-phase microextraction gas chromatography/mass spectrometry, and was accompanied by a 2.12 log(10) increase in active bacterial numbers as measured using the BacLight(TM) bacterial viability kit and flow cytometric enumeration. During the onset of geosmin degradation, the predominance of three bacteria, most similar to previously cultured species of Sphingopyxis alaskensis, Novosphingobium stygiae and Pseudomonas veronii based on 16S rRNA gene sequences was detected by denaturing gradient gel electrophoresis. Subsequent isolation of these organisms revealed that degradation of geosmin, when present as either the sole carbon source (ranging from 40 ng l(-1) to 20 mg l(-1)) or when spiked into sterile reservoir water (37 and 131 ng l(-1)), occurred only when all three isolates were present. None of the isolates was shown to be capable of degrading geosmin either individually or in any combination of two. This study has reported, for the first time, the cooperative degradation of geosmin by a consortium comprising three gram-negative bacteria isolated from a biologically active sand filter column. These results are important for researchers currently employing molecular-based approaches to further understand the biodegradation of geosmin by bacteria, as such studies may be complicated by the discovery of geosmin degradation occurring by a consortium. This study also advances the knowledge surrounding the types of bacteria capable of degrading the taste and odour compound, as investigations to date regarding this are limited.

Analysis of aggregative behavior of Pseudomonas sp. 30-3 isolated from Antarctic soil

Pseudomonas sp. 30-3, a toluene degrading microorganism isolated from oil-contaminated Antarctic soils, was shown to form aggregated flocs of cells when exposed to temperatures of 22 and 4 °C, with an increase in aggregation at 4 °C. This was speculated to be due to the secretion of an extracellular polymeric substance (EPS), thus protecting the organism from cold or frost damage. The flocs of cells were stained with the Live/Dead BacLight Bacterial Viability kit and found to be viable cells. The EPS was identified by lectin binding analysis to consist of N-acetyl- d-glucosamine and N-acetylneuraminic acid. An enzyme-linked lectinosorbent assay was also carried out to quantify the amount of EPS produced at 37, 22 and 4 °C. Results showed that at 37 °C the amount of EPS secreted was low, but there was little difference in the amount of EPS secreted at 22 and 4 °C by Pseudomonas sp. 30-3.

Fluorescence microscopy for studying the viability of micro-organisms in natural whey starters

The aim of this work was to study the viability and cultivability of microbial populations of different natural whey starters and to evaluate their resistance to thermal treatments (such as exposure to high or low temperatures). Twenty-three natural whey starters for Grana Padano cheese were investigated and subsequently pH measurement, plate count agar using Man-Rogasa-Sharpe (MRS) pH 5.4 agar and whey agar medium (WAM) were performed using these samples. LIVE/DEAD BacLight bacterial viability kit was used. Total count and viability of all the 23 samples were high and similar to each other (CV 20%). However, the cultivable population was lower in terms of cfu ml(-1) and number of cells per millilitre than the viable fraction and highly variable, although its count value was higher in WAM than in MRS pH 5.4. The heating (60 degrees C for 5 min and 54 degrees C for 1 h) and freezing (-20 and -80 degrees C) treatments affected the cultivability and viability of the microbial population. This study demonstrated the effectiveness of LIVE/DEAD BacLight bacterial viability kit, which has already been used to evaluate bacterial populations, in investigating microbial viability in a complex ecosystem such as a natural whey starter. The aim of this study was to quantify the presence of damaged nonviable bacterial cells in natural whey starters. The Thoma Glass is a useful method to obtain fluorescence microscopy counts to evaluate the technological performance of natural whey starters.

New methodology for viability testing in environmental samples

Environmental samples can be complex and are comprised of microorganisms and a matrix of decaying organic matter as well as an inorganic phase such as sand or precipitated material (waste water, sludge, soils, etc.). Nucleic acid dyes have recently been developed to address the growing need for environmental analyses (cell staining, counting, viability testing and specific organism identification). However, certain dyes may not be ideally suited for testing of environmental samples, because they readily adhere to the substrate material as well as their target molecule, resulting in increased non-specific binding and background fluorescence. The aim of this study was to address the limitations of the widely used and commercially available Live/Dead BacLight Bacterial Viability kit (Molecular Probes, Eugene, OR). A new combination of nucleic acid dyes, i.e. SYTO13 and SYTOX Orange (Molecular Probes, Eugene, OR), was proposed as an alternative. The dyes were carefully chosen for their spectral separation and increase of fluorescence quantum yield. A protocol for this combination was first designed and optimized and the two staining assays were compared against suspensions of live and dead E. coli, mixed in different proportions and it was shown that both protocols performed equally on pure cultures. However, when testing activated sludge samples, the commercial kit showed greater background fluorescence and non-specific binding than the alternate combination. Therefore, the proposed dye combination and its corresponding protocol are deemed more suitable for use on complex environmental samples than the Live/Dead BacLight Bacterial Viability kit.

Rapid method for total, viable and non-viable acetic acid bacteria determination during acetification process

A rapid epifluorescence staining method using the LIVE/DEAD ® BacLight Bacterial Viability Kit ( BacLight™) and direct counts in Neubauer chamber were applied to estimate both viable and total counts of bacteria in different stages of vinegar making. BacLight kit is composed of a mixture of two nucleic acid binding stains: SYTO 9™ and propidium iodide. These stains differ both in their spectral characteristics and in their ability to penetrate viable bacterial cells. SYTO 9 penetrates all bacterial membranes and stains the cells green, while propidium iodide only penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal dilution and incubation conditions were found to be 1:5 and 15 min at room temperature in dark respectively. Total (red plus green) and viable (green) cells can be obtained in one staining step and hence counted simultaneously. Results obtained with this technique were compared with those from other measurement techniques (colony counts and flow cytometry).

Bacterial Abundance, Activity, and Viability in the Eutrophic River Warnow, Northeast Germany

The River Warnow is the drinking water source for the city of Rostock. Its eutrophic status is accompanied by high amounts of bacteria, which may reach up to 24 x 10(6) cells mL(-1) as recorded during a seasonal study in 2002. Because the river is eutrophic and also heavily loaded with organic matter, this burden is a problem for drinking water purification, as it must be removed completely to not trigger new bacterial growth in the pipeline network. Therefore, restoration measures in the river have to be planned, and bacteria have to be favored as decomposers. That includes the investigation of the physiological state of bacteria in situ. Viable and active cells in the lower reaches of River Warnow were estimated using a broad set of methods. Intact bacteria were investigated by the LIVE/DEAD BacLight bacterial viability kit, containing a mixture of permeant and impermeant nucleic acid stains. Cells with ribosomes were visualized by fluorescence in situ hybridization with the EUB338 oligonucleotide probe. Intact cells and ribosome-containing bacteria represented 24% of total numbers stained by 4'6,-diamidino-2-phenylindole (DAPI) or 66 and 62%, respectively, in relation to all bacteria visualized by the LIVE/DEAD kit. Both fractions were considered as viable, although the fraction of RIB + bacteria is most likely underestimated by the protocol applied. 5-Cyano-2,3-ditolyltetrazolium chloride (CTC) was applied to mark respiring bacteria. The esterase substrate CellTracker Green 5-chloromethylfluorescein diacetate showed cells with intracellular hydrolytic activity. Whereas 1.5% of DAPI-stained bacteria were observed as respiring, 3.8% exhibited intracellular hydrolytic activity on average. If these active fractions were calculated as the percentages of intact cells, much higher fractions of 5.4% were respiring and 16% hydrolytic. Temperature was a main factor influencing total and viable cell numbers simultaneously. The results confirm that there are different states of viable and active cells in natural bacterioplankton communities. However, it remains unclear why fractions of viable and active cells were rather low in this eutrophic river in comparison to similar waters. We recommend to carefully address cells as viable in contrast to nonviable, i.e., dead. As viable cells may be active or inactive with respect to many different activities, e.g., substrate uptake, respiration, hydrolysis, and cell deviation, it is necessary to choose the method to visualize active cells according to the question to be answered.