The optimization and application of two direct viable count methods for bacteria in distributed drinking water

The optimal incubation conditions for the direct viable count method with nalidixic acid were determined. They do not differ from those proposed in the literature for a laboratory strain and a mixed bacterial population isolated from drinking water. The direct viable count method with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was performed under in situ conditions. The bacteria were incubated with CTC at a concentration of 1 mM for 4–6 h at the temperature of the water in the pipes and without the addition of an exogenous substrate. The results obtained for a laboratory strain using the two direct count methods were similar. However, for a mixed bacterial population, the counts were always higher with the CTC method than with the nalidixic acid method.Key words: drinking water, CTC, nalidixic acid, direct viable count.

Download Full-text

Inactivation of indigenous bacteria in water by ozone and chlorine

Water Science & Technology ◽

10.2166/wst.1997.0714 ◽

1997 ◽

Vol 35 (11-12) ◽

pp. 81-86 ◽

Cited By ~ 8

Author(s):

J. C. Joret ◽

V. Mennecart ◽

C. Robert ◽

B. Compagnon ◽

P. Cervantes

Keyword(s):

Drinking Water ◽

Distribution System ◽

Treatment Plant ◽

Drinking Water Treatment ◽

Culturable Bacteria ◽

Tetrazolium Chloride ◽

Indigenous Bacteria ◽

Culture Techniques ◽

Active Bacteria

The aim of this study was to compare the level of removal and inactivation of indigenous bacteria during drinking water production as evaluated by culture techniques and epifluorescent microscopic counts of metabolically active bacteria (in situ respiring bacteria i.e. able to metabolise CTC: cyano 2,3-ditolyl tetrazolium chloride). Two sets of experiments were designed: a) bacterial counts through a full scale drinking water treatment plant (multibarrier treatment including coagulation-flocculation-settling, sand filtration, ozonation, biological GAC filtration, post-chlorination) and distribution system; b) benchscale disinfection studies in order to re-evaluate the C.t values necessary to inactivate laboratory grown E. coli or indigenous bacteria from water by ozone and chlorine. Main conclusions of this study are: a) significant amounts of in situ respiring bacteria (undetected by the classical culture techniques) are detected in finished water; b) the efficiency of ozone and chlorine recorded by microscopic counts of active bacteria is much less than supposed by classical enumerations of culturable bacteria; c) previous results reported in the literature may have largely overestimated the bactericidal efficiency of disinfectants used for producing drinking water.

Download Full-text

Comparing electrochemical disinfection with chlorination for inactivation of bacterial spores in drinking water

Water Science & Technology Water Supply ◽

10.2166/ws.2013.182 ◽

2013 ◽

Vol 14 (1) ◽

pp. 158-164 ◽

Cited By ~ 6

Author(s):

L. Mezule ◽

M. Reimanis ◽

V. Krumplevska ◽

J. Ozolins ◽

T. Juhna

Keyword(s):

Drinking Water ◽

Large Scale ◽

Viable Count ◽

Chloride Ions ◽

Water Disinfection ◽

Active Chlorine ◽

Cell Potential ◽

Electrochemical Disinfection ◽

Ct Value

Drinking water disinfection techniques without the dosage of chemicals are regarded as more advantageous in terms of costs and practical use. Here we investigated the efficacy of electrochemical disinfection for inactivation of Bacillus subtilis spores – a model microorganism of highly resistant pathogens. The effect of electrochemical disinfection with TinO2n−1 ceramic electrodes which generate active chlorine from chloride in situ, was compared to the traditional chlorination in which active chlorine was produced from addition of sodium hypochlorite. Research was performed on a batch scale with a synthetic buffered drinking water containing 35.5 mg/l of chloride ions. Spore viability was analysed with both cultivation and cell potential for dividing (direct viable count method). The results showed that at similar residual disinfectant concentrations x contact time (CT value), electrochemical disinfection was over three times more effective in neutralizing both cultivable B. subtilis spores and those with cell potential for dividing than traditional chlorination. As in chlorination, electrochemical disinfection was shown to be water-pH dependent and the lowest CT value of 112 mg/l min−1 (2-log removal) was obtained at pH 6. The lowest efficiency for both techniques was observed at pH 8. In conclusion, electrochemical disinfection is a viable in situ method even at low levels of chlorides in drinking water and appears to be more effective than simple chlorination with the addition of the active chlorine species when highly resistant microbial forms are analysed, however, to apply the technology on a large scale additional studies on potential formation of disinfection by-products must be performed.

Download Full-text

Effects of Starvation on Physiological Activity and Chlorine Disinfection Resistance in Escherichia coliO157:H7

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4658-4662.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4658-4662 ◽

Cited By ~ 89

Author(s):

John T. Lisle ◽

Susan C. Broadaway ◽

Annette M. Prescott ◽

Barry H. Pyle ◽

Colin Fricker ◽

...

Keyword(s):

Respiratory Activity ◽

Physiological Activity ◽

Membrane Integrity ◽

Viable Count ◽

Starvation Period ◽

Tetrazolium Chloride ◽

E Coli ◽

Chlorine Disinfection ◽

Significant Difference

ABSTRACT Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEADBacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.

Download Full-text

Bacterial Activity at −2 to −20°C in Arctic Wintertime Sea Ice

Applied and Environmental Microbiology ◽

10.1128/aem.70.1.550-557.2004 ◽

2004 ◽

Vol 70 (1) ◽

pp. 550-557 ◽

Cited By ~ 277

Author(s):

Karen Junge ◽

Hajo Eicken ◽

Jody W. Deming

Keyword(s):

Sea Ice ◽

Bacterial Abundance ◽

Ice Cores ◽

Bacterial Activity ◽

Dapi Staining ◽

Tetrazolium Chloride ◽

Subzero Temperatures ◽

In Situ Conditions ◽

Active Bacteria

ABSTRACT Arctic wintertime sea-ice cores, characterized by a temperature gradient of −2 to −20°C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4′,6′-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O2-based respiration), the abundances of total, particle-associated (>3-μm), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (−2 to −20°C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (−20°C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to −20°C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.

Download Full-text

Rock Surface Fungi in Deep Continental Biosphere—Exploration of Microbial Community Formation with Subsurface In Situ Biofilm Trap

Microorganisms ◽

10.3390/microorganisms9010064 ◽

2020 ◽

Vol 9 (1) ◽

pp. 64

Author(s):

Maija Nuppunen-Puputti ◽

Riikka Kietäväinen ◽

Lotta Purkamo ◽

Pauliina Rajala ◽

Merja Itävaara ◽

...

Keyword(s):

Significant Proportion ◽

Debaryomyces Hansenii ◽

Fungal Communities ◽

Fungal Colonization ◽

Rock Surface ◽

Mica Schist ◽

Deep Subsurface ◽

Bacterial Phyla ◽

In Situ Conditions

Fungi have an important role in nutrient cycling in most ecosystems on Earth, yet their ecology and functionality in deep continental subsurface remain unknown. Here, we report the first observations of active fungal colonization of mica schist in the deep continental biosphere and the ability of deep subsurface fungi to attach to rock surfaces under in situ conditions in groundwater at 500 and 967 m depth in Precambrian bedrock. We present an in situ subsurface biofilm trap, designed to reveal sessile microbial communities on rock surface in deep continental groundwater, using Outokumpu Deep Drill Hole, in eastern Finland, as a test site. The observed fungal phyla in Outokumpu subsurface were Basidiomycota, Ascomycota, and Mortierellomycota. In addition, significant proportion of the community represented unclassified Fungi. Sessile fungal communities on mica schist surfaces differed from the planktic fungal communities. The main bacterial phyla were Firmicutes, Proteobacteria, and Actinobacteriota. Biofilm formation on rock surfaces is a slow process and our results indicate that fungal and bacterial communities dominate the early surface attachment process, when pristine mineral surfaces are exposed to deep subsurface ecosystems. Various fungi showed statistically significant cross-kingdom correlation with both thiosulfate and sulfate reducing bacteria, e.g., SRB2 with fungi Debaryomyces hansenii.

Download Full-text

Discrete element analysis of the punching behaviour of a secured drapery system: from laboratory characterization to idealized in situ conditions

Acta Geotechnica ◽

10.1007/s11440-020-01119-z ◽

2021 ◽

Author(s):

Antonio Pol ◽

Fabio Gabrieli ◽

Lorenzo Brezzi

Keyword(s):

Mechanical Response ◽

Steel Wire ◽

Discrete Element ◽

Wire Mesh ◽

Steel Plates ◽

Loading Conditions ◽

Element Analysis ◽

In Situ Conditions ◽

Wire Meshes

AbstractIn this work, the mechanical response of a steel wire mesh panel against a punching load is studied starting from laboratory test conditions and extending the results to field applications. Wire meshes anchored with bolts and steel plates are extensively used in rockfall protection and slope stabilization. Their performances are evaluated through laboratory tests, but the mechanical constraints, the geometry and the loading conditions may strongly differ from the in situ conditions leading to incorrect estimations of the strength of the mesh. In this work, the discrete element method is used to simulate a wire mesh. After validation of the numerical mesh model against experimental data, the punching behaviour of an anchored mesh panel is investigated in order to obtain a more realistic characterization of the mesh mechanical response in field conditions. The dimension of the punching element, its position, the anchor plate size and the anchor spacing are varied, providing analytical relationships able to predict the panel response in different loading conditions. Furthermore, the mesh panel aspect ratio is analysed showing the existence of an optimal value. The results of this study can provide useful information to practitioners for designing secured drapery systems, as well as for the assessment of their safety conditions.

Download Full-text

Detection and Potential Virulence of Viable but Non-Culturable (VBNC) Listeria monocytogenes: A Review

Microorganisms ◽

10.3390/microorganisms9010194 ◽

2021 ◽

Vol 9 (1) ◽

pp. 194

Author(s):

Nathan E. Wideman ◽

James D. Oliver ◽

Philip Glen Crandall ◽

Nathan A. Jarvis

Keyword(s):

Public Health ◽

Listeria Monocytogenes ◽

Viable Count ◽

Vbnc State ◽

Testing Methods ◽

Tetrazolium Chloride ◽

Definitive Evidence ◽

Virulence Potential ◽

Vbnc Bacteria ◽

The Impact

The detection, enumeration, and virulence potential of viable but non-culturable (VBNC) pathogens continues to be a topic of discussion. While there is a lack of definitive evidence that VBNC Listeria monocytogenes (Lm) pose a public health risk, recent studies suggest that Lm in its VBNC state remains virulent. VBNC bacteria cannot be enumerated by traditional plating methods, so the results from routine Lm testing may not demonstrate a sample’s true hazard to public health. We suggest that supplementing routine Lm testing methods with methods designed to enumerate VBNC cells may more accurately represent the true level of risk. This review summarizes five methods for enumerating VNBC Lm: Live/Dead BacLightTM staining, ethidium monoazide and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), direct viable count (DVC), 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and carboxy-fluorescein diacetate (CDFA) staining. Of these five supplementary methods, the Live/Dead BacLightTM staining and CFDA-DVC staining currently appear to be the most accurate for VBNC Lm enumeration. In addition, the impact of the VBNC state on the virulence of Lm is reviewed. Widespread use of these supplemental methods would provide supporting data to identify the conditions under which Lm can revert from its VBNC state into an actively multiplying state and help identify the environmental triggers that can cause Lm to become virulent. Highlights: Rationale for testing for all viable Listeria (Lm) is presented. Routine environmental sampling and plating methods may miss viable Lm cells. An overview and comparison of available VBNC testing methods is given. There is a need for resuscitation techniques to recover Lm from VBNC. A review of testing results for post VBNC virulence is compared

Download Full-text

Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽

10.3390/foods10071502 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1502

Author(s):

Jorge García-Hernández ◽

Manuel Hernández ◽

Yolanda Moreno

Keyword(s):

In Situ Hybridization ◽

Vibrio Parahaemolyticus ◽

Fluorescent In Situ Hybridization ◽

Potential Method ◽

Viable Count ◽

Hybridization Technique ◽

Fish Technique ◽

Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

Download Full-text

In-situ tryptophan-like fluorescence: A real-time indicator of faecal contamination in drinking water supplies

Water Research ◽

10.1016/j.watres.2015.05.035 ◽

2015 ◽

Vol 81 ◽

pp. 38-46 ◽

Cited By ~ 48

Author(s):

J.P.R. Sorensen ◽

D.J. Lapworth ◽

B.P. Marchant ◽

D.C.W. Nkhuwa ◽

S. Pedley ◽

...

Keyword(s):

Drinking Water ◽

Real Time ◽

Water Supplies ◽

Faecal Contamination

Download Full-text

Porcine Repeat Element DNA: In Situ Detection of Xenotransplanted Cells

Cell Transplantation ◽

10.1177/096368979500400209 ◽

1995 ◽

Vol 4 (2) ◽

pp. 253-256 ◽

Cited By ~ 9

Author(s):

Henry F. Oettinger ◽

Amelie Rodrigue-Way ◽

Joyce J. Bousquet ◽

Albert S.B. Edge

Keyword(s):

Southern Blotting ◽

Host Tissue ◽

Repeat Element ◽

Tissue Analysis ◽

Specific Staining ◽

Tetrazolium Chloride ◽

Tissue Sections ◽

Blue Tetrazolium Chloride ◽

In Situ Detection

Using a digoxygenin-labelled DNA probe derived from the porcine repeat element PRE-1, we have developed a protocol for the detection of transplanted porcine islets and hepatocytes against a background of murine host tissue. Analysis of this probe by Southern blotting indicated that PRE-1 hybridizes to pig genomic DNA but not to human or mouse DNA. On tissue sections, hybridizing probe was detected using alkaline phosphatase-conjugated anti-digoxygenin antibody visualized with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride (BCIP/ NBT) substrate. We have demonstrated sensitive and highly specific staining of porcine nuclei in fixed, paraffin embedded tissue sections, and have applied the technique to detect porcine pancreatic islets and hepatocytes transplanted into murine kidney and spleen. Applications of this technique include detection of transplanted cells or organs across a variety of xenogeneic barriers.

Download Full-text