A new method to determine initial viability of entrapped cells using fluorescent nucleic acid staining (original) (raw)
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By this experiment we will demonstrate the possibility to obtain genetically modified microbial strains that can be used as markers in different studies. The trait transferred in this study is the fluorescence in UV light expressed by a gene isolated from jellyfish. This gene was insered into a plasmid carrying ampiciline resistance and in the operon for arabinose fermentation. The plasmid was called pGLO. E coli HB101 K-12, ampicillin resistant colonies has been obtained. The colonies on the LB/amp/ara plate fluoresce green under UV light and the transformed colonies can grow on ampicillin. Transformation efficiency = 362 transformed colonies/ µg DNA. The cells where immobilized by entrapment in alginate gel to study the phenomenon involved in cells immobilization. After immobilization in alginate gel, 5x10 4 cells of E. coli pGLO / capsule and 1,4 x 10 5 cells of E. coli HB101/capsule has been found. Fluorescent microscopy revealed the presence of pGLO carrying cells into the caps...
The effects of cell entrapment on nucleic acid content, cell morphology, cell surface property, and stress of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different entrapment media (alginate, carrageenan, and polyvinyl alcohol) were examined. Results indicated that the entrapment and type of entrapment media affected nucleic acid content, cell morphology, cell surface property, and stress of the three representative species (Alcaligenes faecalis, Comamonas testosteroni, and Pseudomonas putida) studied. The highest deoxyribonucleic acid and ribonucleic acid increases were observed with the alginate and polyvinyl alcohol (PVA) entrapment, respectively. A cell morphological change from bacilli to coccoidal was observed in the case of alginate entrapment while the PVA-entrapped cells had a slim morphology when compared to non-entrapped cells and formed putative nanowires. The entrapment increased or decreased the surface roughness of cells depending on the type of entrapment media. Expression of a nitrosative stress gene, which is linked to oxygen deprivation, was observed more in the alginate-entrapped cells. These research findings advance the fundamental understanding of the entrapped cell physiology which can lead to more efficient entrapped cell-based wastewater treatment.
Biochemical Engineering Journal, 2007
A technique to distinguish viable and dead cells has long been considered necessary in various fields such as sterilization, toxicity assessment, sanitary evaluation and so on. A bacterial staining method using fluorescent dye is a popular tool, although the weakness of fluorescence intensity and its fading over time constitute notable drawbacks. In the process of esterase-active bacteria staining with carboxyfluorescein diacetate (CFDA), we have reported glutaraldehyde (GTA) affected the discriminative recognition of bacteria due to prevention of fluorescence leakage from the cell. In this study, CFDA was applied to four pure bacterial strains (two Gram-negative strains and two Gram-positive strains) during the exponential growth phase and to activated sludge as an indicator of microbial viability. GTA concentration was also optimized and the effect of GTA addition was compared to the conventional method using ethylenediamine tetraacetic acid (EDTA) and the control without pretreatment. At higher concentrations of GTA, microbial viability decreased because of GTA toxicity. In the case of all conditions where CFDA staining was carried out in the assay of microbial viability, the highest viability was achieved by using of 1 g/L GTA.
Measurement of cell viability in in vitro cultures
Methods in Molecular Biology, 2006
An overview of the methods for assessing cell viability is presented. Different protocols of the most commonly used assays are described in detail so that the readers may be able to determine which assay is suitable for their own projects in plant biotechnology.
Applied Microbiology and Biotechnology, 2002
A new method was developed to detect and quantify two strains, Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707, immobilized separately and co-immobilized in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy. The establishment of biomass concentration profiles for each strain was measured during colonization of beads using successive pH-controlled batch fermentations. Growth occurred preferentially in 200-and 300-µm peripheral layers of the beads for L. diacetylactis and B. longum, respectively. Repeated-batch cultures with immobilized cells permitted the production of a mixed culture containing a non-competitive strain of bifidobacteria, as a result of immobilized-cell growth and high cell-release activity from the beads. During co-immobilized fermentations, there were no apparent interactions between the strains.
Enzyme and Microbial Technology, 2010
Entrapped bacteria are used in several applications including food and beverage production, antibiotic production, and wastewater treatment. To date in order to determine the viability of entrapped bacteria, they have to be de-entrapped from the matrix first. However, the cell de-entrapment procedures such as matrix dissolution by acid or heating at high temperatures, may affect the viability of the cells. In this study, the uses of two quantitative approaches for in situ viability estimation of calcium alginate and phosphorylated poly(vinyl) alcohol (PPVA) entrapped Escherichia coli were investigated. Bioreducible tetrazolium salt (XTT) and adenosine triphosphate (ATP) based assays were used to determine microbial viability without the dissolution of the matrices (spherical beads). The data from both assays showed linearity and strong correlations between the viability signals and number of beads in which each bead contained a similar number of live cells. An application of XTT assay on the PPVA entrapped bacterial beads was an exception to these results. Effects of the acid and heat dissolution de-entrapment procedures on cell viability were also evaluated by using both assays and a traditional plate count method. The heating process showed the greatest reduction in bacterial viability when compared to the other de-entrapment procedures. The ATP assay is a more sensitive and less time consuming approach for viability estimation when compared to the XTT assay and traditional plate count method. Both XTT and ATP assays have potential for use in quantifying the viability of entrapped bacteria.
Microbial cell encapsulation as a strategy for the maintenance of stock cultures
A cellular immobilization technique was developed as a new proposal for the maintenance and preservation of microbial cultures. Cells of enterobacteria, Escherichia coli (ATCC 25922) and Enterobacter aerogenes (ATCC 13048) were entrapped in calcium alginate beads, which were subjected to different storage conditions. Nondehydrated beads crude devoid of bacterial cells were kept under refrigeration at 5°C, or frozen at −60°C, and freeze-dried beads were stored at −18°C. The viability and biochemical stability of the entrapped cells during storage time was assessed by the Petrifilm™ procedure and VITEK 2 system. Morphological aspects of the beads were evaluated by optical and scanning electron microscopy. Cell entrapment within alginate beads was a promising technique for maintaining E. coli and E. aerogenes stock cultures. E. aerogenes cells entrapped within calcium alginate beads and maintained under refrigeration or frozen at −18°C were viable for 240 days, and maintained their biochemical characteristics. Encapsulated E. aerogenes cells maintained at −60°C remained viable for up to 150 days. The viability of the E. coli cells was maintained throughout the storage period under all storage conditions studied, but this was accompanied by a loss of synthesis capacity of the enzymes, β-galactosidase and β-glucuronidase.
Monitoring of the Viability of Cells Immobilized by Sol-Gel Process
Journal of Sol-gel Science and Technology, 2004
Three different types of cells, Pseudomonas fluorescens HK44, Saccharomyces cerevisiae strain SP4 and plant cells Nicotiana tabacum L. BY-2, were immobilized by entrapment in tetramethoxysilane prepolymer (TMOS) gel or in composite gel containing prepolymer TMOS and alginate in various ratios. Their growth and viability were monitored by bioluminescence and 2-D fluorescence spectra, which are fast and do not need the dissolution of a matrix. The resulting biocomposite gels were obtained by gelation of the mixtures of TMOS prep. or TMOS/alginate sols and the particular cells in proper media on glass supports to provide films ∼1 mm thick. The effect of the following parameters on the growth and viability of the cells was studied: (a) the composition of the biocomposites, (b) the preparation conditions of TMOS and (c) the conditions of the procedure of entrapment. All three types of cells were tested in TMOS gel and the composite TMOS/alginate = 1:1 (v/v). The sensitivity of the cells to the changes of conditions increased in the sequence: P. fluorescence HK44 < S. cerevisiae strain SP4 < N. tabacum L. BY-2. Cell viability decreased with the increasing content of Si in biocomposites. The entrapment into alginate–silica composites resulted in the leakage of microbial and yeast cells. However, it had positive effects on the growth and metabolic activity of plant cells.
The ideal scenario in most applications of microbial diagnostics is that only viable cells are detected. Bacteria were traditionally considered viable when they could be cultured, whereas today’s viability concept tends to be alternatively based on the presence of some form of metabolic activity, a positive energy status, responsiveness, detection of RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion, although conservative, was the focus of one of the most successful recent approaches to detect viable cells in combination with DNA amplification techniques. The technology is based on sample treatment with the photoactivatable, and cell membrane impermeant, nucleic acid intercalating dyes ethidium monoazide (EMA) or propidium monoazide (PMA) followed by light exposure prior extraction of DNA and amplification. Light activation of DNA-bound dye molecules results in irreversible DNA modification and subsequent inhibition of its amplification. Sample pretreatment with viability dyes has so far been mainly used in combination with PCR (leading to the term viability PCR, v-PCR), and increasingly with isothermal amplification method. The principle is not limited to bacteria, but has also successfully been applied to fungi, protozoa and viruses. Despite the success of the method, some practical limitations have been identified, especially when applied to environmental samples. In part they can be minimized by choice of experimental parameters and conditions adequate for a particular sample. This review summarizes current knowledge and presents aspects which are important when designing experiments employing viability dyes.