Chemiluminescent assay for detection of viable microorganisms

The redox reaction between quinone and viable microorganisms produces active oxygen species. In this study, the production rates of active oxygen species were determined by a luminol chemiluminescent assay, and the luminescence intensity was found to be proportional to the viable cell number. The high sensitivity of the luminol chemiluminescent assay was achieved with Mo-ethylenediaminetetraacetate complex and menadione or coenzyme Q1. The detectable cell densities of bacteria and yeasts were found to be approximately several thousand colony-forming units (CFU/ml) when assays were performed with a 96-well microplate luminometer. The chemiluminescent assay requires 10 min for incubation of quinone and microorganisms and 2s for photon counting. Single Escherichia coli was detected after 4h of cultivation and centrifugation (5 min x 2). This simple chemiluminescent assay is expected to be useful for the rapid detection of viable bacteria and yeast.     

Advantage of menadione-catalyzed chemiluminescent assay for the determination of viable mammalian cell number

A chemiluminescent assay composed of TCPO [bis(2,4,6-trichlorophenyl)oxalate] and harmless rhodamine B is proposed to be superior in the determination of menadione-catalyzed hydrogen peroxide (H₂O₂) production by viable mammalian cells to that composed of TCPO and harmful pyrene [Anal. Biochem. 207 (1992) 255–260]. In tests, the proposed assay showed that the measurable concentration of H₂O₂ and the viable cell number ranged from 10^?9 to 10^?3 M and from 2 × 10² to 2 × 10⁶ cells/100 μl/well in the presence of 10% bovine serum, respectively. The measuring time was approximately 10 min. On the other hand, the measurable cell numbers by the colorimetric WST-1 and MTT assays requiring several hours ranged only from 10³ to 10⁴ cells/100 μl/well and from 10⁴ to 10⁵ cells/100 μl/well, respectively. The cytotoxicity of sodium dodecyl sulfate was also observed at intervals of 1 min by the proposed assay, but not by the above colorimetric assays.     

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L ‐lysine) (Ca‐alg‐PLL), and a novel calcium alginate‐poly(ethylene glycol) hybrid material (Ca‐alg‐PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca‐alg‐PLL capsules, while a resazurin‐based assay was used for comparison in case of the Ca‐alg‐PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter‐assay relative standard deviations below 10%. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:986–993, 2013     

Rapid assay of cell activity of yeast cells

Yeast cells produce H₂O₂ in the presence of exogenous menadione, and the extracellular concentration of H₂O₂ produced is proportional to the viable cell number or cell activity. Chemiluminescent assay of menadione-catalyzed H₂O₂ production requires only a few minutes, and is useful for rapid determination of the viability of yeast cells.     

Autofluorescence of viable cultured mammalian cells.

The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.     

Microplate assay for quantitative determination of cathepsin activities in viable cells using derivatives of 4-methoxy-beta-naphthylamide

A method is described allowing the selective determination of four cathepsins (B, H, K, and L) in live cells. Adherently growing cells are incubated with partially selective substrates for each cathepsin (peptidic derivatives of 4-methoxy-beta-naphthylamine) in microtiter plates together with nitrosalicylaldehyde. Using an appropriate reader accumulating fluorescent products may be detected continously or by end point measurement. Selectivity is achieved by running parallel assays containing inhibitors that are partially selective for each of the cathepsins (in case of cathepsin H, the nonlysosomal aminopeptidases are inhibited by bestatin). Individual cathepsin activities can then be calculated by the difference between the uninhibited and the inhibited assay. The method was validated by measurements in cells isolated from cathepsin B(-/-)-, K(-/-)-, and L(-/-)- mice. This strategy suggests that the combination of two partially selective reaction partners, substrate and inhibitor can yield selective cathepsin assays.     

Immobilization of yeast cells in plant cell wall frameworks

Summary Cell-structured support materials (CSM) representing the cell framework of denaturated and extracted mosses, duckweeds or parenchyma tissue particles have been used for the immobilization of Saccharomyces cerevisiae cells. The method consists of inoculation by soaking the dehydrated materials in a yeast suspension and propagation of the yeast cells that reach the relatively closed inner volumes of the cell-structured particles (inter- or intracellular spaces). In spite of high cell densities (up to 2.5 × 10⁹ cells/g wet immobilizate) the velocity of microaerobic glucose consumption was little influenced by intraparticular diffusion resistances, when yeast loaded CSM made from Wolffia arrhiza was incubated in 100 mM glucose at room temperature.     

A rapid in situ,colorimetric assay for the determination of mammalian cell viability in alginate-immobilized and encapsulated systems

A convenient, rapid colorimetric assay system has been developed in order to determine cell viability of populations of mammalian cells encapsulated using a poly-L-lysine/ alginate encapsulation system. The method is based on the use of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and involves direct incubation of the capsules with the reagent. This is followed by direct solubilization of the resulting formazan product in dimethyl sulfoxide (DMSO). In order to demonstrate that this assay method may be generally applicable to alginate-based carrier systems, viability of cells immobilized in alginate beads, as distinct from encapsulation in the poly-L-lysine/alginate system, was also determined using the in situ method.     

Comparison of viable cell concentration estimation methods for a mammalian cell cultivation process

Various mechanistic and black-box models were applied for on-line estimations of viable cell concentrations in fed-batch cultivation processes for CHO cells. Data from six fed-batch cultivation experiments were used to identify the underlying models and further six independent data sets were used to determine the performance of the estimators. The performances were quantified by means of the root mean square error (RMSE) between the estimates and the corresponding off-line measured validation data sets. It is shown that even simple techniques based on empirical and linear model approaches provide a fairly good on-line estimation performance. Best results with respect to the validation data sets were obtained with hybrid models, multivariate linear regression technique and support vector regression. Hybrid models provide additional important information about the specific cellular growth rates during the cultivation.     

A pilot plant for mammalian cell culture

Studies of the possible viral etiology of human leukemia have required large quantities of cultured cells derived from human hematopoietic tissues. Since cultures sufficiently large and free from contamination could not readily be produced according to existing methods, a pilot, cell culture plant has been constructed for the production of mammalian cells in mass quantity. 500-ml to 20-liter trophocell units have already proved to be scientifically and economically practical, as they provide good reliability, excellent growth rates, and sustained yield of human cells. 200-liter stainless steel culture units have now been added to the trophocell system. Five complete 200 liter units are now in operation. The design of the original stainless steel unit was based on that of a stainless steel, jacketed soup kettle. There are no openings in the vessel other than those in the lid, which provide convenient access points for sampling, sensor probes, etc. Environmental parameters, e.g., liquid level, temperature, and pH, are monitored and controlled with commercially available apparatus. Many initial problems connected with the new 200 liter units have been resolved, but operational and design problems remain in the areas of stable instrumentation, cell harvesting, salvaging and reuse of unspent media components, establishment of physiologic steady stale, recovery of virus-containing cells with reculture of the remaining unaffected cells, and the recovery and separation of cell components and special products such as immunoglobulins, interferons, and hormones. A definitive cell plant with culture units of 20, 50, 250, and 1250 liters is now being constructed.     

Comparison between nuclear localization of nopaline- and octopine-specific Agrobacterium VirE2 proteins in plant, yeast and mammalian cells

In a unique case of trans-kingdom DNA transfer, Agrobacterium genetically transforms plants by transferring its DNA segment into the host cell nucleus and integrating it into the plant genome. One of the central players in this process is the bacterial virulence protein, VirE2, which binds the transported DNA molecule and facilitates its nuclear import. Nuclear import of VirE2 proteins encoded by two major Agrobacterium strains, nopaline and octopine, has been hypothesized to occur by different mechanisms, i.e. the nopaline VirE2 was imported only into the nuclei of plant cells while the octopine VirE2 also accumulated in the nuclei of animal cells. Here, this notion was tested by a systematic comparison of nuclear import of nopaline- and octopine-specific VirE2 in dicotyledonous and monocotyledonous plants and in living mammalian and yeast cells. These experiments showed that nuclear import of both nopaline and octopine VirE2 proteins is plant-specific, occurring in plant but not in non-plant systems.     

The use of plant cell vesicles for immobilization of yeast cells producing ethanol

The preparation of immobilized living yeast cells adsorbed into or onto delipided specimens of the dwarf duckweed Wolffia arrhiza (Fam. Lemnaceae) is reported. These yeast cell-plant cell conjugates were used for the repeated batch production of ethanol from glucose (143 to 246 g/l) or saccharose (150 g/l). Up to 25 fermentation cycles at 30°C were performed. The cycle time for complete substrate conversion to ethanol was reduced 10-fold by a 5-fold increase of the yeast cell Wolffia conjugate concentration (ε = 0.08 to ε =0.4) ε = volume of cell conjugate/totnl reaction volume. The corresponding ethanol production was 11.5 to 13.5 vol% and 9 vol% respectively. The reported results on the discontinuous ethanol fermentation with Wolffia-immobilized yeast cells open the field for their application in continuous ethanol production processes.     

Chemiluminescent determination of choline in cerebrospinal fluid and red blood cells

The concentration of choline in the cerebrospinal fluid (CSF) of patients affected by primary dementia and in red blood cells (RBC) of depressed patients before and after treatment with lithium salts was determined using a chemiluminescent assay. The mean CSF concentration of choline was found to be 60 pmoles/ml (SD = 20 pmoles/ml) and this was lower than values obtained previously by spectrophotometric-colorimetric methods. Mean RBC choline concentrations before and after therapy with lithium salts were 20 nmoles/ml (SD = 16 nmoles/ml and 328 nmoles/ml (SD = 206 nmoles/l) respectively and these are similar to those reported previously (obtained by chemiluminescent and non-chemiluminescent methods).     

Immobilization of viable yeast cells within polyaldehyde-hardened gelatin gel

Summary A gel-entrapment method particularly suitable for viable cells is described. The gel matrix is gelatin insolubilized by interaction with polymeric aldehydes (polyaldehydes) prepared by periodate oxidation of polysaccharides such as starch and dextran. The viability of the entrapped cells is evidenced by growth measurement and by SEM analysis of the immobilizate.     

Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

Determination of viable mammalian cells by luminol chemiluminescence using microperoxidase

Chemiluminescent assay for menadione-catalyzed H₂O₂ production by mammalian cells was modified by luminol chemiluminescence with microperoxidase instead of peroxyoxalate chemiluminescence with carcinogenic fluorescent materials. Luminol can be used as a common chemiluminescent reagent for the determination of viable mammalian cells and bacteria.