Enrichment and Recovery of Mammalian Cells from Contaminated Cultures Using Aqueous Two-Phase Systems.

This Article describes a density-based method for removing contaminants, including microorganisms and nonviable cells, from mammalian cell cultures using an aqueous two-phase system (ATPS). The properties of a 7% w/w polyethylene glycol (PEG)-11% w/w Ficoll ATPS can be tuned to prepare a biocompatible system that removes contaminants with little to no adverse effects on the viability or growth of the cultured cells after treatment. This system can be used to enrich cell culture populations for viable cells and to reduce the number of microorganism contaminants in a culture, which increases the chances of subsequent antibiotic treatments being successful. We test the effectiveness of our method in model contaminated cultures of both adherent (HeLa) and suspension (HL-60 II) mammalian cells contaminated with bacteria (E. coli) and yeast (S. cerevisiae). An average of 70.2 ± 4.6% of HeLa cells added to the system are subsequently recovered, and 55.9 ± 2.1% of HL-60 II cells are recovered. After sedimenting to the interface of the ATPS, these cells have an average viability of 98.0 ± 0.2% and 95.3 ± 2.2%, respectively. By removing unwanted cells, desired cell populations can be recovered, and cultures that would otherwise need to be discarded can continue to be used.

Single step, rapid identification of pathogenic microorganisms in a culture bottle.

Efforts to treat bloodstream infections, which have a relatively high mortality rate, are delayed by the lengthy multi-step process required to identify the causative bacteria. Due to this delay, broad spectrum antibiotics are prescribed on a presumptive basis, leading to the rise of antibiotic resistant microorganisms. Here, as proof of principle, we describe a colourimetric sensor that rapidly identifies opportunistic pathogenic bacteria in a single step in TSB media. The device is composed of a reaction chamber and an array of chemoresponsive dyes deposited on a substrate in a prearranged pattern. This single step, disposable, automated system can detect and identify of eight strains of bacteria, starting with clinically relevant concentrations bacteria in twenty four hours in TSB media. Thus, this technology may be used to streamline the current blood culture process by combining detection and identification in a single step.

Antimicrobial activity of Bacillus sp. strain FAS1 isolated from soil.

During screening for antibiotic producing microorganisms from environmental soil samples, the supernatant of a bacterial isolate was found to have antibacterial and antifungal activity on the standard indicator species. The standard cylinder-plate method was used to determine the inhibitory effect of the crude supernatant of each isolate on 6 bacterial and 3 fungal standard strains by measuring the diameter of inhibition zone. The highest inhibition zone on Aspergillus niger belonged to culture broth of isolate FAS1 by 25 mm, and this isolate was the most efficient microorganism to inhibit standard bacterial and fungal species. Based on morphological and biochemical properties as well as 16S rDNA gene analysis, the selected isolate (isolate FAS(1)) belonged to Bacillus genus. Investigation on the ability of different culture media for antibiotic production led to select Luria-Bertani media for further studies. Treatment of the culture broth of the isolate FAS(1) using typical protease didn't decrease the antimicrobial activity of the supernatant. After extracting of culture broth of the selected isolate by ethyl acetate as an organic solvent, the inhibitory effect was mainly increased. More investigation was done by bioautography method where the ethyl acetate fraction of the broth culture was separated on TLC by chloroform:methanol, 60:40 as mobile phase and R(f) were calculated for inhibition spots.

Enrichment and detection of molecules secreted by tumor cells using magnetic reversed-phase particles and LC-MALDI-TOF-MS.

Tumor cells change their genetic expression pattern as they progress to states of increasing malignancy. Investigations at the DNA and RNA level alone cannot provide all the information resulting after the translation and processing of the corresponding proteins, which is one reason for a poor correlation between mRNA and the respective protein abundance. In diagnostics, differentially expressed peptides or proteins are important markers for the early detection of cancer. Unfortunately, tumor cells secrete peptides and proteins in only very low amounts, making mass spectrometric determination very difficult. In this publication, methods have been developed for the effective enrichment and cleanup of substances secreted by cultivated cancer cells. To obviate peptides from fetal calf serum used in cell culture, a serum surrogate was developed, which maintained growth of the cancer cells. After the binding of substances from cell-culture supernatants to custom-made magnetic reversed-phase particles, the substances were eluted and separated by capillary high-performance liquid chromatography. Fractions were spotted directly on a MALDI target, and MALDI-TOF mass spectrometric data acquisition was performed in automatic mode. This technology was used to detect substances secreted by two mammary carcinoma cell lines differing in their malignancy (MCF-7, MDA-MB 231). Unequivocal differences in the peptide secretion patterns were observed. In conclusion, this system allows the sensitive investigation of peptides secreted by cancer cells in culture and provides a valuable tool for the investigation of cancer cells in different states of malignancy.

Lactic acid recovery from cheese whey fermentation broth using combined ultrafiltration and nanofiltration membranes.

The separation of lactic acid from lactose in the ultrafiltration permeate of cheese whey broth was studied using a cross-flow nanofiltration membrane unit. Experiments to test lactic acid recovery were conducted at three levels of pressure (1.4, 2.1, and 2.8 MPa), two levels of initial lactic acid concentration (18.6 and 27 g/L), and two types of nanofiltration membranes (DS-5DK and DS-5HL). Higher pressure caused significantly higher permeate flux and higher lactose and lactic acid retention (p < 0.0001). Higher initial lactic acid concentrations also caused significantly higher permeate flux, but significantly lower lactose and lactic acid retention (p < 0.0001). The two tested membranes demonstrated significant differences on the permeate flux and lactose and lactic acid retention. Membrane DS-5DK was found to retain 100% of lactose at an initial lactic acid concentration of 18.6 g/L for all the tested pressures, and had a retention level of 99.5% of lactose at initial lactic acid concentration of 27 g/L when the pressure reached 2.8 MPa. For all the tests when lactose retention reached 99-100%, as much as 64% of the lactic acid could be recovered in the permeate.

Recovery of organic acids from fermentation broths.

Rising concerns over the use of fossil resources have generated renewed interest in the production of commodity chemicals via fermentation. Organic acids are a particularly attractive target because their functionality enables downstream catalytic upgrading to a variety of compounds. In this article, we survey how common technical issues are addressed in the recovery schemes for several organic acids. We present results for the recovery of acetate using a new method based on amine complexation. Our reactive separation scheme produces a high-purity product, is energy efficient, and avoids the coproduction of a waste salt coproduct, all prerequisites for a large-scale production process.

Removal of transmissible spongiform encephalopathy prion from large volumes of cell culture media supplemented with fetal bovine serum by using hollow fiber anion-exchange membrane chromatography.

Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco's modified Eagle's medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth media supplemented with FBS.

Re-use of spent cell culture medium in pilot scale and rapid preparative purification with membrane chromatography.

Based on experiments in bench scale, a recycling of spent cell culture medium was performed in a 100-1 pilot scale bioreactor. The cell cultivation has been done as a repeated batch procedure after the initial batch in the following four repeated batches spent medium from the previous batch was partially re-used. After microfiltration and ultrafiltration a part of the filtrate was mixed with a concentrate of amino acids and glucose, sterile filtered and subsequently filled back into the bioreactor. Up to 65% of the harvested cell- and product-free spent medium was re-used in each repeated batch. This procedure results in a saving of pure and waste water volume and saving of supplemented proteins as transferrin, insulin and lipoproteins and, therefore, also in a reduction of the production costs. A strongly acidic membrane ion exchanger was evaluated for the ability to purify the monoclonal antibodies from the pilot scale cultivation. Within minutes, gram quantities of product could be purified in a high flux system, especially developed for this purpose, achieving purities of 80%. The capacity of the acidic membrane ion exchanger was found in former investigations to be 1 mg cm-2 with recoveries up to 96%. Final purification was carried out by gel column filtration.

Recovery of ammonium lactate and removal of hardness from fermentation broth by nanofiltration.

Nanofiltration (NF) was investigated as an alternative to desalting electrodialysis (ED) and ion exchange for the recovery of ammonium lactate from fermentation broth. Three commercial NF membranes, NF45, NF70, and NTR-729HF, were characterized with 50 mM NaCl, MgSO(4), and glucose solutions. NF45 membrane was selected because it showed the lowest rejection of monovalent ion, the highest rejection of divalent ion, and the highest rejection of nonpolar molecule. Effects of the operating pressure were investigated in a range of 100-400 psig, on the flux, lactate recovery, and glucose and magnesium removal from a real fermentation broth containing about 1.0 M of ammonium lactate. The flux and recovery rate increased linearly with the pressure. However, lactate rejection also increased with the pressure, lowering the recovery yield. More magnesium ions and glucose were rejected as the pressure was increased, and at 400 psig, for example, magnesium ion was almost completely rejected, highlighting the chance of obviating the necessity of ion exchange to remove hardness, by using NF instead of desalting ED. Membrane fouling was not so severe as expected, considering the complex nature and a rather high concentration of the fermentation broth treated.

Theoretical investigation of axial and local particle size distribution on expanded bed adsorption process.

The general rate model was developed and solved to describe protein adsorption in an expanded bed. The model takes into account axial variation of bed porosity, particle size distribution (PSD), external and intraparticle mass transfer, and dispersion in liquid and solid phase. The analysis of the influence of the model parameters on dynamic capacity (DC) was investigated. The simulation results showed that major impact on dynamic capacity is exerted by intraparticle mass transfer (particle diameter and pore diffusivity). The external mass transfer resistance and dispersion parameters have secondary effect on DC. The replacement of axial PSD by the mean particle diameter results in error in calculation of DC, which increases remarkably with the increase of mean particle diameter. The PSD can promote a very slow approaching of plateau concentration by breakthrough curves. It was shown also that axial bed porosity variation could be replaced by average porosity with negligible error for DC calculations.

Use and optimization of a dual-flowrate loading strategy to maximize throughput in protein-a affinity chromatography.

The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein-A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein-A system. A gradient-based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two-step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc-fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual-flowrate strategy was found to be superior.

The influence of incubation conditions in sterility tests.

Studies were conducted to evaluate the effects of media, incubation temperature and duration on the detection of bacteria and fungal growth using British, United States and Brazilian compendial sterility test methods. 5 to 50 CFU of nine different microorganisms (including both anaerobic and aerobic bacteria and molds) were used to contaminate test units containing various growth media (sobean-casein digest, thioglycollate, Sabourand and Causen broths). Test units were incubated at temperatures ranging from 12 to 42 degrees C for 1 to 28 days. Inoculations were conducted according to compendial procedures. Optimal detection conditions were obtained at 22 to 32 degrees C over 14 days using soybean casein digest and thioglycollate broths.

[The production of a protein base for nutrient media from a dairy industry byproduct]. / Poluchenie belkovoi osnovy dlia pitatel'nykh sred iz subprodukta molochnoi promyshlennosti.

The technology of obtaining protein hydrolysate, a by-product of butter making, has been developed. This hydrolysate has been successfully tested as protein base for the preparation of culture media used in the quality control of foodstuffs and the diagnosis of infectious diseases.

[Highly purified agar for virological, immunochemical and microbiological research]. / Vysokoochishchennyi agar dlia virusologicheskikh, immunokhimicheskikh i mikrobiologicheskikh issledovaniiakh.

A new effective method for the preparation of highly purified agar for microbiological, virological and immunochemical investigations has been obtained and the technology for its production has been developed. The study revealed that the physico-chemical characteristics of newly obtained highly purified agar are much better than those of microbiological agar currently produced in Russia and not inferior to those of the best kinds of agar produced by Difco (USA), Serva and Ferak (Germany). The method of purification is protected by an author's certificate. The production of this agar for medical purposes has been permitted. The manufacturing plant "Medbioagar", created under the auspices of the Research Institute of Epidemiology and Microbiology has now started the serial production of highly purified agar under the name "Primagar". Further increase of the production of the preparation will facilitate its introduction into laboratory practice with the use of modern research methods in the fields of microbiology and virology, molecular biology and genetics, immunology and biochemistry.

[The possibility of using a bacteriological peptone from whole blood as the nutrient base for growing microorganisms]. / Izuchenie vozmozhnosti ispol'zovaniia bakteriologicheskogo peptona iz tsel'noi krovi kak pitatel'noi osnovy dlia vyrashchivaniia mikroorganizmov.

Study of some parameters of the process of hydrolysis in addition to other experiments has made it possible to develop a method for obtaining a nutrient base for the cultivation of microorganisms with the use of cattle whole blood as protein substrate and papain as a hydrolyzing agent. The product thus obtained is characterized by high levels of amino and total nitrogen (4% and 14% respectively). Biological studies with the use of this product in different culture media and observations on the growth and characteristics of many microbial strains have shown the possibility of its use for the diagnosis of infectious diseases, for the control of the quality of foodstuffs and water.

[Membrane-active properties of a preparation from a bacterial culture broth possessing autoregulation action]. / Membranoaktivnye svoistva preparata iz kul'tural'noi zhidkosti bakterii, obladaiushchego autoreguliatornym deistviem.

The effect of a preparation obtained by butanol extraction of the culture fluid of B. cereus and Ps. carboxydoflava and previously termed an autoregulatory factor d1 on the respiration chain within intact bacterial cells and isolated membranes was investigated. In comparable concentrations this factor d1 inhibits the endogenous respiration of B. cereus and M. lysodeikticus as well as oxidase and dehydrogenase activities of isolated membranes from these bacteria and E. coli. The factor-induced decrease of the cell respiration rate is independent from disorders of the cell permeability osmotic barrier for respiration substrates. The factor d1 shows a higher effect at acidic pH values. It is concluded that the above preparation has membrane-active properties. It is also suggested that in the bacterial cell its target is the inner surface of the cytoplasmic membrane.

[The immunosuppressive activity of filtrates of the culture broth from nonvirulent salmonellae]. / Izuchenie immunosupressivnoi aktivnosti fil'tratov kul'tural'noi zhidkosti nevirulentnykh sal'monell.

Cultural filtrates (CF) of avirulent Salmonella virchow and Salmonella dublin have been studied for their influence on the delayed type hypersensitivity (DTH) to test-antigen in mice. It is found that i.p. injection of CF inhibits DTH in mice. The immunosuppressive activity is indicated in lipid fraction of CF and it may be removed from CF by the O-specific immunosorbent. It is heat-stable and disappears after phenol or trichloroacetic acid treatment. Gel filtration data evidence for high molecular weight of the active factor. These facts indicate to the connection of immunosuppressive activity with native lipopolysaccharide of avirulent Salmonella.

[A method for checking the integrity and quality of membrane filters for sterilizing nutrient culture media]. / Sposob proverki tselostnosti, kachestva membrannykh fil'trov dlia sterilizatsii pitatel'nykh kul'tural'nykh sred.

A new method for checking up the quality of membrane filters for culture medium sterilization is proposed. A 50 ml syringe is used instead of "Millipore" system. The proposed method helps do without the expensive "Millipore" system to check the hermeticity, integrity of membrane filters, and to save operation time used for their preparation.

Recuperación de episodios de infección por Clostridium difficile tras la aplicación de un documento de consenso / Recovery from episodes of Clostridium difficile infection following the implementation of a consensus document

No disponible

Actualización en técnicas para diagnóstico microbiológico de infecciones fúngicas / Microbiological diagnosis of fungal infections: an update

Durante los últimos años se ha producido un incremento constante de las infecciones fúngicas, especialmente en pacientes inmunocomprometidos y sometidos a terapias invasivas, en los que un diagnóstico precoz es importante para su tratamiento y evolución debido a la alta mortalidad ocasionada por estas infecciones. Dadas las limitaciones del diagnóstico clásico, basado en la recuperación, cultivo y estudio de las características macroscópicas y microscópicas del hongo, se están desarrollando y aplicando nuevas técnicas basadas en la detección de diferentes metabolitos, antígenos y ácidos nucleicos fúngicos, así como anticuerpos producidos en respuesta a la infección, que facilitan el diagnóstico rápido de estas infecciones, en especial cuando varias de estas técnicas se utilizan combinadas. Algunos de estos nuevos métodos, en general dotados de buena sensibilidad y fiabilidad, requieren metodologías complejas, personal experimentado y están disponibles en muy pocos laboratorios. El presente trabajo muestra una revisión de las técnicas disponibles y otras en desarrollo para el diagnóstico de las infecciones fúngicas, su utilidad clínica y su posibilidad de utilización de forma rutinaria por los laboratorios clínicos y/o la necesidad de contar con laboratorios de referencia (AU)

Recent years have seen an increase in fungal infections, especially in immunocompromised patients undergoing invasive therapy. Conventional diagnosis methods based on culture are labour-intensive, time-consuming, have variable sensitivity, and in some cases are infeasible due to clinical factors. New assays such as detection of different metabolites, antigens, fungal nucleic acids and antibodies play an important role in the diagnosis and monitoring of response to therapy, especially when several of these techniques are used in combination. Some of these new methods with good sensitivity and reliability require complex methodologies, experienced personnel and are available in very few clinical laboratories. We present a review of the available techniques and other investigational platforms that have recently emerged as promising tools for the diagnosis of fungal infections, their clinical utility and their potential routine use in clinical laboratories and/or the need to rely on reference laboratorios (AU)