Contamination Of Cell Cultures Research Articles

Treatment of Microsporidium Nosema bombycis Spores with the New Antiseptic M250 Helps to Avoid Bacterial and Fungal Contamination of Infected Cultures without Affecting Parasite Polar Tube Extrusion.

Microsporidia are a group of widespread eukaryotic spore-forming intracellular parasites of great economic and scientific importance. Since microsporidia cannot be cultured outside of a host cell, the search for new antimicrosporidian drugs requires an effective antiseptic to sterilize microsporidian spores to infect cell lines. Here, we show that a new polyhexamethylene guanidine derivative M250, which is active against fungi and bacteria at a concentration of 0.5-1 mg/L, is more than 1000 times less effective against spores of the microsporidium Nosema bombycis, a highly virulent pathogen of the silkworm Bombyx mori (LC50 is 0.173%). Treatment of N. bombycis spores that were isolated non-sterilely from silkworm caterpillars with 0.1% M250 solution does not reduce the rate of spore polar tube extrusion. However, it completely prevents contamination of the Sf-900 III cell culture medium by microorganisms in the presence of antibiotics. The addition of untreated spores to the medium results in contamination, whether antibiotics are present or not. Since 0.1% M250 does not affect spore discharging, this compound may be promising for preventing bacterial and fungal contamination of microsporidia-infected cell cultures.

Prevention, Diagnosis and Eradication of Mycoplasma Contamination in Cell Culture.

Mycoplasmas, which are the smallest and simplest prokaryotes, lack a cell wall but possess the ability to undergo self-replication. Mycoplasma contamination is a common problem for laboratories engaging in cell culture. Due to their small size, Mycoplasmas can easily permeate filters designed to prevent bacterial and fungal contamination in cell culture. Although Mycoplasma contamination usually does not result in cell death, it can significantly affect cell proliferation, metabolism, and cause chromosomal aberrations. Therefore, it is crucial to detect and eliminate Mycoplasma contamination in cell culture. This step-by-step protocol presents a comprehensive approach to prevent Mycoplasma contamination in cell culture, as well as to detect and eradicate Mycoplasma to ensure accurate experimental and sequencing results.

Comparative genomic analysis of Mycoplasma related to cell culture for infB gene-based loop-mediated isothermal amplification.

Mycoplasma contamination in cell culture affects the properties of cell lines. Gold standard detection by microbiological culture takes days and requires specialists. The polymerase chain reaction and loop-mediated isothermal amplification (LAMP) are fast molecular options, but LAMP only requires one heating block for DNA amplification. This study presents a comparative genomic analysis of Mycoplasma species to identify common target genes different from the rrsA gene, which encodes 16S rRNA. The aim is to implement a LAMP assay to detect Mycoplasma species, reducing the time and specialized equipment required for detection. We performed a comparative genomic analysis through Mauve software and the GView server and selected infB and clpB genes as target candidates for designing LAMP primers. We evaluated both genes by multiple sequence alignment (MSA). The infB gene presented the best score MSA assessment with lower odd-log values (5,480,281) than other genes. We selected the infB gene to design LAMP primers specific to Mycoplasma spp. We used these primers to implement LAMP at 63°C for 30min, which showed 100% positive amplifications for detecting Mycoplasma spp. In conclusion, we present a methodology utilizing the infB gene-based LAMP assay to detect three of the six most prevalent Mycoplasma species in cell culture.

Detection of Microbiological Contamination in Cell Cultures and their Raw Materials: a 6 years report of this activity.

Contaminations in cell cultures are a source of concern for biotechnology laboratories, bio products manufacturers and cell banks as they involve economical loss, time loss and non-valid results. The main issues are fungi, bacteria (including mycoplasmas) and viruses. In this study, we present a retrospective of cell cultures quality control and their raw materials carried out at INMETRO throughout 6 years. From 2014 to 2019, different cell lines and raw materials derived from LABIO, from partner institutions of research projects, or from cooperation agreements were assessed. The detection of mycoplasmas were made using the biochemical method MycoAlert Plus. Contamination by bacteria and fungi were detected by a microbiological culturing according to the Brazilian Pharmacopoeia. Among the 944 assays carried out to detect bacteria and fungi; 5.6% presented contamination. Among 1143 assays of mycoplasmas detection; 10.8% presented contamination. Regarding raw materials assessment, 0.7% and 7.7% were contaminated with mycoplasmas and bacteria/fungi, respectively. In total, 8% of all samples presented contamination: 5.6% by mycoplasmas, 2.4% by bacteria/fungi and 0.1% by both. The dissemination of Good Practices on Cells Culture, the availability of these assays and the support of the implementation given to laboratories which are interested have been our contribution to improve control quality in the activity of cell cultures.

Classifying contaminated cell cultures using time series features

We examine the use of time series data, derived from Electric Cell-substrate Impedance Sensing (ECIS), to differentiate between standard mammalian cell cultures and those infected with a mycoplasma organism. With the goal of easy visualization and interpretation, we perform low-dimensional feature-based classification, extracting application-relevant features from the ECIS time courses. We can achieve very high classification accuracy using only two features, which depend on the cell line under examination. Initial results also show the existence of experimental variation between plates and suggest types of features that may prove more robust to such variation. Our paper is the first to perform a broad examination of ECIS time course features in the context of detecting contamination; to combine different types of features to achieve classification accuracy while preserving interpretability; and to describe and suggest possibilities for ameliorating plate-to-plate variation.

Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures.

In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a significant problem since such contamination can cause disastrous effects on eukaryotic cells by altering their cellular parameters, which, in turn, can lead to unreliable experimental results. For this reason, it is mandatory to carry out continuous testing for the presence of Mycoplasma in cell culture and the development of appropriate methodologies for this purpose. An ideal detection methodology should be fast, sensitive, and reliable. In this study, we propose an alternative detection method based on real-time PCR in conjunction with a novel combination of primers and probes that have been improved to increase their efficiency. The new PCR method demonstrates 100% sensitivity and specificity results in the detection of common Mycoplasma species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for Mycoplasma (24.4%) using the new PCR method (corresponding to 5 of the 14 lines tested (35.71%)), only 10 of 45 supernatants showed positive results with the commercial Venor®GeM qEP and Plasmotest® kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent Mycoplasma species, such as those related to the M. mycoides cluster. The use of an alternative Mycoplasma-detection method in cell culture labs can guarantee the detection of Mycoplasma contamination, especially in cases when dubious results are recorded.

A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures

Purpose This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures. Methods Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts. Results Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination. Conclusions Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.

Ultrasensitive qPCR platform for rapid detection of bacterial contamination of raw biological samples at the point of care

Contamination of cell cultures can result in a significant loss of precious biological material, particularly in long-term processes including amplification of chimeric antigen receptors (CAR)-T cells and differentiation of patient-derived stem cells, for therapeutic purposes. Bacterial contamination can also lead to more complex conditions such as sepsis which can cause morbidity and mortality, despite strict controls and good laboratory/manufacturing practices in the manipulation of complex biological samples such as blood used in autologous and allogeneic stem cells transplantation.The current standard method to identify biological risk is the set-up of microbial cultures, which can be time consuming with the likelihood of wasting large amounts of reagents in the event of contamination.Real-Time Polymerase Chain Reaction (qPCR) is a molecular method able to detect biological agents in a highly sensitive and specific way and in a short time. However, qPCR assays require complex DNA/RNA purification steps and expensive benchtop instruments, which may not always be available.This paper reports an extraction-free and low-volume protocol for qPCR in a standard instrument, which has been demonstrated to be effective on both Gram-positive (Gram+) and Gram-negative (Gram-) bacteria. Detection has been obtained from spiked cell culture samples, reaching a limit of detection (LOD) of 1 colony forming unit (CFU)/ml. To demonstrate the high potential of this optimized procedure, the same samples were also tested on a Point-Of-Care platform, which includes a cartridge with micro-chambers and a compact instrument, capable of performing qPCR with the same efficiency. Staphylococcus aureus (Gram+) was selected as the target for a proof of concept, achieving a LOD of 1 CFU/ml also on the portable device. The availability of these results paves the way for a simplified protocol for DNA extraction and amplification.

Comparison of Five Commercial Molecular Assays for Mycoplasma Testing of Cellular Therapy Products.

Testing of cellular therapy products for Mycoplasma is a regulatory requirement by the United States Food and Drug Administration (FDA) to ensure the sterility and safety of the product prior to release for patient infusion. The risk of Mycoplasma contamination in cell culture is high. Gold standard testing follows USP 63 which requires a 28-day agar and broth cultivation method that is impractical for short shelf-life biologics. Several commercial molecular platforms have been marketed for faster raw material and product release testing; however, little performance data are available in the literature. In this study, we performed a proof-of-principle analysis to evaluate the performance of five commercial molecular assays, including the MycoSEQ Mycoplasma detection kit (Life Technologies), the MycoTOOL Mycoplasma real-time detection kit (Roche), the VenorGEM qOneStep kit (Minerva Biolabs), the ATCC universal Mycoplasma detection kit, and the Biofire Mycoplasma assay (bioMérieux Industry) using 10 cultured Mollicutes spp., with each at four log-fold dilutions (1,000 CFU/mL to 1 CFU/mL) in biological duplicates with three replicates per condition (n = 6) to assess limit of detection (LOD) and repeatability. Additional testing was performed in the presence of tumor infiltrating lymphocytes (TILs). Based on LOD alone, the Biofire Mycoplasma assay was most sensitive followed by the MycoSEQ and MycoTOOL which were comparable. We showed that not all assays were capable of meeting the ≤10 CFU/mL LOD to replace culture-based methods according to European and Japanese pharmacopeia standards. No assay interference was observed when testing in the presence of TILs.

Identification of cell culture contamination by an unusual species of Mycoplasma related to the M. mycoides cluster.

Mycoplasma contamination is a significant problem in cell culture replication and maintenance. From more than 200 known species, a limited number of Mycoplasma species have been detected in cell cultures, representing new species or variants that can escape detection systems. A qPCR commercial kit was used for Mycoplasma detection in cell cultures. Furthermore, an amplified Mycoplasma species was sequenced and summited for sequence assembly, clustering, and evolutionary analysis study. Our work has identified a new and unusual variant or species of Mycoplasma that possesses a high degree of homology with species related with M. mycoides cluster. This variant is usually associated with cattle but has been detected contaminating a cell culture. Mycoplasma testing (even for unusual species) in cell cultures is essential to ensure the validity and reproducibility of research that uses cell cultures and to ensure the quality of cell line deposits in biobanks. For this reason, it is necessary to perform continuous checks for the absence of Mycoplasma in cell cultures and engage in the continuous adaptation of relevant detection systems.

IDENTIFICATION OF MYCOPLASMA CONTAMINATION IN CELL CULTURE

Mycoplasmas are the smallest and simplest prokaryotes wich were found in the endosomes of mammalian cells. They are widespread contaminants in cell cultures. It was identified a mycoplasma infection in a human breast cancer cell line. It was used fluorescence microscopy and nuclear affinity 4,6-diamidino-2-phenylindole (DAPI) staining. For the treatment of mycoplasma infection in cell culture, two antibiotics of the macrolide series (Tiamulin) and tetracyclines (Minocycline) was used. The effectiveness of combined antibiotic therapy against mycoplasmas has been proven, which was confirmed by the microscopic method. Therefore, treatment with combined antibiotics can completely eradicate mycoplasma infection from cultured cells.

Constant testing for Pestivirus in cell lines reveals different routes of contamination.

Pestivirus can contaminate cell cultures and sera and cause serious problems that evolve the integrity of studies, confidence in diagnostic results, and safety of human and animal vaccines. Contaminations by Pestivirus and other viruses may occur at any time and regular assays of monitoring in cell cultures and your supplies are necessary. This study aimed to analyze the phylogeny of Pestivirus detected from cell cultures, calf serum, and standard strains of three laboratories in Brazil that carry out frequent tests for the monitoring of cellular contaminations. These samples were submitted to phylogenetic analysis to understand the genetic relationship between contaminants occurring in these facilities. As result, the Pestivirus found in samples were Bovine viral diarrhea virus (BVDV-1 and BVDV-2), Hobi-like viruses (often named BVDV-3), and Classical swine fever virus (CSFV), and the phylogenetic analysis help us to infer at three possible routes of contamination in this work.

Mycoplasma testing: regulatory guidance and strategies for cGMP cell and gene therapy manufacturing

Mycoplasma, the smallest known free-living, self-replicating microorganisms, are potential contaminants of mammalian cell cultures, including biotherapeutic manufacturing cell cultures. As such, these cultures must be tested and are required to be free of Mycoplasma. Regulatory guidance globally for Mycoplasma testing has evolved, from strictly requiring culture-based testing methods to now accepting rapid molecular methods, including real-time quantitative PCR-based methods. This poster details a highly sensitive, specific, and robust method for rapid Mycoplasma detection for lot-release and in-process testing

Elucidating the Effectiveness of Antibiotic Treatments in Eliminating the Black Dots Contamination from Vero Cell Line Culture

The present study was conducted to identify the effectiveness of various antibiotics and antibiotic combinations in eliminating the black dots contamination in cell culture and to identify its effect on cell growth kinetics. Isolation of contaminant organism (Black dots) observed in our laboratory, was attempted by inoculation of harvested cell supernatant and pellet into Nutrient broth, Luria broth, and Pleuropneumonia-like organisms’ broth but no visible growth was observed. To analyze the effect of antibiotics in elimination of contamination, the cell monolayer was incubated with different antibiotics and combination of antibiotics till 120 hrs. Withdrawal effect of antibiotics was observed by further incubation cells in maintenance media for another 48 hrs. To investigate the effect of different antibiotics on cell growth kinetics, cell seeding was done in media containing antibiotics. A subset of cells was harvested every 24 hrs and counted till 120 hrs. Among all antibiotics, Ampicillin reduced the contamination, the Piperacillin and Tazobactam combination diminished the contamination to a great extent. At the studied concentrations of the antibiotic trio of piperacillin, tazobactam, and ampicillin, black dot contamination was eliminated and found 100% effective in inhibiting contamination within 120 hrs of the study period; but, after 48 hrs of antibiotic withdrawal, contamination reappears in cell culture. The growth kinetics showed no significant impact of antibiotics treatment on the cell growth curve. The growth kinetics showed that cell growth was not interfering significantly by individual or combination of antibiotics. Therefore, in the presence of black dot, a combination of three antibiotics (piperacillin, tazobactam, and ampicillin) should be effective in cell culture.

Optimization of in vitro culture conditions of common carp germ cells for purpose of surrogate production.

Common carp (Cyprinus carpio) is the fourth most-produced fish species in aquaculture and frequently used model species with significant effort invested in development of biotechnological applications. In present study, we attempted to establish an in vitro germ cell culture condition for short term cell culture, which could facilitate further applications such as surrogacy or gene manipulation. Basal media and different types of feeder cells were investigated to optimize carp germ cell culture condition to favor maintenance of mitotic proliferation. Results indicated that germ cells cultured with hESC media and RTG2 cell line as feeder possessed significantly higher proliferation and survival rate compared to that cultured with StemPro media and Sertoli cell line as feeder. In addition, we compared two dissection strategies to compare risk of cell culture contamination and body cavity was open from dorsal part or from ventral part. As a result, carp open from the dorsal side can minimize the risk of contamination. In summary, this is the first study to optimize the cultivation of germ cells in common carp. This opens up new opportunities for the application of specific techniques in the breeding of those species with high commercial value and frequent use as a model fish. Results obtained in this study are important for implementation of new strategies in common carp breeding, conservation of genetic resources, restoration of lines or development of clonal and isogenic carp lines.

MP06-06 ESTABLISHMENT AND EVALUATION OF A SIMPLIFIED APPROACH TO PATIENT DERIVED BLADDER CANCER ORGANOIDS USING URINE

You have accessJournal of UrologyCME1 May 2022MP06-06 ESTABLISHMENT AND EVALUATION OF A SIMPLIFIED APPROACH TO PATIENT DERIVED BLADDER CANCER ORGANOIDS USING URINE Paul Pollehne, Ivonne Aidee Montes Mojarro, Johannes Schneider, Bastian Amend, Falko Fend, Arnulf Stenzl, Wilhelm K. Aicher, and Niklas Harland Paul PollehnePaul Pollehne More articles by this author , Ivonne Aidee Montes MojarroIvonne Aidee Montes Mojarro More articles by this author , Johannes SchneiderJohannes Schneider More articles by this author , Bastian AmendBastian Amend More articles by this author , Falko FendFalko Fend More articles by this author , Arnulf StenzlArnulf Stenzl More articles by this author , Wilhelm K. AicherWilhelm K. Aicher More articles by this author , and Niklas HarlandNiklas Harland More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patient derived organoids (PDO) are a promising tool for research and individualized therapy. The cells that are used to culture organoids are commonly retrieved from invasive surgery, requiring general anesthesia. In this study we evaluate a simplified approach through PDOs out of rinsing urine from patients with urothelial carcinoma. METHODS: Urine cells were isolated and after using an erythrocyte lysis buffer were resuspended with Matrigel® (BME) and covered with an PDO medium containing the wnt-agonist r-spondin, fibroblast-growth- factors 2, 7 and 10, the ROCK-inhibitor Y-27632 dihydrochloride and epidermial growth factor. The growth of the PDOs was observed by measuring the diameters by light microscopy. When the PDOs ceased to grow, the cells were passaged. After 5 passages the PDOs were confirmed as established organoid cell lines. For characterization the cells were transferred into chamber slides and stained using primary antibodies against urothelial and epithelial markers (GATA-3, AE1/AE3, CK5, CK7), tumor markers (p53, TP63, CD276, CK20, FGFR-3, Vimentin, S-100-P, Ki67) and stem cell markers (CD24, CD44). RESULTS: In 35 out of 35 samples (100%), cells were isolated without any contamination in cell cultures. Spheroid growth was observed in 27 samples in primary culture (77%), in 18 samples in 1st passage (51%) and in 10 samples in 2nd passage (29%). One of the samples (3%) reached the 5th passage (UCO#33). In 23 associated solid tumor samples were retrieved during transurethral resection of a bladder tumor. Out of these, PDOs were established in 19 samples in primary culture (83%), 9 samples in 1st passage (39%) and 3 samples in 2nd passage (13%)). None of the samples (0%) reached the 5th passage. Compared tocell cultures which ceased growth before the 5th passage UCO#33 increased in diameter in later passages and showed a significant higher expression of CD276 and as the only culture a coexpression of CD24 and CD44. The expression of CK7 and Ki67 increased from 3rd to 5th passage while the expression of TP63, CK20, Vimentin and CD24 decreased. CONCLUSIONS: In this project we were able to grow PDOs out of rinsing urine from patients with bladder cancer, whose growth are comparable to the ones out of solid tumor samples. We identified immune checkpoint CD276 and stem cell markers CD24/CD44 as potential markers for the success of organoid growth out of urine. PDOs out of rinsing urine are promising tools for research and as well for clinical use. Source of Funding: The work was funded by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 2543/I) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e79 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Paul Pollehne More articles by this author Ivonne Aidee Montes Mojarro More articles by this author Johannes Schneider More articles by this author Bastian Amend More articles by this author Falko Fend More articles by this author Arnulf Stenzl More articles by this author Wilhelm K. Aicher More articles by this author Niklas Harland More articles by this author Expand All Advertisement PDF DownloadLoading ...

Effect of Faradarmani Consciousness Field on Cell Culture, Bacterial Contamination of Cell Culture and SARS-CoV-2 Replication in vitro

The highly contagious and life-threatening coronavirus SARS-CoV-2 that has caused a global outbreak since December 2019 (COVID-19) is a beta coronavirus, which has spread rapidly and become a worldwide pandemic. So far, there is no definitive treatment for this disease. The Faradarmani Consciousness Field as one of many Taheri Consciousness Fields (TCFs) introduced by Mohammad Ali Taheri, is a novel field that is neither matter nor energy. Therefore, it is non-quantifiable and cannot be directly observed or measured. However, it is possible to demonstrate and measure the effects of these fields through standard scientific experiments. The present work aimed to study the effect of the Faradarmani CF on SARS-CoV-2 replications and contaminated cell culture flasks by the bacteria. According to the results, in virus culture, Faradarmani CF caused induction of virus proliferation. In contaminated cell culture flasks by the bacteria; significant differences were found in the color, turbidity, and viability of the cultured Vero cells between TCF treatment and control groups, which demonstrated the significant effect of Faradarmani CF. Further studies are highly recommended to investigate the effect of TCFs in vivo.

The ratio of nicotinic acid to nicotinamide as a microbial biomarker for assessing cell therapy product sterility

Controlling microbial risks in cell therapy products (CTPs) is important for product safety. Here, we identified the nicotinic acid (NA) to nicotinamide (NAM) ratio as a biomarker that detects a broad spectrum of microbial contaminants in cell cultures. We separately added six different bacterial species into mesenchymal stromal cell and T cell culture and found that NA was uniquely present in these bacteria-contaminated CTPs due to the conversion from NAM by microbial nicotinamidases, which mammals lack. In cells inoculated with 1 × 104 CFUs/mL of different microorganisms, including USP <71> defined organisms, the increase in NA to NAM ratio ranged from 72 to 15,000 times higher than the uncontaminated controls after 24 h. Importantly, only live microorganisms caused increases in this ratio. In cells inoculated with 18 CFUs/mL of Escherichia coli, 20 CFUs/mL of Bacillus subtilis, and 10 CFUs/mL of Candida albicans, significant increase of NA to NAM ratio was detected using LC-MS after 18.5, 12.5, and 24.5 h, respectively. In contrast, compendial sterility test required >24 h to detect the same amount of these three organisms. In conclusion, the NA to NAM ratio is a useful biomarker for detection of early-stage microbial contaminations in CTPs.

Integrated microfluidic device to monitor unseen Escherichia coli contamination in mammalian cell culture

Microbial contamination in in vitro cell culture technologies is a common problem endangering the reliability of the collected data and biosafety of the biological products. Conventionally, microbial contamination can be detected by monitoring the pH and turbidity of cell culture media when using light microscope and/or molecular probes. These methods have limitations such as incompatibility with some cell lines, requirement for human intervention, and long experimental times. Here we present a new integrated microfluidic device (IMD) for the detection of the presence of Escherichia coli (E. coli) in benign prostatic hyperplasia (BPH-1) cell culture. Cell culture media acidity was monitored using an optofluidic sensor, and the cellular glucose metabolism using an electrochemical sensor having in-line continuous monitoring capability. Using this IMD, detection of a bacterial contamination at stages in which it is undetectable using a light microscope and plate counting methods is achieved. Additionally, since even the short-term (less than 8 h) presence of E. coli can change the cellular metabolism in the long term (at least up to 24 h), the use of non-invasive continuous cell culture monitoring platforms significantly reduce the risk of an undetected contamination, increasing the reliability of the conducted experiments with no interference to the cell culture process.

Mycoplasma decontamination in Chlamydia trachomatis culture: a curative approach.

Mycoplasma contamination of cell culture represents a serious problem in research and decontamination from cell-propagated obligate intracellular bacteria has proven challenging. Here, we presented an optimized protocol to remove Mycoplasma from contaminated Chlamydia trachomatis culture. A stepwise procedure of Mycoplasma removal entails (i) incubation in nonionic detergent-containing solution and (ii) separation of viable chlamydial organisms by fluorescence-activated cell sorting (FACS), followed by subcloning using a focus-forming assay. We also adapted a polymerase chain reaction (PCR) assay using paired universal and Mycoplasma-specific primers, which are distinguishable from the C. trachomatis counterparts, in combination with Sanger sequencing to determine the presence of mycoplasmas' 16S rRNA genes. These integrated approaches allow for full removal of Mycoplasma, as verified by the improved PCR assay, without compromising the capacity of viable C. trachomatis to adapt to new infection in epithelial cells. Some pitfalls during the Mycoplasma decontamination process are discussed.