Media Recipes Research Articles

Integrated bioprocessing and genetic strategies to enhance soluble expression of anti-HER2 immunotoxin in E. Coli

Immunotoxins are widely applied for cancer therapy. However, bacterial expression of immunotoxins usually leads to the formation of insoluble and non-functional recombinant proteins. This study was aimed to improve soluble expression of a novel anti-HER2 immunotoxin under the regulation of the trc promoter in Escherichia coli by optimization of the cultivation conditions using response surface methodology (RSM). To conduct RSM, four cultivation variables (i.e., inducer concentration, post-induction time, post-induction temperature, and medium recipe), were selected for statistical characterization and optimization using the Box-Behnken design and Design Expert software. Based on the developed model using the Box-Behnken design, the optimal cultivation conditions for soluble expression of anti-HER2 immunotoxin were determined to be 0.1 mM IPTG for induction in the LB medium at 33 °C for 18 h. The expressed immunotoxin was successfully purified using affinity chromatography with more than 90% purity and its bioactivity was confirmed using cell-based ELISA. Technical approach developed in this study can be generally applied to enhance the production yield and quality of recombinant proteins using E. coli as the gene expression system.

Improved production of RNA-inhibiting antimicrobial peptide by Bacillus licheniformis MCC 2514 facilitated by a genetic algorithm optimized medium.

One of the significant challenges during the purification and characterization of antimicrobial peptides (AMPs) from Bacillus sp. is the interference of unutilized peptides from complex medium components during analytical procedures. In this study, a semi-synthetic medium was devised to overcome this challenge. Using a genetic algorithm, the production medium of AMP is optimized. The parent organism, Bacillus licheniformis MCC2514, produces AMP in very small quantities. This AMP is known to inhibit RNA biosynthesis. The findings revealed that lactose, NH4Cl and NaNO3 were crucial medium constituents for enhanced AMP synthesis. The potency of the AMP produced was studied using bacterium, Kocuria rhizophila ATCC 9341. The AMP produced from the optimized medium was eightfold higher than that produced from the unoptimized medium. Furthermore, activity was increased by 1.5-fold when cultivation conditions were standardized using the optimized medium. Later, AMP was produced in a 5 L bioreactor under controlled conditions, which led to similar results as those of shake-flask production. The mode of action of optimally produced AMP was confirmed to be inhibition of RNA biosynthesis. Here, we demonstrate that improved production of AMP is possible with the developed semi-synthetic medium recipe and could help further AMP production in an industrial setup.

High-level expression of leghemoglobin in Kluyveromyces marxianus by remodeling the heme metabolism pathway.

Soy leghemoglobin, when bound to heme, imparts a meat-like color and flavor and can serve as a substitute for animal-derived proteins. Enhancing cellular heme synthesis improves the recombinant expression of leghemoglobin in yeast. To achieve high-level expression of leghemoglobin A (LBA) in Kluyveromyces marxianus, a food-safe yeast, large-scale heme synthesis modules were transferred into K. marxianus using yeast artificial chromosomes (KmYACs). These modules contained up to 8 native and heterologous genes to promote the supply of heme precursors and downstream synthesis. Next, eight genes inhibiting heme or LBA synthesis were individually or combinatorially deleted, with the lsc1Δssn3Δ mutant yielding the best results. Subsequently, heme synthesis modules were combined with the lsc1Δssn3Δ mutant. In the resulting strains, the module genes were all actively expressed. Among these module genes, heterologous S. cerevisiae genes in the downstream heme synthesis pathway significantly enhanced the expression of their counterparts in K. marxianus, resulting in high heme content and LBA yield. After optimizing the medium recipe by adjusting the concentrations of glucose, glycine, and FeSO4·7H2O, a heme content of 66.32mg/L and an intracellular LBA titer of 7.27g/L were achieved in the engineered strain in a 5L fermentor. This represents the highest intracellular expression of leghemoglobin in microorganisms to date. The leghemoglobin produced by K. marxianus can be utilized as a safe ingredient for plant-based protein products.

The unbearable ignorance of the composition of IVF culture media

Culture media play an essential role in the success of IVF. Their composition has undergone major modifications over the 45 years since the birth of Louise Brown. Most IVF programmes now rely on commercially produced media, which they buy in small vials, guaranteed to be sterile and non-embryotoxic. Unfortunately, information about the components of the culture media and their concentrations is no longer available. Arguing that culture media recipes are proprietary, relevant commercial interests have stopped labelling their products with this vital information. Given the critical role that is played by culture media in the success of IVF, as well as the subsequent health of the children who are born after IVF, this information should not remain a ‘company secret’. Clinicians and scientists working in IVF must insist that the labelling of culture media includes all of the constituents and their concentrations. Only in this way can we monitor the influence of culture media on IVF outcomes, innovate and continue to advance the field of IVF.

The Effectiveness of Different Types of PDA Culture Media on the Mycelial Growth of Shimeji Mushroom

This experiment aims to compare different types of PDA culture media recipes, including potato, sweet potato, pumpkin, and sugar banana, for their effects on the growth of Shimeji mushroom mycelium. It also aims to provide alternative options for formulating culture media for Shimeji mushroom spawn cultivation. A completely randomized design (CRD) was employed for the experiment, with four treatments and five replicates each, as follows: (T1) potato-based media; (T2) sweet potato-based media; (T3) pumpkin-based media; (T4) sugar banana-based media. Data collection involved daily measurements of the length (in centimeters) and width (in centimeters) for a period of seven days. After the experiment concluded, data analysis was conducted to determine the variation and compare the mean values of the treatments using Duncan's Multiple Range Test (DMRT) and package software. The experimental results showed that over a period of 3, 5, and 7 days, the potato-based media (T1) exhibited the highest growth of Shimeji mushroom mycelium and the highest mycelium density values, measuring 6.2000, 3.2000, and 0.0000a (density scale +++). The pumpkin-based media (T3) had lower mean values of 0.9000, 3.1600, and 0.0000a (density scale +) for mycelium growth and density. Therefore, it can be concluded that among the four PDA media recipes tested over a period of 3, 5, and 7 days, the potato-based media (T1) demonstrated the highest mycelium growth and density values of approximately 6.2000, 3.2000, and 0.0000a (density scale +++).

Ragam Bahan Untuk Pembuatan Pakan Khusus Yang Diberikan Kepada Pasangan Kerbau Sumbawa Karapan Sebelum Bertanding (Studi Di Kecamatan Empang Kabupaten Sumbawa)

A survey was conducted in Empang sub-district, which has many Sumbawa buffalo racing enthusiasts. Before the animals are competed, are they given special feed/herbs? What are their chances of winning? This is what the research will seek to answer, as there is no written information on this subject. The survey found that all farmers give special feed/herbs to their raced sumbawa buffaloes, but the ingredients/recipes differ, ranging from the most complete recipe consisting of 17 ingredients, the medium recipe consisting of 12 ingredients, and the minimalist recipe consisting of 9 ingredients. All of them have won. The complete recipe won first place (43 pairs of buffalo), the medium recipe won second place (48 pairs of buffalo), and the minimalist recipe (29 pairs of buffalo) won third place.

OptimizedPrimary Culture of Neuronal Populations for Subcellular OmicsApplications.

Primary cell culture is an invaluable method frequently used to overcome challenges associated with in vivo experiments. In zebrafish research, in vivo live imaging experiments are routine owing to the high optical transparency of embryos, and, as a result, primary cell culture has been less utilized. However, the approach still boasts powerful advantages, emphasizing the importance of sophisticated zebrafish cell culture protocols. Here, we present an enhanced protocol for the generation of primary cell cultures by dissociation of 24 hpf zebrafish embryos. We include a novel cell culture medium recipe specifically favoring neuronal growth and survival, enabling relatively long-term culture. We outline primary zebrafish neuronal culture on glass coverslips, as well as in transwell inserts which allow isolation of neurite tissue for experiments such as investigating subcellular transcriptomes.

Effect of Methionine on Gene Expression in Komagataella phaffii Cells.

Komagataella phaffii yeast plays a prominent role in modern biotechnology as a recombinant protein producer. For efficient use of this yeast, it is essential to study the effects of different media components on its growth and gene expression. We investigated the effect of methionine on gene expression in K. phaffii cells using RNA-seq analysis. Several gene groups exhibited altered expression when K. phaffii cells were cultured in a medium with methanol and methionine, compared to a medium without this amino acid. Methionine primarily affects the expression of genes involved in its biosynthesis, fatty acid metabolism, and methanol utilization. The AOX1 gene promoter, which is widely used for heterologous expression in K. phaffii, is downregulated in methionine-containing media. Despite great progress in the development of K. phaffii strain engineering techniques, a sensitive adjustment of cultivation conditions is required to achieve a high yield of the target product. The revealed effect of methionine on K. phaffii gene expression is important for optimizing media recipes and cultivation strategies aimed at maximizing the efficiency of recombinant product synthesis.

Fed intestinal solubility limits and distributions applied to the Developability classification system

For solid oral dosage forms drug solubility in intestinal fluid is an important parameter influencing product performance and bioavailability. Solubility along with permeability are the two parameters applied in the Biopharmaceutics and Developability Classification Systems (DCS) to assess a drug’s potential for oral administration. Intestinal solubility varies with the intestinal contents and the differences between the fasted and fed states are recognised to influence solubility and bioavailability. In this study a novel fed state simulated media system comprising of nine media has been utilised to measure the solubility of seven drugs (ibuprofen, mefenamic acid, furosemide, dipyridamole, griseofulvin, paracetamol and acyclovir) previously studied in the fasted state DCS. The results demonstrate that the fed nine media system provides a range of solubility values for each drug and solubility behaviour is consistent with published design of experiment studies conducted in either the fed or fasted state. Three drugs (griseofulvin, paracetamol and acyclovir) exhibit very narrow solubility distributions, a result that matches published behaviour in the fasted state, indicating that this property is not influenced by the concentration of simulated media components. The nine solubility values for each drug can be utilised to calculate a dose/solubility volume ratio to visualise the drug’s position on the DCS grid. Due to the derivation of the nine media compositions the range and catergorisation could be considered as bioequivalent and can be combined with the data from the original fed intestinal fluid analysis to provide a population based solubility distribution. This provides further information on the drugs solubility behaviour and could be applied to quality by design formulation approaches. Comparison of the fed results in this study with similar published fasted results highlight that some differences detected match in vivo behaviour in food effect studies. This indicates that a combination of the fed and fasted systems may be a useful in vitro biopharmaceutical performance tool. However, it should be noted that the fed media recipes in this study are based on a liquid meal (Ensure Plus) and this may not be representative of alternative fed states achieved through ingestion of a solid meal. Nevertheless, this novel approach provides greater in vitro detail with respect to possible in vivo biopharmaceutical performance, an improved ability to apply risk-based approaches and the potential to investigate solubility based food effects. The system is therefore worthy of further investigation but studies will be required to expand the number of drugs measured and link the in vitro measurements to in vivo results.

Biofouling Control in an Anaerobic Membrane Bioreactor with Indigenous Quorum Quenching Bacterium Micrococcus luteus Strain AnQ-m1

This study isolated and screened indigenous strains quenching the acyl homoserine lactone (AHL)-mediated quorum sensing system and investigated the effectiveness of a selected strain for biofouling control in a laboratory-scale anaerobic membrane bioreactor (AnMBR). Among the 11 facultative anaerobes isolated from anaerobic sludge, Micrococcus luteus strain AnQ-m1 was screened out as the most potent AHL degrader. Its AHL degradability was further examined while encapsulating the cells in polysulfone–alginate beads to optimize the encapsulation medium recipe. The QQ beads with immobilized AnQ-m1 cells retarded the apparent membrane fouling rate by 2.2 times compared with the AnMBR with vacant beads. C8-HSL, the dominant AHL in AnMBRs, was effectively (∼70%) quenched in the QQ-AnMBR before the membrane was fouled; however, its concentration was increased to the normal level when the membrane was fouled. Polysaccharides in a loosely bound extracellular polymeric substance were gradually reduced at a constant rate during the QQ stage, while the indigenous QQ strain barely affected the microbial community structure of the bulk sludge and the water treatment performance of the AnMBR. These findings provide new insights into the QQ-based biofouling control strategy and highlight the beneficial effects of indigenous QQ strains on AnMBR stability.

Small scale in vitro method to determine a potential bioequivalent equilibrium solubility range for fed human intestinal fluid

Intestinal drug solubility is a key parameter controlling oral absorption but varies both intra and inter individuals and between the fasted and fed states, with food intake known to alter the bioavailability of many compounds. Intestinal solubility can be measured in vitro either using sampled fed human intestinal fluid (FeHIF) or simulated fed intestinal fluid (SIF) but neither approach is optimal. FeHIF is difficult to obtain and variable, whilst for fed SIF multiple recipes are available with no consensus on the ideal version. A recent study characterised FeHIF aspirates using a multidimensional approach and calculated nine simulated media recipes that covered over ninety percent of FeHIF compositional variability. In this study the equilibrium solubility of thirteen drugs have been measured using the nine simulated media recipes and compared to multiple previous design of experiment (DoE) studies, which have examined the impact of fed SIF media components on solubility. The measured nine media solubility data set is only statistically equivalent to the large scale 92 media DoE in 4 out of 13 drug comparisons, but has improved equivalence against small scale DoEs (9 or 10 media) with 6 out of 9 or 10 out of 12 (9 and 10 media respectively) equivalent. Selective removal of non-biorelevant compositions from the 92 media DoE improves statistical equivalence to 9 out of 13 comparisons. The results indicate that solubility equivalence is linked to media component concentrations and compositions, the nine media system is measuring a similar solubility space to previous systems, with a narrower solubility range than the 92 point DoE but equivalent to smaller DoE systems. Phenytoin and tadalafil display a narrow solubility range, a behaviour consistent with previous studies in fed and fasted states and only revealed through the multiple media approach. Custom DoE analysis of the nine media results to determine the most statistically significant component influencing solubility does not detect significant components. Indicating that the approach has a low statistical resolution and is not appropriate if determination of media component significance is required. This study demonstrates that it is possible to assess the fed intestinal equilibrium solubility envelope using the nine media recipes obtained from a multi-dimensional analysis of fed HIF. The derivation of the nine media compositions coupled with the results in this study indicate that the solubility results are more likely to reflect the fed intestinal solubility envelope than previous DoE studies and highlight that the system is worthy of further investigation.

An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies.

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.

A Two-Step Process for Improved Biomass Production and Non-Destructive Astaxanthin and Carotenoids Accumulation in Haematococcus pluvialis

Carotenoids extracted from microalgae have a considerable economic interest in numerous high-value markets. Natural astaxanthin has gained much interest in its powerful antioxidant properties, however, its commercial-scale production is still challenging. In this study, a simple and economical way to cultivate Haematococcus pluvialis (CCAP 34/1D) by a two-step process was investigated by exploring alternative strategies to maximise algal growth and astaxanthin yield. During the first step—improving biomass production—four nitrogen sources were tested (NH4Cl, NaNO3, Fe(III)NO3, and urea). The second step—carotenogenesis induction—was achieved by using a mix of moderate stressors that worked in synergy (i.e., mild light, nitrogen limitation, the addition of sodium acetate at 0.25% w/v), thereby minimising potential losses of the accumulated biomass caused, for example, by photobleaching or nitrogen starvation. Results showed that urea was the nitrogen source, allowing the highest cell density and growth rate. In terms of carotenogenesis induction, the use of mild stressors resulted in three out of four treatments having a relative increase in cell number (13.8–26.7%) and a concomitant increase in astaxanthin yield. Simple low-cost strategies, such as small adjustments to media recipes and synergism between mild stressors, could bring a disproportionate effect on the future successes of making algal biotechnology a widespread reality.

Green sorption media for the removal of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) from water

This study investigated the removal of selected per- and polyfluoroalkyl substances (PFAS) from water via two green sorption media (IFGEM-7 and AGEM-2). Both selected green sorption media recipes contain sand (85–91%) and clay (3–4%), in addition to recycled iron (Fe) (5–7.5%) or aluminum (Al) (4.5% in AGEM-2 only). Batch and column studies were integrated and performed using the prescribed green sorption media recipes to determine their efficiencies in removing two most targeted PFAS, including perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). In the batch test, while the removal efficiencies of PFOS ranged from 27 to 46% and 23 to 42%, those for PFOA ranged from 6 to 16% and 5 to 18% when using IFGEM-7 and AGEM-2, respectively. The higher removal of PFOS than PFOA observed in both IFGEM-7 and AGEM-2 batch tests could be attributed to higher media affinity for sulfonate groups of PFOS when compared to the carboxylate groups of PFOA. In the column study, the initial removal (within 1 h) by IFGEM-7 was greater than 99% for PFOS and 28% for PFOA. When comparing different dynamic adsorption models, it appears that the non-linear equations could better describe the trend of experimental data compared to the linear forms of the Modified Dose Response model. Life expectancy calculations, performed for demonstration purposes of field applications, suggested that if IFGEM-7 were to be applied in a downflow filter box to treat a hypothetical volume of 60,000 L of water during an emergency response, and it may last for 1506 h (62.8 d) and 4.2 h for a target removal of 80% of PFOS and PFOA, respectively.

Efficient Method to Differentiate Mouse Embryonic Stem Cells into Macrophages in vitro.

Macrophages are key cells in the innate immune system and play a role in a variety of diseases. However, macrophages are terminally differentiated and difficult to manipulate genetically via transfection or through CRISPR-Cas9 gene editing. To overcome this limitation, we provide a simplified protocol for the generation of mouse embryonic stem cells-derived macrophages (ESDM). Thus, genetic manipulation can be performed using embryonic stem cells, selecting for the desired changes, and finally producing macrophages to study the effects of the previous genetic manipulation. These studies can contribute to many areas of research, including atherosclerosis and inflammation. Production of ESDM has been previously achieved using embryoid body (EB) intermediates. Here, we optimized the EB method using a simplified medium, reducing the number of recombinant proteins and medium recipes required. Our EB-based differentiation protocol consists of three stages: 1) floating EB formation; 2) adherence of EBs and release of floating macrophage progenitors; and, 3) terminal differentiation of harvested macrophage progenitors. The advantages of this protocol include achieving independent floating EBs in stage 1 by using a rocker within the tissue culture incubator, as well as the exclusion of small EBs and cell clusters when harvesting macrophage progenitors via cell filtration.

Transcriptome Analysis Unveils the Effects of Proline on Gene Expression in the Yeast Komagataella phaffii.

Komagataella phaffii yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast K. phaffii was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when K. phaffii was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by K. phaffii even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of AOX1 gene promoter with proline can be partially reversed by the deletion of the KpPUT4.2 gene.

Small scale in vitro method to determine a bioequivalent equilibrium solubility range for fasted human intestinal fluid

Drug solubility is a key parameter controlling oral absorption, but intestinal solubility is difficult to assess in vitro. Human intestinal fluid (HIF) aspirates can be applied but they are variable, difficult to obtain and expensive. Simulated intestinal fluids (SIF) are a useful surrogate but multiple recipes are available and the optimum is unknown. A recent study characterised fasted HIF aspirates using a multi-dimensional approach and determined nine bioequivalent SIF media recipes that represented over ninety percent of HIF compositional variability. In this study these recipes have been applied to determine the equilibrium solubility of twelve drugs (naproxen, indomethacin, phenytoin, piroxicam, aprepitant, carvedilol, zafirlukast, tadalafil, fenofibrate, griseofulvin, felodipine, probucol) previously investigated using a statistical design of experiment (DoE) approach. The bioequivalent solubility measurements are statistically equivalent to the previous DoE, enclose literature solubility values in both fasted HIF and SIF, and the solubility range is less than the previous DoE. These results indicate that the system is measuring the same solubility space as literature systems with the lower overall range suggesting improved equivalence to in vivo solubility, when compared to DoEs. Three drugs (phenytoin, tadalafil and griseofulvin) display a comparatively narrow solubility range, a behaviour that is consistent with previous studies and related to the drugs’ molecular structure and properties. This solubility behaviour would not be evident with single point solubility measurements. The solubility results can be analysed using a custom DoE to determine the most statistically significant factor within the media influencing solubility. This approach has a lower statistical resolution than a formal DoE and is not appropriate if determination of media factor significance for solubilisation is required. This study demonstrates that it is possible to assess the fasted intestinal equilibrium solubility envelope using a small number of bioequivalent media recipes obtained from a multi-dimensional analysis of fasted HIF. The derivation of the nine bioequivalent SIF media coupled with the lower measured solubility range indicate that the solubility results are more likely to reflect the fasted intestinal solubility envelope than previous DoE studies and highlight that intestinal solubility is a range and not a single value.

Methods for Isolation and Recovery of Bifidobacteria.

Since their discovery, bifidobacteria have been considered to represent cornerstone commensal microorganisms in the host-microbiome interface at the intestinal level. Bifidobacteria have therefore enjoyed increasing scientific and commercial interest as a source of microorganisms with probiotic potential. However, since functional and probiotic traits are strictly strain-dependent, there is a constant need to isolate, cultivate, and characterize novel strains, activities that require the utilization of appropriate media, as well as robust isolation, cultivation, and preservation techniques. Besides, effective isolation of bifidobacteria from natural environments might require different manipulation and cultivation media and conditions depending on the specific characteristics of the sample material, the presence of competitive microbiota, the metabolic state in which bifidobacteria might be encountered within the sample and the particular metabolic traits of the bifidobacterial species adapted to such inhabitation.A wide array of culture media recipes have been described in the literature to routinely isolate and grow bifidobacteria under laboratory conditions. However, there is not a single and universally applicable medium for effective isolation, recovery, and cultivation of bifidobacteria, as each growth medium has its own particular advantages and limitations. Besides, the vast majority of these media formulations was not specifically formulated for these microorganisms, and thus information on bifidobacterial cultivation options is scarce while being scattered throughout literature. This chapter intends to serve as a resource summarizing the options to cultivate bifidobacteria that have been described to date, highlighting the main advantages and limitations of each of them.

Combination of soluble factors and biomaterial scaffolds enhance human adipose-derived stem/stromal cell myogenesis

Volumetric muscle loss and muscle degeneration are conditions for which there are currently no effective treatment options. Human adipose stem cells (hASCs) offer promise in cell-based regenerative therapies to treat muscle damage due to their ability to self-renew and differentiate. However, in the absence of universal culture conditions that yield greater than 15% myogenic differentiation, the clinical potential of these cells is limited. Here we report on the evaluation of two different media recipes, three extracellular matrix (ECM) proteins, and a poly (ethylene glycol) (PEGDMA) hydrogel with a physiologically relevant elasticity to determine how the extracellular chemical and physical environment work together to enhance myogenic differentiation of hASCs. Our results identify a combination of unique biochemical and physical factors that promote myogenesis, laying the groundwork for creating a scaffold and culture medium that will effectively and efficiently direct myogenic differentiation of adult stem cells for clinical applications in the future.

Vibrio fischeri: Laboratory Cultivation, Storage, and Common Phenotypic Assays.

Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species that can be readily grown and stored with common laboratory equipment. In this article, protocols for routine growth, storage, and phenotypic assessment of V. fischeri, as well as recipes for useful media, are included. Specifically, this article describes procedures and considerations for growth of this microbe in complex and minimal media. It also describes assays for biofilm formation, motility, and bioluminescence, three commonly assessed phenotypes of V. fischeri. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth of V. fischeri from frozen stocks Basic Protocol 2: Growth of V. fischeri in rich, undefined liquid medium Alternate Protocol 1: Growth of V. fischeri in minimal medium Basic Protocol 3: Storage of V. fischeri in frozen stocks Basic Protocol 4: Biofilm assay on solid agar Alternate Protocol 2: Biofilm assay in shaking liquid culture Alternate Protocol 3: Biofilm assay in static liquid culture Basic Protocol 5: Motility assay Basic Protocol 6: Luminescence assay.