Low Culture Temperature Research Articles

Comparing the Effects of Two Culture Methods to Determine the Total Heterotrophic Bacterial Colony Count in Hospital Purified Water

BackgroundAccurately detecting the quantity of microorganisms in hospital purified water is of significant importance for early identification of microbial contamination and reducing the occurrence of water-borne hospital infections. The choice of detection method is a prerequisite for ensuring accurate results. Traditional Plate Count Agar (PCA) belongs to a high-nutrient medium, and there may be limitations in terms of accuracy or sensitivity in detecting microorganisms in hospital purified water. On the other hand, Reasoner’s 2A agar (R2A) has characteristics, such as low-nutrient levels, low cultivation temperature, and extended incubation time, providing advantages in promoting the growth of aquatic microorganisms. This study, through comparing the differences in total colony counts between two detection methods, aims to select the method more suitable for the growth of aquatic microorganisms, offering new practical insights for accurately detecting the total count of heterotrophic bacteria in hospital purified water.MethodsThe most commonly used plate count agar (PCA) method, and the R2A agar culture were adopted to detect microorganisms and determine the total number of bacterial colonies in the water for oral diagnosis and treatment water and terminal rinse water for endoscopes in medical institutions. The two water samples were inoculated by pour plate and membrane filtration methods, respectively. Using statistical methods including Spearman and Pearson correlation, Wilcoxon signed-rank sum test, paired-Chi-square test, and linear regression, we analyze the differences and associations in the bacterial counts cultivated through two different methods.ResultsIn 142 specimens of the water, the median and interquartile range of the heterotrophic bacterial colony number under the R2A culture method and under the PCA culture method were 200 (Q1–Q3: 25–18,000) and 6 (Q1–Q3: 0–3700). The total number of heterotrophic bacteria colonies cultured in R2A medium for 7 days was more than that cultured in PCA medium for 2 days (P < 0.05). The linear regression results showed a relatively strong linear correlation between the number of colonies cultured by the R2A method and that cultured by the PCA method (R2 = 0.7264). The number of bacterial species detected on R2A agar medium is greater than that on PCA agar medium.ConclusionThe R2A culture method can better reflect the actual number of heterotrophic bacterial colonies in hospital purified water. After logarithmic transformation, the number of colonies cultured by the two methods showed a linear correlation.

Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis.

Introduction: Autologous transplantation of spermatogonial stem cells (SSCs) isolated from cryopreserved testicular biopsies obtained before oncological treatment could restore fertility in male childhood cancer survivors. There is a clear necessity for in vitro propagation of the limited SSCs from the testicular biopsy prior to transplantation due to limited numbers of spermatogonia in a cryopreserved testicular biopsy. Still, there is no consensus regarding their optimal culture method. Methods: We performed a systematic review and meta-analysis of studies reporting primary testicular cell cultures of human and non-human primate origin through use of Pubmed, EMBASE, and Web of Science core collection databases. Of 760 records, we included 42 articles for qualitative and quantitative analysis. To quantify in vitro spermatogonial propagation, spermatogonial colony doubling time (CDT) was calculated, which measures the increase in the number of spermatogonial colonies over time. A generalized linear mixed model analysis was used to assess the statistical effect of various culture conditions on CDT. Results: Our analysis indicates decreased CDTs, indicating faster spermatogonial propagation in cultures with a low culture temperature (32°C); with use of non-cellular matrices; use of StemPro-34 medium instead of DMEM; use of Knockout Serum Replacement; and when omitting additional growth factors in the culture medium. Discussion: The use of various methods and markers to detect the presence of spermatogonia within the reported cultures could result in detection bias, thereby potentially influencing comparability between studies. However, through use of CDT in the quantitative analysis this bias was reduced. Our results provide insight into critical culture conditions to further optimize human spermatogonial propagation in vitro, and effectively propagate and utilize these cells in a future fertility restoration therapy and restore hope of biological fatherhood for childhood cancer survivors.

Enhanced cold tolerance mechanisms in Euglena gracilis: comparative analysis of pre-adaptation and direct low-temperature exposure.

Microalgae, known for their adaptability to extreme environments, are important for basic research and industrial applications. Euglena, unique for its lack of a cell wall, has garnered attention due to its versatility and the presence of bioactive compounds. Despite its potential, few studies have focused on Euglena's cold adaptation mechanisms. This study investigates the cold adaptation mechanisms of Euglena gracilis, a microalga found in highly diverse environmental habitats, by comparing its growth, photosynthetic performance, and physiological and biochemical responses under two low-temperature cultivation modes: pre-adaptation to 16°C followed by exposure to 4°C (PreC) and direct exposure to 4°C (DirC). In this study, the PreC group exhibited superior growth rates, higher photosynthetic efficiency, and more excellent antioxidant activity compared to the DirC group. These advantages were attributed to higher levels of protective compounds, enhanced membrane stability, and increased unsaturated fatty acid content. The PreC group's ability to maintain higher cell vitality under cold stress conditions underscores the significance of pre-adaptation in enhancing cold tolerance. The findings from this research provide valuable insights into the mechanisms underlying cold adaptation in E. gracilis, emphasizing the benefits of pre-adaptation. These insights are crucial for optimizing the cultivation of algal species under cold stress conditions, which is essential for both biotechnological applications and ecological studies. This study not only advances our understanding of Euglena's adaptive responses to low temperatures but also contributes to the broader field of algal research and its industrial exploitation.

Medium optimization for enhanced production of recombinant lignin peroxidase in Pichia pastoris.

Different cultivation conditions and parameters were evaluated to improve the production and secretion of a recombinant Phanerochaete chrysosporium lipH8 gene in Komagataella phaffii (Pichia pastoris). The recombinant lipH8 gene with its native secretion signal was successfully cloned and expressed in Komagataella phaffii (Pichia pastoris) under the control of the alcohol oxidase 1 promoter (PAOX1). The results revealed that co-feeding with sorbitol and methanol increased rLiP secretion by 5.9-fold compared to the control conditions. The addition of 1mM FeSO4 increased LiP activity a further 6.0-fold during the induction phase. Moreover, the combination of several optimal conditions and parameters yielded an extracellular rLiP activity of 20.05Ul-1, which is more than ten-fold higherrelative to standard growth conditions (BMM10 medium, pH 6 and 30°C). Extracellular activity of a recombinant LiP expressed in P. pastoris increased more than ten-fold when co-feeding sorbitol and methanol as carbon sources, together with urea as nitrogen source, FeSO4 supplementation, lower pH and lowercultivation temperature.

Multi-omics profiling reveals potential mechanisms of culture temperature modulating biosynthesis of carotenoids, lipids, and exopolysaccharides in oleaginous red yeast Rhodotorula glutinis ZHK

Rhodotorula glutinis is a biotechnologically important oleaginous red yeast that has piqued interest as a promising platform strain to industrially synthesize numerous valuable compounds such as carotenoids, lipids, and exopolysaccharides. Here, the effects of low and high culture temperatures on carotenoids, lipids, and exopolysaccharides production in R. glutinis ZHK were investigated. The results indicated that low temperature (16 °C) significantly increased crude exopolysaccharides production, while high temperature (32 °C) significantly increased certain carotenoids (torulene and torularhodin) and total lipids production as compared to control (25 °C) after 5 days of cultivation. Multi-omics analysis suggested that low temperature promotes exopolysaccharide synthesis probably because more exopolysaccharide precursors are made when the expression levels of related protein-encoding genes and activities of their encoded enzymes in the TCA cycle pathway are reduced. Furthermore, high temperature enhances the yield of torulene, torularhodin, and total lipids might be attributed to the increased production of acetyl-CoA derived from the up-regulation of the TCA cycle pathway, as well as the up-regulation of related pathways directly related to carotenogenesis and lipogenesis. This work should uncover a feasible fermentation process for improving carotenoids, lipids, and exopolysaccharides production in R. glutinis ZHK, and provide molecular foundation to further improve their yield by genetic engineering.

Cold stress stimulates algae to produce value-added compounds

Two cold-tolerant microalgae, Chlorella vulgaris and Scenedesmus sp., were grown at 22 and 5 °C. At the lower temperature, the microalgae showed substantial biochemical and morphological changes. The soluble sugar profile in response to low-temperature cultivation was very different in the two strains. C. vulgaris increased both the sucrose and raffinose family oligosaccharides (RFOs) content at 5 °C while Scenedesmus sp. drastically reduced the sucrose content. Both strains increased the total fatty acid methyl ester (FAME) content when grown at 5 °C. However, the FAME profiles were very different: C. vulgaris mainly increased C18:1 and less so C18:3, while Scenedesmus sp. decreased C18:1 but greatly increased C18:3. The morphology of C. vulgaris changed slightly at the lower temperature, while Scenedesmus sp. showed substantial changes in the size and shape. Low temperature triggered the synthesis of unsaturated fatty acids that are essential for human nutrition.

Intensifying Continuous Production of Gag-HA VLPs at High Cell Density Using Stable Insect Cells Adapted to Low Culture Temperature.

Protein production processes based on stable insect cell lines require intensification to be competitive with the insect cell-baculovirus expression vector system (IC-BEVS). High cell density (HCD) cultures operate continuously, capable of maintaining specific production rates for extended periods of time which may lead to significant improvements in production yields. However, setting up such processes is challenging (e.g., selection of cell retention device and optimization of dilution rate), often demanding the manipulation of large volumes of culture medium with associated high cost. In this study, we developed a process for continuous production of Gag virus–like particles (VLP) pseudotyped with a model membrane protein (influenza hemagglutinin, HA) at HCD using stable insect cells adapted to low culture temperature. The impact of the cell retention device (ATF vs. TFF) and cell-specific perfusion rate (CSPR) on cell growth and protein expression kinetics was evaluated. Continuous production of Gag-HA VLPs was possible using both retention devices and CSPR of 0.04 nL/cell.d; TFF induces higher cell lysis when compared to ATF at later stages of the process (kD = 0.009 vs. 0.005 h−1, for TFF and ATF, respectively). Reducing CSPR to 0.01–0.02 nL/cell.d using ATF had a negligible impact on specific production rates (rHA = 72–68 titer/109 cell.h and rp24 = 12–11 pg/106 cell.h in all CSPR) and on particle morphology (round-shaped structures displaying HA spikes on their surface) and size distribution profile (peaks at approximately 100 nm). Notably, at these CSPRs, the amount of p24 or HA formed per volume of culture medium consumed per unit of process time increases by up to 3-fold when compared to batch and perfusion operation modes. Overall, this work demonstrates the potential of manipulating CSPRs to intensify the continuous production of Gag-HA VLPs at HCD using stable insect cells to make them an attractive alternative platform to IC-BEVS.

Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the β-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins.

Challenges in Expression and Purification of Functional Fab Fragments in E. coli: Current Strategies and Perspectives

Microbial host systems remain the most efficient and cost-effective chassis for biotherapeutics production. Escherichia coli is often the preferred host due to ease of cloning, scale-up, high product yields, and most importantly, cost-effective cultivation. E. coli often experience difficulties in producing biologically active therapeutics such as Fab fragments, which require protein folding and subsequent three-dimensional structure development. This paper outlines the recent improvements in upstream and downstream unit operations for producing Fab fragments in E. coli. Monoclonal antibody fragments (Fab) are a rising class of biotherapeutics and their production has been optimised using coexpression of molecular chaperones such as DsbC or DnaK–DnaJ–GrpE, as well as strain engineering for post-translational modifications such as disulphide bridging. Different media systems such as EnBase and combining nitrogen source supplementation with low-temperature cultivation have resulted in improvement in cell integrity, protein expression, and protein refolding. The recovery of native proteins from insoluble inclusion bodies can be improved by adjusting refolding conditions, as well as by incorporating multimodal and affinity chromatography for achieving high product yields in purification. Recent developments summarised in this review may tune the E. coli expression system to produce more complex and glycosylated proteins for therapeutic use in the near future.

Co-Expression of Chaperones for Improvement of Soluble Expression and Purification of An Anti-HER2 scFv in Escherichia Coli.

Single-chain fragment variable (scFv) is one of the most commonly used antibody fragments. They offer some advantages over full-length antibodies, including better penetration to target tissues. However, their functional production has been a challenge for manufacturers due to the potential misfolding and formation of inclusion bodies. Here we evaluated the soluble expression and purification of molecular chaperone co-expression. E. coli BL21(DE3) cells were co-transformed with the mixture of plasmids pKJE7 and pET22b-scFv by the electroporation method. First, L-arabinose was added to induce the expression of molecular chaperones, and then IPTG was used as an inducer to start the expression of anti-HER2 scFv. The effect of cultivation temperature and IPTG concentration on soluble expression of the protein with or without chaperones was evaluated. The soluble expressed protein was subjected to native purification using the Ni-NTA affinity column. SDS-PAGE analysis confirmed the successful co-expression of anti-HER2-scFv and DnaK/DnaJ/GrpE chaperones. Co-expression with chaperones and low-temperature cultivation synergistically improved the soluble expression of anti-HER2 scFv. Co-expression with chaperone also exhibited an approximately four-fold increase in the final yield of purified soluble protein. The combination of co-expression with chaperones and low temperature presented in this work may be useful for the improvement of commercial production of other scFvs in E. coli as functionally bioactive and soluble form.

Contrary to Red, Blue Monochromatic Light Improves the Bioactive Compound Content in Broccoli Sprouts

Broccoli sprouts are rich in health-promoting bioactive compounds. Their content depends on both cultivation light quality and temperature. However, these effects have been previously addressed in isolation. Here, the dual inputs of cultivation light quality [blue (B), red (R), mixture of R and B (R+B), mixture of R and UVA (R+UVA)] and air temperature (15, 19, and 23 °C) on determining growth, external quality, and the cotyledon and hypocotyl content of five major bioactive compounds were investigated. The carbohydrate status at harvest and postharvest ratio of variable to maximum fluorescence (Fv/Fm) were also assessed. Hypocotyl length was generally enhanced under monochromatic light (R or B) and elevated temperature. Total phenolic, total flavonoid, and glucoraphanin contents were generally higher in cotyledon as compared to hypocotyl. Hypocotyl anthocyanin, total phenolic, total flavonoid, and ascorbic acid contents were generally enhanced by R+B, and were decreased by R. Cotyledon content in these metabolites was generally stimulated by B, and reduced under R or R+UVA. Temperature affected metabolite content depending on the metabolite, organ, and light quality. Lower temperatures, R (23 °C) or R+UVA (15, 19, and 23 °C) were associated with decreased postharvest Fv/Fm. In conclusion, low cultivation temperature (&lt;23 °C), as well as R or R+UVA ought to be avoided. Instead, B and R+B are suitable, with B being preferable, owing to better external quality and enhanced metabolite content in cotyledon which generally holds higher content than hypocotyl.

Effects of Low Temperature, Nitrogen Starvation and Their Combination on the Photosynthesis and Metabolites of Thermosynechococcus E542: A Comparison Study.

Both low temperature and nitrogen starvation caused chlorosis of cyanobacteria. Here, in this study, for the first time, we compared the effects of low temperature, nitrogen starvation, and their combination on the photosynthesis and metabolites of a thermophilic cyanobacterium strain, Thermosynechococcus E542. Under various culture conditions, the growth rates, pigment contents, and chlorophyll fluorescence were monitored, and the composition of alkanes, lipidomes, and carbohydrates were determined. It was found that low temperature (35 °C) significantly suppressed the growth of Thermosynechococcus E542. Nitrogen starvation at 45 °C and 55 °C did not affect the growth; however, combined treatment of low temperature and nitrogen starvation led to the lowest growth rate and biomass productivity. Both low temperature and nitrogen starvation caused significantly declined contents of pigments, but they resulted in a different effect on the OJIP curves, and their combination led to the lowest pigment contents. The composition of fatty acids and alkanes was altered upon low-temperature cultivation, while nitrogen starvation caused reduced contents of all lipids. The low temperature did not affect carbohydrate contents, while nitrogen starvation greatly enhanced carbohydrate content, and their combination did not enhance carbohydrate content, but led to reduced productivity. These results revealed the influence of low temperature, nitrogen starvation, and their combined treatment for the accumulation of phycobiliproteins, lipids, and carbohydrates of a thermophilic cyanobacterium strain, Thermosynechococcus E542.

The Effect of Long-Term Storage on Mycobacterium bovis.

It was established that when stored for many years (10–13 years) in low-temperature conditions (3°C), without sub-culture on a nutrient medium, Mycobacterium bovis grew as visible colonies along the line of inoculation. However, due to long-term storage in conditions of low temperature (3°C) morphology of mycobacteria differed significantly from initial cultures formed by rod-shaped bacteria. Some of them became pigment-forming and smooth on the surface. Unlike the initial strain of mycobacteria, a perennial bacteria stored under hard conditions did not cause the death of guinea pigs or their sensitization to a purified protein derivative for mammals. Morphological forms of the perennial mycobacteria had the following changes: pigment forming, L-forms of the vesicular type, non-acid-fast thread-like (filamentous) bacillary forms, and elementary bodies when compared to the initial strain. There were also some genetic changes in the target DNA due to the long-term storage of M. bovis. It may indicate a mutation in the pathogen’s DNA. These mycobacteria had altered biochemical activity during storage. The number of passages on the solid nutrient medium did not affect their fermentative activity. However, the low cultivation temperature increases mycobacterial catalase activity and the ability to hydrolyze Tween-80.

Growth and fatty acid composition of Acutodesmus obliquus under different light spectra and temperatures.

The combined impact of temperature and light spectra on the fatty acid (FA) composition in microalgae has been sparsely investigated. The aim of this study was to investigate the interactions of light and temperature on the FA composition in Acutodesmus obliquus. For this purpose, A. obliquus was cultivated with different temperatures (20, 30, and 35°C), as well as broad light spectra (blue, green, and red light). Growth and FA composition were monitored daily. Microalgal FA were extracted, and a qualitative characterization was done by gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS). Compared to red light, green and blue light caused a higher percentage of the polyunsaturated fatty acids (PUFA) 16:4, 18:3, and 18:4, at all temperatures. The highest total percentage of these PUFA were observed at the lowest cultivation temperature and blue and green light. These data imply that a combination of lower temperatures and blue-green light (450-550 nm) positively influences the activity of specific FA-desaturases in A. obliquus. Additionally, a lower 16:1 trans/cis ratio was observed upon green and blue light treatment and lower cultivation temperatures. Remarkably, green light treatment resulted in a comparably high growth under all tested conditions. Therefore, a higher content of green light, compared to blue light might additionally lead to a higher biomass concentration. Microalgae cultivation with low temperatures and green light might therefore result in a suitable FA composition for the food industry and a comparably high biomass production.

Maturation of bovine oocytes under low culture temperature decreased glutathione peroxidase activity of both oocytes and blastocysts.

It is known that the basic variable in the cellular environment is temperature and low temperature decreases cellular metabolism rate. Also, low cellular metabolic activity reduces oxidative stress, resulting in low ROS production. The aim of this study was therefore to investigate the effect of 36.5°C (low) and 38.5°C (conventional) incubation temperatures during IVM on glutathione peroxidase activity of oocytes and blastocysts following fertilization. Bovine oocytes were matured in medium-199 for 22 hours at either 36.5°C or 38.5°C and they were subjected to in vitro fertilization (IVF). Putative zygotes were then transferred randomly into SOFaa embryo culture media with or without antioxidant (a mixture of GSH and SOD) until development to the blastocyst stage. Glutathione peroxidase enzyme (GSH-Px) activity was lower (p⟨0.05) in oocytes matured at low temperature than those of conventional temperature. Similarly, GSH-Px activity was lower (p⟨0.05) in blastocysts, which were obtained from oocytes matured at low temperature and cultured in antioxidants-supplemented embryo media. The GSH-Px activity of blastocysts, obtained from oocytes matured in low temperature, cultured in antioxidants-free embryo media was similar to blastocysts obtained from oocytes matured in conventional temperature, cultured in antioxidants-supplemented embryo media. The results of the present study show that decreasing the in vitro maturation temperature decreases antioxidant enzyme activity in both oocyte and blastocyst. Additionally, maturation of bovine oocytes at 36.5°C incubation temperature may provide an optimal thermal condition for the enzymatic antioxidant system of both oocytes and blastocyst.

Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli

BackgroundViolaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers.ResultsThrough four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA−) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins.ConclusionsTo our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

Bacterial cell recovery after hollow fiber microfiltration sample concentration: Most probable bacterial composition in frozen vegetables

A commercial “concentrating pipette” was scrutinized from the perspective of sample volume reduction to augment native bacterial quantitation and identification within food samples. Aspects related to the elution protocol were evaluated for their impact on bacteria recovery. The results demonstrated there was no significant effect from different diluents/eluents on the relative recovery rate. Further, results from a complementary technique, dynamic light scattering, imply that recoveries less than unity are likely due to an inability to completely elute the bacterial cells from the capillary filter as opposed to bacterial injury caused by sample processing. When concentrating PBS washes from thawed frozen vegetables and enumerating after culturing at either 30 or 37 °C, the recovery rates were only slightly lower than the simplified experimental conditions. However, in food wash samples, significantly more mass-normalized bacteria were recovered at the lower culture temperature. Nevertheless, the vegetable wash concentration process did not change the most probable isolate composition relative to that of the controls at either culture temperature. The study demonstrates the potential for the system to serve as an effective tool for bacterial recovery and concentration within water-based food systems such as equipment rinses, process wash water, and other samples coming from food processing plants.

Comparison of Reasoner’s 2A Agar and Muller Hinton Agar Media for Microbiological Monitoring of Dialysis Water

Background and Aim: Microbiological culture of dialysis water is a routine safety measure. In, Khorramabad laboratories perform these cultures on Muller Hinton Agar (MHA) at 35–378C for 48 h, not on the Reasoner’s 2A agar (R2A agar) at 17–238oC for 7 days recommended by international standards, the objective of the present study was the comparison of the efficiency of R2A and MHA media in the counting of heterotrophic bacteria in the samples of water collected in dialysis centers from 2 hospitals in Khorramabad, from September to November 2019. Methods: A total of 165 samples of treated water in dialysis centers were collected aseptically and then transported in ice‑packs to the Department of Medical Microbiology of the Lorestan University of Medical Sciences and the pour plate technique was carried out for the enumerating of heterotrophic bacteria. Finally, bacterial colonies were counted after incubation at 34±2oC for 48 hours on MHA and 25oC for 1 week on R2A. Results: Results showed heterotrophic bacterial counts in R2A were greater than those in MHA in 89% of the samples, so enumeration of heterotrophic bacteria should be carried out in R2A agar associated with longer incubation times, because of the greater sensitivity. The proportion of water samples yielding colony counts ≥200 CFU/mL by R2A -7d was significantly different from the proportion by MHA-48h (p<0.001). Conclusion: The results proposed using R2A agar combined with relative low culture temperature (20-25°C), and an extended incubation time (7-10 days) is more efficient. However, as the spectrum of bacterial contamination is not similar for dialysis centers and countries, many studies using different media and culture parameters are required to confirm this. *Corresponding Author: Faranak Rezaei; Email: Rezaei.f@lums.ac.ir Please cite this article as: Pouladi I, Delfani S, Hadian B, Soroush S, Anbari K, Rezaei F. Comparison of Reasoner’s 2A Agar and Muller Hinton Agar Media for Microbiological Monitoring of Dialysis Water. Arch Med Lab Sci. 2020;6:1-5 (e10). https://doi.org/10.22037/amls.v6.32905

Interrogation of chemical changes on, and through, the bacterial envelope of Escherichia coli FabF mutant using time‐of‐flight secondary ion mass spectrometry

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) using a primary ion beam of (CO2)6k+ was used to analyse chemical changes in the bacterial envelope of a fabF knock‐out Escherichia coli strain. fabF is the gene coding for FabF, the enzyme involved in the elongation of FA(16:1) to FA(18:1) and has been associated with plasmid transfer that can lead to acquired multiantibiotic resistance. Comparison of the membrane composition between fabF mutant E. coli and wild type E. coli during the logarithmic and stationary growth phases at two culture temperatures (37°C and 30°C) revealed substantial depletion of FA(18:1) in the fabF mutant during logarithmic growth that resulted in a correlated reduction in FA(cp19:0) during stationary phase. While no clear temperature dependence on the effect of the fabF mutation was found, a reduction in cyclopropanation was observed at lower culture temperature in the wild type strain. Additionally, depth profile analysis revealed a ‘thickening’ of the lipid A layer on the surface of the bacteria during stationary phase and also the appearance of cyclic enterobacterial common antigen (ECACYC) below the surface of the bacteria upon the shift from logarithmic to stationary growth phase.

Dynamic flux balance analysis of biomass and lipid production by Antarctic thraustochytrid Oblongichytrium sp. RT2316-13.

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.