In situ characterisation and manipulation of biological systems with Chi.Bio.

The precision and repeatability of in vivo biological studies is predicated upon methods for isolating a targeted subsystem from external sources of noise and variability. However, in many experimental frameworks, this is made challenging by nonstatic environments during host cell growth, as well as variability introduced by manual sampling and measurement protocols. To address these challenges, we developed Chi.Bio, a parallelised open-source platform that represents a new experimental paradigm in which all measurement and control actions can be applied to a bulk culture in situ. In addition to continuous-culturing capabilities, it incorporates tunable light outputs, spectrometry, and advanced automation features. We demonstrate its application to studies of cell growth and biofilm formation, automated in silico control of optogenetic systems, and readout of multiple orthogonal fluorescent proteins in situ. By integrating precise measurement and actuation hardware into a single low-cost platform, Chi.Bio facilitates novel experimental methods for synthetic, systems, and evolutionary biology and broadens access to cutting-edge research capabilities.

Culturing periprosthetic tissue in blood culture bottles results in isolation of additional microorganisms.

Despite low sensitivity, culture of periprosthetic tissue (PPT) specimens on agars and in broths has traditionally been used for the detection of causative microorganisms in patients suspected for prosthetic joint infection (PJI). The aim of this study was to evaluate the added diagnostic value of culturing PPT in blood culture bottles (BCB) over the conventional combination of standard agar and broth alone. This prospective cohort study was conducted over a 12-month period and included consecutive patients undergoing revision arthroplasty. Overall, 113 episodes from 90 subjects were studied; 45 subjects (50.0%) met the Infectious Diseases Society of America (IDSA) criteria for PJI, of whom the majority (75.6%) had an acute infection. Sensitivity and specificity of culture were assessed using IDSA criteria for PJI as gold standard. Although the increase in sensitivity from 84.44 (CI 70.54; 93.51) to 93.33% (81.73; 98.60) was not significant, added diagnostic value of culturing PPT in BCBs was demonstrated by the significantly higher number of detected pathogens in culture sets with BCBs compared to culture without BCBs (61 pathogens in conventional set versus 89 when BCBs were included for 57 PJI episodes, P = <0.0001). In 17 (29.8%) episodes, microorganisms were cultured from BCBs only, and in 9 (52.9%) of these episodes, virulent pathogens were found. This study demonstrates that PPT culture in BCBs leads to isolation of additional microorganisms, both virulent and low-virulent, which were not cultured with use of agars and broths alone. Isolation of additional causative microorganisms has serious consequences for the treatment strategy in PJI.

Effects of metoprolol on aquatic invertebrates in artificial indoor streams.

Aquatic organisms are impacted by various biotic and abiotic stressors such as current, inter- and intraspecific competition for food resources and habitat, neobiota as well as an increasing number of chemicals. The latter also include pharmaceuticals, which are increasingly being detected in surface waters due to their growing use. The aim of our study was to determine effect data for metoprolol as a model compound for beta-blockers under an environmentally realistic exposure scenario on aquatic invertebrates inhabiting lotic environments. To this end we performed a 40-day experiment in artificial indoor streams (AIS) located in a greenhouse. We focussed on three autochthonous invertebrate species with high relevance in stream ecology: the amphipod Gammarus fossarum, the gastropod Potamopyrgus antipodarum, and the oligochaete Lumbriculus variegatus. Effects on reproduction were found with EC10 (40 days) values of 0.092 mg L-1 (G. fossarum), 0.253 mg L-1 (P. antipodarum), and 0.596 mg L-1 (L. variegatus). Considering environmental data, metoprolol seems to pose no hazard for aquatic invertebrates at present exposure levels.

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.

Study of the behavior of Euglena viridis, Euglena gracilis and Lepadella patella cultured in all-glass microaquarium.

In the paper, the microaquarium fabricated in a form of entirely glass lab-on-a-chip for culturing and microscale study of microorganisms has been presented. A new approach towards cellular studies that brings a significant improvement over commonly utilized - polymer-based solutions has been shown. For the first time, all-borosilicate glass chip was applied for the culturing of the selected microorganisms and enabled notable population growth and behaviorism investigation. The chip fabrication method in comparison to typical glass chip technology was notably simplified, including quick patterning and low temperature bonding in 80 °C. In the studies, both a single-cell (Euglena gracilis and Euglena viridis) and multi-cell microorganisms (Lepadella patella) were cultured in the microaquarium. Behaviorism of the selected microorganisms was investigated by supplying various proportions of carbon dioxide, nitrogen and air into the chip. Tests included studies of microorganisms chemotaxis, viability (mostly based on photosynthesis process) and coexistence in the lab-on-a-chip environment. The experiments confirmed that the developed chip is a tool that fits the requirements for the culturing and behavioral studies of microorganisms and constitute ground-works to propel its further application in broadly defined cellular study field.

Medical Devices; Obstetrical and Gynecological Devices; Classification of the Intravaginal Culture System. Final order.

The Food and Drug Administration (FDA) is classifying the intravaginal culture system into class II (special controls). The special controls that will apply to the device are identified in this order and will be part of the codified language for the intravaginal culture system's classification. The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

[Effects of salinity and adhesive materials on the growth and adhesion of Amphora sp. HN08 under static conditions].

OBJECTIVE: To optimize the cultural conditions including adhesive materials and salinity for one tropical benthic diatom (Amphora sp. HN08). METHODS: Two experiments were performed: (1) five adhesive materials including agar, glass, PVC plate, plastic film and nylon net were used to culture Amphora sp. HN08 HN08; (2) Amphora sp. HN08 was cultured under 6 salinity levels from 1 through 6%. The algal cells were harvested after 9 days treatments, and the biomass productivity, adhesive strength and pigments content of cells were examined. RESULTS: For the adhesive materials experiment, cultures with glass and plastic plate showed the highest biomass production and the dry weight of cells reached to 3.64 g/m2. The adhesion strength level of cells on the glass plate was III degree, which means the cells were easy to be separated from the glass and dewatered by centrifugation. Salinity did not contribute significantly to the biomass production, while it significantly influenced the cells adhesion and pigments concentration. The adhesive strength levels of the cells under 3% or 4% salinity was IV degree, which led to an easy harvest process. While the. cells cultured under high salinity (5% and 6%) usually suspended in the medium and were difficult to be dewatered by centrifugation. Both chlorophyll a and carotenoid content of cells cultured with higher salinity (≥ 3%) are extremely higher (p < 0.01) than that with lower salinity (1% and 2%). Chlorophyll a and carotenoid content of cells under 5% salinity was 26.27% and 11.11% by dry weight, respectively. CONCLUSION: Taking the harvest and biomass production together, we think the optimal salinity for Amphora sp. HN08 was between 3% and 4%, and the glass plate was suitable for adhesive material. However, considering the cost and safety, the PVC plate is suggested to be used for biomass production.

Use of an automated blood culture system (BD BACTEC™) for diagnosis of prosthetic joint infections: easy and fast.

BACKGROUND: For the diagnosis of prosthetic joint infection (PJI) automated BACTEC™ blood culture bottle methods have comparable sensitivity, specificity and a shorter time to positivity than traditional cooked meat enrichment broth methods. We evaluate the culture incubation period required to maximise sensitivity and specificity of microbiological diagnosis, and the ability of BACTEC™ to detect slow growing Propionibacteria spp. METHODS: Multiple periprosthetic tissue samples taken by a standardised method from 332 patients undergoing prosthetic joint revision arthroplasty were cultured for 14 days, using a BD BACTEC™ instrumented blood culture system, in a prospective study from 1st January to 31st August 2012. The "gold standard" definition for PJI was the presence of at least one histological criterion, the presence of a sinus tract or purulence around the device. Cases where > =2 samples yielded indistinguishable isolates were considered culture-positive. 1000 BACTEC™ bottle cultures which were negative after 14 days incubation were sub-cultured for Propionibacteria spp. RESULTS: 79 patients fulfilled the definition for PJI, and 66 of these were culture-positive. All but 1 of these 66 culture-positive cases of PJI were detected within 3 days of incubation. Only one additional (clinically-insignificant) Propionibacterium spp. was identified on terminal subculture of 1000 bottles. CONCLUSIONS: Prolonged microbiological culture for 2 weeks is unnecessary when using BACTEC™ culture methods. The majority of clinically significant organisms grow within 3 days, and Propionibacteria spp. are identified without the need for terminal subculture. These findings should facilitate earlier decisions on final antimicrobial prescribing.

Culture of the green microalga Botryococcus braunii Showa with LED irradiation eliminating violet light enhances hydrocarbon production and recovery.

The green microalga Botryococcus braunii (B. braunii), race B, was cultured under light-emitting diode (LED) irradiation with and without violet light. This study examined the effect of violet light on hydrocarbon recovery and production in B. braunii. C34 botryococcene hydrocarbons were efficiently extracted by thermal pretreatments at lower temperatures when the alga was cultured without violet light. The hydrocarbon content was also higher (approximately 3%) in samples cultured without violet light. To elucidate the mechanism of effective hydrocarbon recovery and production, we examined structural components of the extracellular matrix (ECM). The amounts of extracellular carotenoids and water-soluble polymers extracted by thermal pretreatment from the ECM were decreased when the alga was cultured without violet light. These results indicate that LED irradiation without violet light is more effective for hydrocarbon recovery and production in B. braunii. Furthermore, structural ECM components are closely involved in hydrocarbon recovery and production in B. braunii.

Development of a selective culture medium for bifidobacteria, Raffinose-Propionate Lithium Mupirocin (RP-MUP) and assessment of its usage with Petrifilm™ Aerobic Count plates.

This study aimed to develop a selective culture media to enumerate bifidobacteria in fermented milk and to assess this medium when used with Petrifilm™ AC plates. For this purpose, Bifidobacterium spp., Lactobacillus spp. and Streptococcus thermophilus strains were tested to verify their fermentation patterns for different carbohydrates. All bifidobacteria strains were able to use raffinose. Based on these characteristic, a selective culture medium was proposed (Raffinose-Propionate Lithium Mupirocin, RP-MUP), used with Petrifilm™ AC plates, and was used to enumerate bifidobacteria in fermented milk. RP-MUP performance was assessed by comparing the results with this medium to reference protocols and culture media for bifidobacteria enumeration. RP-MUP, whether used or not with Petrifilm™ AC, presented similar performance to TOS-MUP (ISO 29981), with no significant differences between the mean bifidobacteria counts (p < 0.05) and with high correlation indices (r = 0.99, p < 0.05). As an advantage, reliable results were obtained after just 48 h of incubation when RP-MUP was used with Petrifilm™ AC, instead of the 72 h described in the ISO 29981 protocol.

Actinobacillus pleuropneumoniae genes expression in biofilms cultured under static conditions and in a drip-flow apparatus.

BACKGROUND: Actinobacillus pleuropneumoniae is the Gram-negative bacterium responsible for porcine pleuropneumonia. This respiratory infection is highly contagious and characterized by high morbidity and mortality. The objectives of our study were to study the transcriptome of A. pleuropneumoniae biofilms at different stages and to develop a protocol to grow an A. pleuropneumoniae biofilm in a drip-flow apparatus. This biofilm reactor is a system with an air-liquid interface modeling lung-like environment. Bacteria attached to a surface (biofilm) and free floating bacteria (plankton) were harvested for RNA isolation. Labelled cDNA was hybridized to a microarray to compare the expression profiles of planktonic cells and biofilm cells. RESULTS: It was observed that 47 genes were differentially expressed (22 up, 25 down) in a 4 h-static growing/maturing biofilm and 117 genes were differentially expressed (49 up, 68 down) in a 6h-static dispersing biofilm. The transcriptomes of a 4 h biofilm and a 6 h biofilm were also compared and 456 genes (235 up, 221 down) were identified as differently expressed. Among the genes identified in the 4 h vs 6h biofilm experiment, several regulators of stress response were down-regulated and energy metabolism associated genes were up-regulated. Biofilm bacteria cultured using the drip-flow apparatus differentially expressed 161 genes (68 up, 93 down) compared to the effluent bacteria. Cross-referencing of differentially transcribed genes in the different assays revealed that drip-flow biofilms shared few differentially expressed genes with static biofilms (4 h or 6 h) but shared several differentially expressed genes with natural or experimental infections in pigs. CONCLUSION: The formation of a static biofilm by A. pleuropneumoniae strain S4074 is a rapid process and transcriptional analysis indicated that dispersal observed at 6 h is driven by nutritional stresses. Furthermore, A. pleuropneumoniae can form a biofilm under low-shear force in a drip-flow apparatus and analyses indicated that the formation of a biofilm under low-shear force requires a different sub-set of genes than a biofilm grown under static conditions. The drip-flow apparatus may represent the better in vitro model to investigate biofilm formation of A. pleuropneumoniae.

[Artificial cultivation modes for Dendrobium officinale].

Since the beginning of the new century, the artificial cultivation of Dendrobium officinale has made a breakthrough progress. This paper systematically expounds key technologies, main features and cautions of the cultivation modes e.g. bionic-facility cultivation, the original ecological cultivation, and potting cultivation for D. officinale, which can provide useful information for the development and improvement of D. officinale industry.

A new larval tray and rack system for improved mosquito mass rearing.

The requirement for efficient mosquito mass rearing technology has been one of the major obstacles preventing the large scale application of the Sterile Insect Technique against mosquitoes. At the Food and Agriculture Organization/International Atomic Energy Agency (FAO/ IAEA) Insect Pest Control Laboratories we developed a larval rearing unit based on the use of a stainless steel rack that operates 50 thermoformed ABS plastic trays and is expected to be able to successfully rear 140,000-175,000 Anopheles arabiensis (Patton) adult mosquitoes per rack. The mechanized rearing unit is simple to handle, maintains minimal water temperature variation and negligible water evaporation and allows normal larval development. The mosquito mass-rearing tray was designed to provide a large surface area of shallow water that would closely mimic natural breeding sites. The trays stack into a dedicated rack structure and filling and draining were easily performed. The close stacking of the trays in the rack and the possibility to tightly line up several racks makes this rearing unit a valid solution for maximal use of the space thus reducing construction, heating, and cooling costs. The low amount of labor required to operate the system also reduces labor costs that represent one of the main expenditures in any mass rearing facility operation. Preliminary experiments performed on Aedes albopictus (Skuse) also confirm the possibility of successfully extending the use of this technology to other mosquito species. Our larval rearing unit could enhance any mosquito control strategy in which large-scale releases of mosquitoes are needed to suppress or replace natural populations.

Integrated microbioreactor for culture and analysis of bacteria, algae and yeast.

We introduce a micro-scale bioreactor for automated culture and density analysis of microorganisms. The microbioreactor is powered by digital microfluidics (DMF) and because it is used with bacteria, algae and yeast, we call it the BAY microbioreactor. Previous miniaturized bioreactors have relied on microchannels which often require valves, mixers and complex optical systems. In contrast, the BAY microbioreactor is capable of culturing microorganisms in distinct droplets on a format compatible with conventional bench-top analyzers without the use of valves, mixers or pumps. Bacteria, algae and yeast were grown for up to 5 days with automated semi-continuous mixing and temperature control. Cell densities were determined by measuring absorbances through transparent regions of the devices, and growth profiles were shown to be comparable to those generated in conventional, macro-scale systems. Cell growth and density measurements were integrated in the microbioreactor with a fluorescent viability assay and transformation of bacteria with a fluorescent reporter gene. These results suggest that DMF may be a useful new tool in automated culture and analysis of microorganisms for a wide range of applications.

Electrical cell-substrate impedance sensing as a non-invasive tool for cancer cell study.

Cell-substrate interactions are investigated in a number of studies for drug targets including angiogenesis, arteriosclerosis, chronic inflammatory diseases and carcinogenesis. One characteristic of malignant cancerous cells is their ability to invade tissue. Cell adhesion and cytoskeletal activity have served as valuable indicators for understanding the cancer cell behaviours, such as proliferation, migration and invasion. This review focuses on bio-impedance based measurement for monitoring the behaviours in real time and without using labels. Electric cell-substrate impedance sensing (ECIS) provides rich information about cell-substrate interactions, cell-cell communication and cell adhesion. High sensitivity of the ECIS method allows for observing events down to single-cell level and achieving nanoscale resolution of cell-substrate distances. Recently, its miniaturization and integration with fluorescent detection techniques have been highlighted as a new tool to deliver a high-content platform for anticancer drug development.

Cells in space

How does one treat in a seriously injured astronaut in outer space or even another planet? To answer such a question, the US National Aeronautical Space Administration (NASA) has embarked on a program of growing tissues--and possibly whole organs--in space. NASA has developed a unique rotating bioreactor that allow cells to be grown in a microgravity environment that eliminates almost all shear forces placed upon a cell culture system while entering space. Back on earth, this novel bioreactor has led to exciting discoveries and applications by scientists trying to get cells to differentiate and form their natural three-dimensional tissue matrices--the holy grail of tissue engineers. NASA's bioreactor has allowed various labs to culture cells and even viruses previously impossible to grow using traditional methods. These successes are attributed to the bioreactor's ability to provide an unique environment that closely resembles tissue differentiation during embryogenesis, and thus allowing cellular expression of surface epitopes similar to that of intact tissues. It also appears that cells grown in a microgravity, low-shear environment allows for greater chemical signaling, probably as a result of more surface contact between cells. Realizing the bioreactor's commercial potential, Santa Monica, California-based VivoRx licensed exclusive rights from NASA for both therapeutic and diagnostic commercial applications. VivoRx has, in the past, successfully transplanted encapsulated islet cells from cadavers and porcine pancreas into insulin-dependent diabetics, perhaps a major breakthrough in the treatment of diabetes. However, pancreas from cadavers are in very short supply. The bioreactor may be the answer; VivoRx hopes the bioreactor will allow them to propagate enough human islet cells to use their cell-based approach to treat a large diabetic population. The company has already successfully grown islet cells generated from the bioreactors, and is beginning FDA-approved Phase I/II clinical trials.

In vitro maintenance of a human proximal colon microbiota using the continuous fermentation system P-ECSIM.

Ethical and technical difficulties for in vivo studies on gut microbiotas argue for the development of alternative in vitro models: here, we describe a system simulating the proximal part of a human colon both nutritionally and physico-chemically with a procedure aimed to limit experimental variations over the time (Proximal Environmental Control System For Intestinal Microbiota--P-ECSIM). The continuous culture system P-ECSIM is first inoculated by a -20 °C glycerol stock established from the batch culture of a stool-inoculated medium. The anaerobic atmosphere is self-maintained by the gases produced in the ordinary metabolism of fermentations. The monitoring of metabolic activities and microbial constitutions indicates that different steady states are obtained according to the dilution rate. Finally, the glycerol conservation of the batch culture-derived inoculum gives a similar differential response between the two dilution rates (D = 0.08 h⁻¹ and D = 0.04 h⁻¹) after a 1-year storage time as well for their metabolism and constitution in steady states, but with a lower abundance. Molecular fingerprints of the microbiota reveal however alterations over the time. Further efforts are needed concerning the preservation of standardized inoculums in order to improve the process for intra- and inter-lab comparison. Combined with appropriate analytical techniques, this system provides an efficient alternative means of studying functionally human microbiota in its constitution, metabolism and adaptation to environmental changes, particularly nutritional.

Platotex: an innovative and fully automated device for cell growth scale-up of agar-supported solid-state fermentation.

Among various factors that influence the production of microbial secondary metabolites (MSM), the method of cultivation is an important one that has not been thoroughly investigated. In order to increase microbial throughput and simplify the extraction and workup steps, we performed a study to compare liquid-state fermentation (LSF) with agar-supported solid-state fermentation (AgSF). We found that AgSF is not only more suitable for our applications but offers, for some microbial strains, a higher yield and broader diversity of secondary metabolites. The main limitation of AgSF is the lack of a system to allow production scale-up. In order to overcome this obstacle we developed Platotex, an original fermentation unit offering 2 m(2) of cultivation surface that combines automatic sterilization, cultivation, and drying steps. Platotex is also able to support both LSF and solid-state fermentation (SSF). Platotex conforms to international security and quality requirements and benefits from total remote automation through industrial communication and control standards.

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae.

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.

Paddy straw as a substrate for the cultivation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.) P. Karst. in India.

Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, is generally cultivated on hardwood logs or sawdust/woodchips based formulations. More than 100 million tonnes of paddy straw is being produced in India per year, and almost 50% of the straw is potentially available for growing mushrooms. In the present study an attempt was made to use paddy straw as a substrate to cultivate G. lucidim. Different proportions of paddy straw were mixed with 0, 22.5%, 45%, and 67.5% sawdust and 10% rice bran. Spawn run period, fruiting initiation period, yield, moisture content, dry recovery, and fruiting body characteristics were recorded and compared. Fructification was observed with all the substrate formulations and they did not show any significant difference in yield. The highest biological efficiency (BE) (29.9%) was observed with the combination sawdust:paddy straw:rice bran 22.5:67.5:10, followed by saw dust:paddy straw:rice bran 45:45:10 with BE 27.3%. The current study demonstrated for the first time that the cultivation of G. lucidum is possible with paddy straw as the base substrate and indicated the enormous potential of paddy straw for the cultivation of G. lucidum.