Monitoring Of Bacterial Growth Research Articles

Tetrazole-based organoselenium bi-functionalized corrosion inhibitors during oil well acidizing: Experimental, computational studies, and SRB bioassay

Three tetrazole-based organoselenium compounds (TOS1, TOS2, and TOS3) were synthesized via Ugi and sequential Ugi/nucleophilic substitution approaches. The synthesized agents were evaluated against the corrosive effect of 10% HCl solution over J55 steel tubing samples as a simulation of the impact of a well-stimulating acid job. Chemical and electrochemical studies were conducted by means of various test methods, namely, weight loss, potentiodynamic polarization, electrochemical impedance spectroscopy, and electrochemical frequency modulation. Furthermore, surface examinations were also performed using scanning electron microscope and X-ray photoelectron spectroscopy, which confirmed the formation of protective layer of TOS molecules on J55 steel surface. The inhibition efficiencies (%IE) of TOS1 and TOS2 were up to 92.2% and 94.6%, respectively, whereas TOS3 manifested a lower %IE (89.1%). Moreover, the assessment of TOS1, TOS2, and TOS3 inhibitors as a potential biocidal agent was accounted using NACE Standard TM0194-14 (Field Monitoring of Bacterial Growth in Oil and Gas Systems) against well annulus water samples infected with sulfate-reducing bacteria. Interestingly, all of the tetrazole-based organoselenium compounds exhibited a strong biocidal effect toward microbial-induced corrosion. Finally, quantum chemical calculations and Monte Carlo simulations were performed to provide a rationalization for the inhibitory action of the tetrazole-based organoselenium compounds.

Label-Free Pathogen Detection Based on Yttrium-Doped Carbon Nanoparticles up to Single-Cell Resolution.

The capability to detect bacteria at a low cell density is critical to prevent the delay in therapeutic intervention and to avoid the emergence of antibiotic-resistant species. Till date, significant advancement has been made to develop a sensing platform for rapid and reliable bacterial detection. However, critical requirements, that is, limit of detection, fast time of response, ultrasensitivity with high reproducibility, and the ability to distinguish between bacterial strains are yet to be met within a single sensing platform. In this contribution, we present a novel label-free sensor based on pH-sensitive fluorescent yttrium-doped carbon nanoparticles (YCNPs) embedded in agarose that can rapidly and accurately detect and discriminate pathogens in real time. The developed sensor matrix presented pH-triggered aggregation-induced emission quenching of YCNPs in a wide pH range. When the pH decreased from 10.0 to 4.0, the fluorescence of the matrix decreased linearly (R2 = 0.9229). The sensor 's high sensitivity in a physiologically relevant pH range enables the monitoring of the presence of live pathogens to single-cell resolution. In addition, the 3D matrix sensor showed low cytotoxicity and long stability (>30 days). Besides, the YCNP platform is stable for several hours (5 h) in a complex medium and does not alter the bacterial activities, allowing real-time monitoring of bacterial growth with a small sample volume (100 μL) and rapid response time (25 min). Furthermore, using machine learning-assisted tools, different bacterial strains with various cell densities were discriminated with an accuracy of almost 100%. Moreover, blends of pathogens and a real-world sample can also be identified accurately, thus enabling the sensor to provide fast and reliable pathogen information for clinical decisions and allowing continuous monitoring of infectious disease trends.

ODX: A Fitness Tracker-Based Device for Continuous Bacterial Growth Monitoring.

Continuous monitoring of bacterial growth in aqueous media is a crucial process in academic research as well as in the biotechnology industry. Bacterial growth is usually monitored by measuring the optical density of bacteria in liquid media, using benchtop spectrophotometers. Due to the large form factor of the existing spectrophotometers, they cannot be used for live monitoring of the bacteria inside bacterial incubation chambers. Additionally, the use of benchtop spectrometers for continuous monitoring requires multiple samplings and is labor intensive. To overcome these challenges, we have developed an optical density measuring device (ODX) by modifying a generic fitness tracker. The resulting ODX device is an ultraportable and low-cost device that can be used inside bacterial incubators for real-time monitoring even while shaking is in progress. We evaluated the performance of ODX with different bacterial types and growth conditions and compared it with a commercial benchtop spectrophotometer. In all cases, ODX showed comparable performance to that of the standard benchtop spectrophotometer. Finally, we demonstrate a simple and useful smartphone application whereby the user is notified when the bacterial concentration reaches the targeted value. Due to its potential for automation and mass production, we believe that the ODX has a wide range of applications in biotechnology research and industry.

Exploring electrochemistry of carbon nanodots and its application in noninvasive bacterial growth monitoring

Cost effective and miniaturized methods aiming for high throughput monitoring of bacterial growth are of great significance, especially for tracking disease progression in early stage as well as in screening antibiotic resistant species. Here, we demonstrate an electrochemical platform for noninvasive monitoring of bacterial growth by encapsulating bacterial cells and carbon nanodots in alginate microspheres. The synthesized carbon nanodots have been explored for electrochemical properties, and its redox properties have been utilized for developing bacterial growth monitoring platform. These synthesized CDs are sensitive to pH change and respond as change in redox potential over time as pH of the medium changes due to growth and metabolic activities of bacteria. We determined the bacterial growth kinetics by measuring the redox potential changes of the carbon nanodots over time. The developed platform has been demonstrated to detect the presence of bacteria, the difference in growth rates of bacteria and its susceptibility to the antibiotic with low bacterial counts (103 CFU) in 20 min; thus, redox properties of CDs has the potential to provide a sensitive detection platform.

Vertical capacitance aptasensors for real-time monitoring of bacterial growth and antibiotic susceptibility in blood

For the treatment of bacteremia, early diagnosis and rapid antibiotic susceptibility tests (ASTs) are necessary because survival chances decrease significantly if the proper antibiotic administration is delayed. However, conventional methods require several days from blood collection to AST as it requires three overnight cultures, including blood culture, subculture, and AST culture. Herein, we report a more rapid method of sensing bacterial growth and AST in blood based on a vertical capacitance sensor functionalized with aptamers. Owing to their vertical structure, the influence of blood cells sunk by gravity on capacitance measurements were minimized. Thus, bacterial growth in blood at 100–103 CFU/mL was monitored in real-time by measuring changes in capacitance at f = 10 kHz. Moreover, real-time capacitance measurements at f = 0.5 kHz provided information on biofilm formation induced during blood cultures. Bacterial growth and biofilm formation are inhibited above the minimal inhibitory concentration of antibiotics; therefore, we also demonstrated that vertical capacitance aptasensors could be applied to rapid AST from positive blood cultures without a need for the subculture process.

Typical and Atypical Enteroaggregative Escherichia coli Are Both Virulent in the Galleria mellonella Model.

Enteroaggregative Escherichia coli (EAEC) is an emerging pathotype responsible for acute and persistent diarrhea. It can be classified as typical and atypical strains, respectively, based on the presence or absence of the AggR regulon, suggesting a higher virulence for typical EAEC. This study aims to evaluate in the Galleria mellonella model if there are differences in the virulence profiles among clinical strains of typical and atypical EAEC, prototype strains EAEC C1096, 042 and its aggR mutant. The clinical EAEC strains (n = 20) were analyzed for the presence of 22 putative virulence factors of EAEC or extraintestinal E. coli by PCR, as well as phenotypic characteristics of virulence (enzymes, siderophore, and biofilm). The survival of the larvae was analyzed after inoculation of 104–107 CFU/larva; the monitoring of bacterial growth in vivo and hemocyte quantification was determined after inoculation of the prototype strains (105 CFU/larva) at different periods after infection. The strains of typical and atypical EAEC presented the same virulence profile for the larva, regardless of the amount or type of genes and phenotypic aspects of virulence analyzed. In addition, the EAEC 042 aggR mutant strain showed a significant reduction in the mortality of the inoculated larvae compared to the wild-type strain. In conclusion, the results obtained herein demonstrate that the virulence of EAEC seems to be related to the AggR regulon, but not exclusively, and atypical EAEC strains may be as virulent as typical ones in vivo in the G. mellonella model.

The impact of red blood cell manufacturing variables on bacterial growth dynamics: a pilot study.

Bacterial contamination of red blood cells (RBC) remains a rare but serious clinical concern. Despite the low temperature storage of RBC, some bacteria can proliferate. The impact of RBC additive solutions (AS), manufacturing method or donor sex on bacterial growth/survival in RBC was addressed in this pilot study. Using a partial pool-and-split design, bacterial growth/survival was assessed in intentionally inoculated RBC, manufactured separately from male and female donors using three different manufacturing methods (two whole blood [WB] filtration methods; one RBC filtration method), and resuspended in one of four AS: SAGM, PAGGSM, AS-1 or AS-3. At the beginning of storage, RBC were inoculated with 10 CFU/ml of either Klebsiella pneumoniae, Staphylococcus epidermidis, Yersinia enterocolitica or Propionibacterium acnes. Manufacturing, inoculation, storage (until day 42) and monitoring of bacterial growth were conducted at two sites: Canadian Blood Services and Héma-Québec. Yersinia enterocolitica was the only bacterium that proliferated during storage at both sites. RBC tested at Canadian Blood Services had higher bacterial concentrations than those at Héma-Québec (P=0·0044). At Héma-Québec, where two different manufacturing methods were used, Y.enterocolitica reached significantly higher bacterial concentrations in AS-3 RBC (WB filtration method) compared to units prepared in the other three AS (RBC filtration method; P<0·05). Bacterial survival/growth dependent on donor sex was not uniformly noted. Only one of four bacteria grew under RBC storage conditions. The results indicate that RBC manufacturing variables, rather than AS or donor sex, affect bacterial growth in RBC.

Multimodal Tracking of Controlled Staphylococcus aureus Infections in Mice.

There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, 99mTc-UBI29–41-Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with 99mTc-UBI29–41-Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the 99mTc-UBI29–41-Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with 99mTc-UBI29–41-Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies.

Acinetobacter as a potentially important producer of urocanic acid in chub mackerel, a histidine metabolite of emerging health concern

Urocanic acid, deaminated histidine and the first intermediate in the histidine utilization (Hut) pathway in bacteria, has recently been suggested to have adverse health effects by inducing scombrotoxicosis and immunosuppression after trans–cis isomerization. This work aimed to shed some light on the microbiology of its formation in scombroid fish, given that such information is very scarce. The isolation of urocanic acid producers from chub mackerel was basically based on enrichment of bacteria possessing the Hut pathway with histidine as the sole source of carbon, energy, and nitrogen. Morphological and molecular identification revealed that the isolates (n = 12), taken from the skin, gills, and intestine, were comprised entirely of Acinetobacter from at least five species. This implies that the Hut pathway in the microbes can be particularly active and as such conferred a growth advantage. Dynamic monitoring of bacterial growth and urocanic acid production in fish juice broth indicated that urocanic acid is a primary metabolite and its production is a closely growth-associated process. This in turn implies that its production can be controlled directly by suppressing the bacterial growth. Several potential methods are proposed through a study of the growth characteristics and preservative susceptibility of representative isolates. In conclusion, this work suggests that Acinetobacter can constitute a potentially important group of urocanic acid producers in chub mackerel.

Intracellular Assays to Monitor Survival and Growth of Yersinia pestis Within Macrophages.

Yersinia pestis is able to survive and replicate within macrophages, while also being able to live in the extracellular milieu of the host. Assays that facilitate better understanding of how Y. pestis survives intracellularly and subverts normal host antimicrobial defenses require the ability to monitor intracellular Y. pestis survival and replication. In this chapter three different assays for monitoring intracellular survival and replication will be described, along with the formulas and methods to quantify and present the acquired data. These assays are fundamental to answering a multitude of questions pertaining to which bacterial factors are important for intracellular survival. Additionally, these assays can be used, with modifications, for other intracellular pathogens of interest. The first assay discussed will be the conventional bacterial enumeration assay, which quantifies bacterial numbers directly through a classic colony forming units (CFU) assay. Quantifying bacterial burden through CFU determination allows for differentiation between intracellular/cell-associated bacteria and extracellular bacteria. However, CFU determination is laborious, does not allow for direct kinetic monitoring of bacterial growth, and is difficult to adapt to high throughput assays. Bioluminescence bioreporters that use luciferase to monitor bacterial numbers allow for simple, plate reader-based, real-time kinetic monitoring of bacterial growth that is amendable to high throughput techniques. Finally, we will describe live cell microscopy using fluorescent bioreporters, which allows for monitoring of bacterial replication in individual cells and the possibility to visualize interactions between bacterial and host proteins during intracellular infection.

Impedance-Based Detection of Bacteria.

Pathogenic bacteria have always posed one of the most serious threats to public health, and continue to be especially dangerous with the rise in antibiotic resistance. The prevalence of these infectious agents necessitates rapid, point-of-care sensors for their detection, identification, and monitoring. Electrochemical sensors are promising for the low-cost monitoring of bacterial growth and the detection of specific microbial species due to the consistency and ease-of-use of impedance measurements. Though the commercialization of these sensors is currently limited, they offer significant promise for detecting pathogens from real-world environments.

Interference Disturbance Analysis Enables Single-Cell Level Growth and Mobility Characterization for Rapid Antimicrobial Susceptibility Testing.

To support the emerging battle against antimicrobial resistance (AMR), detection methods that allow fast and accurate antimicrobial susceptibility testing (AST) are urgently needed. The early identification and application of an appropriate antibiotic treatment leads to lower mortality rates and substantial cost savings and prevents the development of resistant pathogens. In this work, we present a diffraction-based method, which is capable of quantitative bacterial growth, mobility, and susceptibility measurements. The method is based on the temporal analysis of the intensity of a light diffraction peak, which arises due to interference at a periodic pattern of gold nanostructures. The presence of bacteria disturbs the constructive interference, leading to an intensity decrease and thus allows the monitoring of bacterial growth in very low volumes. We demonstrate the direct correlation of the decrease in diffraction peak intensity with bacterial cell number starting from single cells and show the capability for rapid high-throughput AST measurements by determining the minimum inhibitory concentration for three different antimicrobials in less than 2-3 h as well as the susceptibility in less than 30-40 min. Furthermore, bacterial mobility is obtained from short-term fluctuations of the diffraction peak intensity and is shown to decrease by a factor of 3 during bacterial attachment to a surface. This multiparameter detection method allows for rapid AST of planktonic and of biofilm-forming bacterial strains in low volumes and in real-time without the need of high initial cell numbers.

Real-Time Bioluminescence Analysis of Escherichia coli O157:H7 Survival on Livestock Meats Stored Fresh, Cold, or Frozen

Foodborne bacteria such as Escherichia coli O157:H7 can cause severe hemorrhagic colitis in humans following consumption of contaminated meat products. Contamination with pathogenic bacteria is frequently found in the food production environment, and adequate household storage conditions of purchased foods are vital for illness avoidance. Real-time monitoring was used to evaluate bacterial growth in ground horse, beef, and pork meats maintained under various storage conditions. Various levels of E. coli O157:H7 carrying the luxCDABE operon, which allows the cells to emit bioluminescence, were used to inoculate meat samples that were then stored at room temperature for 0.5 day, at 4°C (cold) for 7 or 9 days, or -20°C (frozen) for 9 days. Real-time bioluminescence imaging (BLI) of bacterial growth was used to assess bacterial survival or load. Ground horse meat BLI signals and E. coli levels were dose and time dependent, increasing during room temperature and -20°C storage, but stayed at low levels during 4°C storage. No bacteria survived in the lower level inoculum groups (101 and 103 CFU/g). With an inoculum of 107 CFU/g, pork meats had higher BLI signals than did their beef counterparts, displaying decreased BLI signals during 7 days storage at 4°C. Both meat types had higher BLI signals in the fat area, which was confirmed with isolated fat tissues in the beef meat. Beef lean and fat tissues contrasted with both pork fat and lean tissues, which had significantly higher BLI signals and bacterial levels. BLI appears to be a useful research tool for real-time monitoring of bacterial growth and survival in various stored livestock meats. The dependence of E. coli O157:H7 growth on meat substrate (fat or lean) and storage conditions may be used as part of an effective antibacterial approach for the production of safe ground horse, beef, and pork meats.

The EcoChip: A Wireless Multi-Sensor Platform for Comprehensive Environmental Monitoring.

This paper presents the EcoChip, a new system based on the state-of-the-art electro-chemical impedance (EIS) technologies allowing the growth of single strain organisms isolated from northern habitats. This portable system is a complete and autonomous wireless platform designed to monitor and cultivate microorganisms directly sampled from their natural environment, particularly from harsh northern environments. Using 96-well plates, the EcoChip can be used in the field for real-time monitoring of bacterial growth. Manufactured with high-quality electronic components, this new EIS monitoring system is designed to function at a low excitation voltage signal to avoid damaging the cultured cells. The high-precision calibration network leads to high-precision results, even in the most limiting contexts. Luminosity, humidity, and temperature can also be monitored with the addition of appropriate sensors. Access to robust data storage systems and power supplies is an obvious limitation for northern research. That is why the EcoChip is equipped with a flash memory that can store data over long periods of time. To resolve the power issue, a low-power micro-controller and a power management unit control and supply all electronic building blocks. Data stored in the EcoChip's flash memory can be transmitted through a transceiver whenever a receiver is located within the functional transmission range. In this paper, we present the measured performance of the system, along with results from laboratory tests in vitro and from two field tests. The EcoChip has been utilized to collect bio-environemental data in the field from the northern soils and ecosystems of Kuujjuarapik and Puvirnituq, during two expeditions, in 2017 and 2018, respectively. We show that the EcoChip can effectively carry out EIS analyses over an excitation frequency ranging from 750 Hz to 10 kHz with an accuracy of 2.35%. The overall power consumption of the system was 140.4 mW in normal operating mode and 81μW in sleep mode. The proper development of the isolated bacteria was confirmed through deoxyribonucleic acid sequencing, indicating that bacteria thrive in the EcoChip's culture wells while the growing conditions are successfully gathered and stored.

Continuous Recirculation of Microdroplets in a Closed Loop Tailored for Screening of Bacteria Cultures.

Emerging microfluidic technology has introduced new precision controls over reaction conditions. Owing to the small amount of reagents, microfluidics significantly lowers the cost of carrying a single reaction. Moreover, in two-phase systems, each part of a dispersed fluid can be treated as an independent chemical reactor with a volume from femtoliters to microliters, increasing the throughput. In this work, we propose a microfluidic device that provides continuous recirculation of droplets in a closed loop, maintaining low consumption of oil phase, no cross-contamination, stabilized temperature, a constant condition of gas exchange, dynamic feedback control on droplet volume, and a real-time optical characterization of bacterial growth in a droplet. The channels (tubing) and junction cubes are made of Teflon fluorinated ethylene propylene (FEP) to ensure non-wetting conditions and to prevent the formation of biofilm, which is particularly crucial for biological experiments. We show the design and operation of a novel microfluidic loop with the circular motion of microdroplet reactors monitored with optical sensors and precision temperature controls. We have employed the proposed system for long term monitoring of bacterial growth during the antibiotic chloramphenicol treatment. The proposed system can find applications in a broad field of biomedical diagnostics and therapy.

Online Monitoring of Bacterial Growth with an Electrical Sensor.

Herein, we developed an automatic electrical bacterial growth sensor (EBGS) based on a multichannel capacitively coupled contactless conductivity detector (C4D). With the use of the EBGS, up to eight culture samples of E. coli in disposable tubes were online monitored simultaneously in a noninvasive manner. Growth curves with high resolution (on the order of a time scale of seconds) were generated by plotting normalized apparent conductivity value against incubation time. The characteristic data of E. coli growth (e.g., growth rate) obtained here were more accurate than those obtained with optical density and contact conductivity methods. And the correlation coefficient of the regression line ( r) for quantitative determination of viable bacteria was 0.9977. Moreover, it also could be used for other tasks, such as the investigation of toxic/stress effects from chemicals and antimicrobial susceptibility testing. All of these performances required neither auxiliary devices nor additional chemicals and biomaterials. Taken together, this strategy has the advantages of simplicity, accuracy, reproducibility, affordability, versatility, and miniaturization, liberating the users greatly from financial and labor costs.

Aptamer-functionalized capacitance sensors for real-time monitoring of bacterial growth and antibiotic susceptibility

To prevent spread of infection and antibiotic resistance, fast and accurate diagnosis of bacterial infection and subsequent administration of antimicrobial agents are important. However, conventional methods for bacterial detection and antibiotic susceptibility testing (AST) require more than two days, leading to delays that have contributed to an increase in antibiotic-resistant bacteria. Here, we report an aptamer-functionalized capacitance sensor array that can monitor bacterial growth and antibiotic susceptibility in real-time. While E. coli and S. aureus were cultured, the capacitance increased over time, and apparent bacterial growth curves were observed even when 10 CFU/mL bacteria was inoculated. Furthermore, because of the selectivity of aptamers, bacteria could be identified within 1h using the capacitance sensor array functionalized with aptamers. In addition to bacterial growth, antibiotic susceptibility could be monitored in real-time. When bacteria were treated with antibiotics above the minimum inhibitory concentration (MIC), the capacitance decreased because the bacterial growth was inhibited. These results demonstrate that the aptamer-functionalized capacitance sensor array might be applied for rapid ASTs.

Non-ureolytic calcium carbonate precipitation by Lysinibacillus sp. YS11 isolated from the rhizosphere of Miscanthus sacchariflorus.

Although microbially induced calcium carbonate precipitation (MICP) through ureolysis has been widely studied in environmental engineering fields, urea utilization might cause environmental problems as a result of ammonia and nitrate production. In this study, many non-ureolytic calcium carbonate-precipitating bacteria that induced an alkaline environment were isolated from the rhizosphere of Miscanthus sacchariflorus near an artificial stream and their ability to precipitate calcium carbonate minerals with the absence of urea was investigated. MICP was observed using a phase-contrast microscope and ion-selective electrode. Only Lysinibacillus sp. YS11 showed MICP in aerobic conditions. Energy dispersive X-ray spectrometry and X-ray diffraction confirmed the presence of calcium carbonate. Field emission scanning electron microscopy analysis indicated the formation of morphologically distinct minerals around cells under these conditions. Monitoring of bacterial growth, pH changes, and Ca2+ concentrations under aerobic, hypoxia, and anaerobic conditions suggested that strain YS11 could induce alkaline conditions up to a pH of 8.9 and utilize 95% of free Ca2+ only under aerobic conditions. Unusual Ca2+ binding and its release from cells were observed under hypoxia conditions. Biofilm and extracellular polymeric substances (EPS) formation were enhanced during MICP. Strain YS11 has resistance at high pH and in high salt concentrations, as well as its spore-forming ability, which supports its potential application for self-healing concrete.

Gulliver Prototype Development and Deployment

The current objective of our project is to develop a technology, coined Gulliver, aimed at enhancing the study of microorganisms. This device will expand upon established in situ technology to cultivate and isolate microorganisms in one step. Our long term goal is for this device is to not only isolate and culture in one step, but also to eventually equip our device with nanosensors to allow in situ monitoring of bacterial growth and activity, and transmit this data directly to the researcher, allowing an unprecedented view into the microbial world. The Gulliver design consists of a flat mounting device with a 4 mm hole in the center to act as the growth chamber. On one side of the device, a polycarbonate membrane with 0.03μm pores is attached, allowing for diffusion of nutrients and growth signals from the environment. On the reverse side, the same membrane is affixed, however it contains an approximately 1 μm entry aperture, to allow entry of a single microbial cell into the growth chamber. Once the microorganism enters the pore, the entrance is blocked. As the organism continues to grow and divide, it is provided with nutrients from the environment through the diffusion pores, and a pure colony will develop inside the device. After a period of in situ incubation, the device is removed from the environment, the growing biomass retrieved from the growth chamber, “domesticated” and grown on a petri dish for further study. Currently, testing is underway using fluorescently labeled bacterial species to prove the concept of autonomous cultivation and isolation in on step. Devices will be incubated in mixed cultures of fluorescently labeled bacteria, and growth will be collected from each chamber. Fluorescence will be used to ensure a pure culture is obtained. Thus far, we have demonstrated an ability to reliably produce 1 μm pores in polycarbonate membrane. Furthermore, we have designed a prototype of the device consisting of the polycarbonate membrane adhered to a silicone or PTFE base. The prototype is capable of withstanding incubation in an aqueous environment, without material degradation, for up to 3 months, providing an extended period of time for environmental study of microorganisms. The overall goal of this project is to gain a fuller picture of the microbial world, allowing researchers to access novel microorganisms, study the important environmental processes they perform, and utilize the valuable bioactive compounds they produce.Support or Funding InformationThis particular segment of the project is supported by the NSF, the Alpha Fund Grant, and an Northeastern Undergraduate Research and Creative Endeavors Award.

Simple and Versatile Turbidimetric Monitoring of Bacterial Growth in Liquid Cultures Using a Customized 3D Printed Culture Tube Holder and a Miniaturized Spectrophotometer: Application to Facultative and Strictly Anaerobic Bacteria.

Here we introduce a novel strategy for turbidimetric monitoring of bacterial growth in liquid culture. The instrumentation comprises a light source, a customized 3D printed culture tube holder and a miniaturized spectrophotometer, connected through optical cables. Due to its small footprint and the possibility to operate with external light, bacterial growth was directly monitored from culture tubes in a simple and versatile fashion. This new portable measurement technique was used to monitor the growth of facultative (Escherichia coli ATCC/25922, and Staphylococcus aureus ATCC/29213) and strictly (Butyrivibrio fibrisolvens JW11, Butyrivibrio proteoclasticus P18, and Propionibacterium acnes DSMZ 1897) anaerobic bacteria. For E. coli and S. aureus, the growth rates calculated from normalized optical density values were compared with those ones obtained using a benchtop spectrophotometer without significant differences (P = 0.256). For the strictly anaerobic species, a high precision (relative standard deviation < 3.5%) was observed between replicates up to 48 h. Regarding its potential for customization, this manifold could accommodate further developments for customized turbidimetric monitoring, such as the use of light-emitting diodes as a light source or flow cells.