Biosafety Cabinet Research Articles

Environment monitoring in a cell therapy manufacturing facility – 3 year data analysis

Background & Aim To study the Environment Monitoring (EM) activities in a hospital-based Cell Therapy Manufacturing Facility (mainly processes islet cells and HPC(s), to ensure compliance with FACT (03/2018), ISO14644 (12/2015), validation standards and facility EM SOP. Methods, Results & Conclusion Methods Available EM data from 08/2014 to 12/2017 analyzed, including initial validation EM by vendor. EM are normally performed on a monthly basis, while some months had partial or no EM done due to facility construction, schedule conflicts, or machine error. EM includes particle count (PC), air sampling (AS), contact plate bacterial & fungal (CP). See Image 1 for routine EM locations. CR: Cleanroom; GR: Gown Room; DR: Degown Room; BSC: Biosafety Cabinet. Results In the 41 months, 37 months of particle counts, 40 months of Air Sampling, 38 months of contact plate data were analyzed. Among them, (1) One was In-Process EM, all the others were At Rest EM. The In-Process particle counts were significantly higher than those At-Rest. (2) All critical ISO 5 areas were all within expected ranges for all three EM parameters. (3) Two deviations filed due to high air sampling counts, and both passed after improving operator technique and after facility cleaning. (4) In this 3 year data, particle counts and contact plates results are trending low or stable, within expected range, while Air Sampling results had some fluctuations, see Image 2 and 3. (5) Besides the regular/routine sampling locations, several non-routine sampling locations were also sampled, which detected “mold” in Incubator, “>100 CFU” in freezer, and both repeated “0 CFU” count after equipment decontamination. See Image 3. Conclusion (1) The facility's EM program monitors particulates, microbial contamination, with proper sampling sites, frequency, investigative and corrective actions when specified limits exceeded. (2) All BSC data were satisfactory and safe for cell therapy manufacturing processes. (3) Gown Room and Degown Room were built per ISO 8, and met ISO 7 requirements. (4) More In-Process EM shall be performed to provide baseline data during manufacturing. (5) Non-routine sampling in ISO unclassified areas helps detect suspected/ at risk areas, and help improve the cleanliness of the facility.

Automated closed systems for cell manufacturing

Background & Aim As more autologous cell therapy products are coming through the FDA approval and clinical trial pipeline, there is a need for validated cell manufacturing system that scales across phases of production, from the development to the manufacturing of novel and existing cell therapies. Currently, the manufacturing process involves culturing patient's cells with open culture vessels, especially in the initial phases. This process is labour-intensive and requires skilled operators working in cleanrooms and with biosafety cabinets. Automated closed systems enable one operator to oversee the manufacture of multiple batches of cell therapy products, reducing the number of and demand in skilled operators. For validated manufacturing processes, automated closed systems also increase the reliability and repeatability of each step, and thus contributing to the principle of Quality by Design (QbD) in the current Good Manufacturing Practice (cGMP) framework. While there are a few closed all-in-one cell manufacturing systems in the market, the common complaint is that these systems are not flexible enough to accommodate different manufacturing protocols. Methods, Results & Conclusion We developed a modular system which automates routine menial steps in a closed environment to reduce overall manufacturing costs with respect to labour and facility, with the flexibility to adjust to different manufacturing protocols. In addition to climate control to maintain temperature, humidity and pCO2, our automated closed system performs routine steps such as removal and top up of the cell culture media; addition of raw and ancillary materials; agitation of the cell culture; sampling of the cell culture; and harvesting of the cells. These steps are programmable to suit the protocol of choice. The current system is validated according to a five-week process for culturing T cells with feeder cells. Each system contains one batch of cell culture which corresponds to one or more therapeutic dose of autologous cell therapy product. With a closed and automated system, resources can be transferred from the manual handling of cell culture to developing new cell therapy products. Our system enables seamless translation of cell therapy products through the clinical trial pipeline, starting from initial research and development and to the final cell manufacturing for administration into patients.

Aseptic Technique

AbstractThis article describes common laboratory procedures that can reduce the risk of culture contamination, collectively referred as “aseptic technique.” Two major strategies of aseptic work are described: using a Bunsen burner and a biosafety cabinet. Both methods are presented in the form of general protocols applicable to a variety of laboratory tasks such as pipetting and aliquotting, preparation of growth media, inoculation, passaging, and spreading microorganisms on petri dishes. © 2019 by John Wiley & Sons, Inc.

Heat Sources in a Biosafety Cabinet Compromise Experimental and User Protection

Introduction: Keeping a contamination free environment in the laboratory has commonly been achieved by one of two ways: a flame or a biosafety cabinet (BSC). However, it has been frequently observed that these two practices have been combined, where a heat source has been used within the BSC. As flames require flammable gasses and cause hot air to rise, it was hypothesized that these could lead to a loss of BSC containment, as BSCs rely on unidirectional downflow air. Objectives: The objective of this study was to determine whether BSCs can maintain containment when a heat source is operated within the work area. Methods: Several heat sources (Bunsen burner, High Heat Bunsen Burner, Spirit Lamp and Bacti-cinerator) were placed within two sizes of BSCs (4-foot and 6-foot), and smoke was used to visualize airflow disturbances, air cleanliness was measured by particle counting , and aerosol microbiological testing was conducted to ascertain containment. The risk of introducing a flammable gas into a BSC was also calculated. Results: Large flamed Bunsen burners were found to have the most detrimental effects on the ability of the BSC to maintain containment, especially in the center of the work area, while the smaller heat sources were more variable. Containment was completely lost in the 4-foot BSC, whereas the 6-foot BSC was capable of maintaining containment in only a few conditions. The BSC was also calculated to be able to maintain the required volume of flammable gas needed to operate the burners, not taking into consideration unintended leaks. Conclusions: Overall, it was determined that BSCs cannot operate safely and reliably while housing a heat source, as it could cause unexpected contamination of the work or the worker, or BSC ignition or explosion.

System to Evaluate Movement of Biological Contaminants in Soil

Soil columns have been utilized in hydrology to study vertical solute transfer through porous material for decades. Soil columns are typically designed as open tubes with soil held in place with meshing. While this open design is sufficient for non-hazardous particles, it is not ideal for hazardous biological contaminants that may be harmful to humans. The design of this study features a closed soil column system for use with potentially hazardous biological components. The apparatus is comprised of a mist nozzle, flow-reducing cap, and meshing to simulate rainfall on each soil column. After percolating through the soil, water and contaminants pass through a funnel coupling and discharge tube into a collection container. For additional safety, the soil column design fits within a standard biosafety cabinet for use with hazardous contaminants. Its modular design allows for simple maintenance, water flowrate adjustment, and versatility that encourages use in multiple applications. These soil columns were created to study the vertical flow of pathogens, pesticides, and other biological agents. Further experimentation with various hazardous components will develop a better understanding of their fate and transport in soil. This paper details the construction processes and testing methods to validate the system’s ability to replicate a desired flowrate, which is a precursor to studying the vertical transport of pathogens and other agents through soil.

Application of next generation sequencing technology on contamination monitoring in microbiology laboratory

The surveillance and prevention of pathogenic microbiological contamination are the most important tasks of biosafety management in the lab. There is an urgent need to establish an effective and unbiased method to evaluate and monitor such contamination. This study aims to investigate the utility of next generation sequencing (NGS) method to detect possible contamination in the microbiology laboratory. Environmental samples were taken at multiple sites at the lab including the inner site of centrifuge rotor, the bench used for molecular biological tests, the benches of biosafety cabinets used for viral culture, clinical sample pre-treatment and nucleic acids extraction, by scrubbing the sites using sterile flocked swabs. The extracted total nucleic acids were used to construct the libraries for deep sequencing according to the protocol of Ion Torrent platform. At least 1G raw data was obtained for each sample. The reads of viruses and bacteria accounted for 0.01 ± 0.02%, and 77.76 ± 12.53% of total reads respectively. The viral sequences were likely to be derived from gene amplification products, the nucleic acids contaminated in fetal bovine serum. Reads from environmental microorganisms were also identified. Our results suggested that NGS method was capable of monitoring the nucleic acids contaminations from different sources in the lab, demonstrating its promising utility in monitoring and assessing the risk of potential laboratory contamination. The risk of contamination from reagents, remnant DNA and environment should be considered in data analysis and results interpretation.

Plasma-Activated Aerosolized Hydrogen Peroxide (aHP) in Surface Inactivation Procedures.

Hydrogen peroxide is a strong oxidant that possesses an antimicrobial activity. It has been successfully used in surface/room decontamination processes either under the form of hydrogen peroxide vapor (HPV) or of vaporized hydrogen peroxide (VHP). Aerosolized hydrogen peroxide (aHP) offers a third alternative. The technology relies on the dispersion of aerosols of a hydrogen peroxide solution often complemented with silver cations. aHP provides an inexpensive and safe approach to treat contaminated rooms but sometimes fails to achieve the 6-log10 reduction limit in the number of viable microorganisms. Here, we used a venturi-based aHP generator that generates 4 mm in size aerosols from a 12% plasma-activated hydrogen peroxide solution free of silver cations. We could successfully and constantly inactivate bacterial growth from biological indicators containing at least 106 spores of Geobacillus stearothermophilus placed on stainless steel discs wrapped in Tyvek pouches. We could also show that the biological indicators placed at various locations in a class II biosafety cabinet were equally inactivated, showing that hydrogen peroxide aerosols migrate through HEPA filters. Considering that our method for aerosol generation is simple, reproducible, and highly effective at inactivating spores, our approach is expected to serve as a relatively cost effective alternative method for disinfecting potentially contaminated rooms or surfaces.

An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus.

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20–25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle (n = 1135) and swine (n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent–free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

Frequency of Instrument, Environment, and Laboratory Technologist Contamination during Routine Diagnostic Testing of Infectious Specimens.

Laboratory testing to support the care of patients with highly infectious diseases may pose a risk for laboratory workers. However, data on the risk of virus transmission during routine laboratory testing conducted using standard personal protective equipment (PPE) are sparse. Our objective was to measure laboratory contamination during routine analysis of patient specimens. Remnant specimens were spiked with the nonpathogenic bacteriophage MS2 at 1.0 × 107 PFU/ml, and contamination was assessed using reverse transcriptase PCR (RT-PCR) for MS2. Specimen containers were exteriorly coated with a fluorescent powder to enable the visualization of gross contamination using UV light. Testing was performed by two experienced laboratory technologists using standard laboratory PPE and sample-to-answer instrumentation. Fluorescence was noted on the gloves, bare hands, and laboratory coat cuffs of the laboratory technologist in 36/36 (100%), 13/36 (36%), and 4/36 (11%) tests performed, respectively. Fluorescence was observed in the biosafety cabinet (BSC) in 8/36 (22%) tests, on test cartridges/devices in 14/32 (44%) tests, and on testing accessory items in 29/32 (91%) tests. Fluorescence was not observed on or in laboratory instrumentation or adjacent surfaces. In contrast to fluorescence detection, MS2 detection was infrequent (3/286 instances [1%]) and occurred during test setup for the FilmArray instrument and on FilmArray accessory equipment. The information from this study may provide opportunities for the improvement of clinical laboratory safety practices so as to reduce the risk of pathogen transmission to laboratory workers.

The Effectiveness of HEPA Filters on DNA

Most laboratory techniques are performed in biosafety cabinets (BSCs) to provide contamination control of the experiments. By using specifically directed airflow and high-efficiency particulate air...

Nondestructive assessment of collagen hydrogel cross-linking using time-resolved autofluorescence imaging.

We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable.

Stabilization of RDT target antigens present in dried Plasmodium falciparum-infected samples for validating malaria rapid diagnostic tests at the point of care

BackgroundMalaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time.MethodsThis study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the stabilized samples using SD Bioline, BinaxNOW, CareStart, and First Response was done at intervals for 53 weeks.ResultsStability of HRP2 at ambient temperature was reported at 21–24 weeks while that of PAN antigens (pLDH and aldolase) was 2–18 weeks of storage at all parasite densities. The ten chemical additives increased the percentage stability of HRP2 and PAN antigens. Sucrose alone and its combinations with Alsever’s solution or biostab significantly increased stability of HRP2 by 56% at 2000 p/µL (p < 0.001). Trehalose and its combinations with biostab, sucrose or glycerol significantly increased stability of HRP2 by 57% (p < 0.001). Unlike sucrose, the stability of the HRP2 was significantly retained by trehalose at lower concentrations (500, and 200 p/µL). Trehalose in combination biostab stabilizer increased the percentage stability of PAN antigens by 42, and 32% at 2000 and 500 p/µL respectively (p < 0.01). This was also the chemical combination with the shortest reconstitution time (~ < 20 min).ConclusionsThese findings confirm that stabilizing RDT target antigens in dried P. falciparum-infected samples using chemical additives provides field-stable positive controls for malaria RDTs.

Cold Plasma Inactivation of Salmonella in Prepackaged, Mixed Salads Is Influenced by Cross-Contamination Sequence

Customer demand for convenient food products has led to an increased production of prepackaged and ready-to-eat food products. Most of these products rely mainly on surface disinfection and other traditional approaches to ensure shelf life and safety. Novel processing techniques, such as cold plasma, are currently being investigated to enhance the safety and shelf life of prepacked foods. The purpose of this study was to determine the effects of cold plasma corona discharge on the inactivation of Salmonella on prepackaged, tomato-and-lettuce mixed salads. Two different inoculation methods were evaluated to address cross-contamination of Salmonella from cherry tomatoes to lettuce and vice versa. In separate studies, a sample of either cherry tomatoes (55 g) or romaine lettuce (10 g) was inoculated with a Salmonella cocktail (6.93 ± 0.99 log CFU/mL), placed into a commercial polyethylene terephthalate plastic container, and thoroughly mixed together with its noninoculated counterpart. Mixed salads were allowed to dry in a biosafety cabinet for 1 h. Samples were treated with 35 kV cold plasma corona discharge inside plastic containers for 3 min. Samples were stomached and serially diluted in buffered peptone water and then were plated onto aerobic plate count Petrifilm and incubated for 18 h at 37°C. When lettuce was the inoculated counterpart, log kill of Salmonella was significantly greater on tomatoes (0.75 log CFU/g) compared with lettuce (0.34 log CFU/g) (P = 0.0001). Salmonella was reduced on mixed salad only when lettuce was the inoculated counterpart (0.29 log CFU/g) (P = 0.002). Cold plasma can kill Salmonella in a prepackaged mixed salad, with efficacy dependent on the nature of contamination, direction of transfer, and the surface topography of the contaminated commodity.

Carpal tunnel syndrome among laboratory technicians in relation to personal and ergonomic factors at work

Work-related carpal tunnel syndrome (CTS) has been reported in different occupations, including laboratory technicians, so this study was carried out to determine the prevalence and the associated personal and ergonomic factors for CTS among laboratory technicians. A cross-sectional study was conducted among 279 laboratory technicians at King Fahd Hospital, Saudi Arabia, who filled in a self-administered questionnaire, including questions regarding their demographic criteria, occupational history, job tasks, workplace tools, ergonomic factors at work, and symptoms suggestive of CTS. Physical examinations and electrodiagnostic studies were carried out for those who had symptoms suggestive of CTS to confirm the diagnosis. Univariate and multivariate analysis were performed for both personal and physical factors in association with confirmed CTS among laboratory technicians. The prevalence of CTS among the laboratory technicians was 9.7% (27/279). The following were the statistically significant risk factors for CTS among them: gender (all cases of CTS were female, P=0.00), arm/hand exertion (OR: 7.96; 95% CI: 1.84-34.33), pipetting (OR: 7.27; 95% CI: 3.15-16.78), repetitive tasks (OR: 4.60; 95% CI: 1.39-15.70), using unadjustable chairs or desks (OR: 3.35; 95% CI: 1.23-9.15), and working with a biosafety cabinet (OR: 2.49; 95% CI: 1.11-5.59). CTS cases had significant longer work duration (17.9 ± 5.6 years) than CTS non-case (11.5 ± 7.4 yeas) with low OR (1.108). This study demonstrates some personal and ergonomic factors associated with CTS among the laboratory technicians, including female gender, arm/hand exertion, pipetting, repetitive tasks, working with a biosafety cabinet, and an unadjusted workstation.

A perfusion incubator liver chip for 3D cell culture with application on chronic hepatotoxicity testing

Liver chips have been developed to recapitulate in vivo physiological conditions to enhance hepatocyte functions for assessing acute responses to drugs. To develop liver chips that can assess repeated dosing chronic hepatotoxicity, we need to ensure that hepatocyte functions be maintained at constant values over two weeks in stable culture conditions of sterility, temperature, pH, fluidic-flow of culture media and drugs. We have designed a perfusion-incubator-liver-chip (PIC) for 3D cell culture, that assures a tangential flow of the media over the spheroids culture. Rat hepatocyte spheroids constrained between a cover glass and a porous-ultrathin Parylene C membrane experienced optimal mass transfer and limited shear stress from the flowing culture media; maintained cell viability over 24 days. Hepatocyte functions were significantly improved and maintained at constant values (urea, albumin synthesis, and CYP450 enzyme activities) for 14 days. The chip act as an incubator, having 5% CO2 pressure-driven culture-media flow, on-chip heater and active debubbler. It operates in a biosafety cabinet, thus minimizing risk of contamination. The chronic drug response to repeated dosing of Diclofenac and Acetaminophen evaluated in PIC were more sensitive than the static culture control.

Development of an elution device for ViroCap virus filters

Environmental surveillance of waterborne pathogens is vital for monitoring the spread of diseases, and electropositive filters are frequently used for sampling wastewater and wastewater-impacted surface water. Viruses adsorbed to electropositive filters require elution prior to detection or quantification. Elution is typically facilitated by a peristaltic pump, although this requires a significant startup cost and does not include biosafety or cross-contamination considerations. These factors may pose a barrier for low-resource laboratories that aim to conduct environmental surveillance of viruses. The objective of this study was to develop a biologically enclosed, manually powered, low-cost device for effectively eluting from electropositive ViroCap™ virus filters. The elution device described here utilizes a non-electric bilge pump, instead of an electric peristaltic pump or a positive pressure vessel. The elution device also fully encloses liquids and aerosols that could contain biological organisms, thereby increasing biosafety. Moreover, all elution device components that are used in the biosafety cabinet are autoclavable, reducing cross-contamination potential. This device reduces costs of materials while maintaining convenience in terms of size and weight. With this new device, there is little sample volume loss due to device inefficiency, similar virus yields were demonstrated during seeded studies with poliovirus type 1, and the time to elute filters is similar to that required with the peristaltic pump. The efforts described here resulted in a novel, low-cost, manually powered elution device that can facilitate environmental surveillance of pathogens through effective virus recovery from ViroCap filters while maintaining the potential for adaptability to other cartridge filters.

Rapid Pathogen detection By Surface Swab Sampling and Wireless Biosensing

This paper presents a method that combines surface swab sampling and wireless biosensing for rapid detection of foodborne pathogens on solid surfaces. The method can be applied to any surfaces, including the surface of food processing tables and fresh produce, for improving food safety. Surface swab sampling is an inexpensive and easy-to-use method of collecting pathogens. A desired area or the whole area of a target surface (if needed) can be swabbed to collect pathogens rapidly. In this study, we first investigated the efficiency of capture and release of a model pathogen, Salmonella Typhimurium, in swab sampling. A Salmonella-contaminated surface (2 × 2 cm2 flat polyethylene slab) was prepared by using a spot-inoculation method. Five drops of a Salmonella solution (20 ul/drop at concentrations from 5X108 cfu/ml to 5X103 cfu/ml) were inoculated on the polyethylene surface and allowed to dry in a biosafety cabinet for 2 hours. Swab sampling was then conducted at a controlled temperature and humidity to capture the pathogen. Finally, the swabs used were dipped in a TBS solution for releasing the captured Salmonella cells. Various swab tip materials were tested, including cotton, rayon, and nylon. The effects of swab force and sonication/vortexing on the capture and release efficiency were also investigated. The released Salmonella cells were finally detected and quantified instantaneously by using a wireless phage-based magnetoelastic biosensor. By comparing the quantities of the initially inoculated cells and detected cells, the efficiency of capture and release was calculated.

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment.

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

Cabinet Decontamination Using Formaldehyde

Formaldehyde fumigation is the most widely used method for terminal decontamination of biosafety cabinets in the UK. International standards define volumes of Formalin and water to be used for an e...