a mobile biosafety microanalysis system for infectious agents - hepa air filter replacement

The biological threat posed by pathogens such as Ebola virus must be quickly diagnosed while protecting the safety of personnel.
Scanning Electron Microscopy and Microanalysis require minimal sample preparation, which helps to identify harmful substances or substances.
Here we report a compact biosafety system for rapid imaging and elemental analysis of samples (including powders, viruses and bacteria) that is easy to transport to the scene of the accident.
The best solution is to package the entire microscope in Class III bio-safe High
Air Efficiency of particles (HEPA)
A filter cabinet with negative internal air pressure to the surrounding room (, ).
This avoids the design problem that contains only one part of the microscope system (
This will make it difficult to seal the joints around the complex parts of the system).
In addition, the vacuum exhaust is inside the containment to avoid any potential aerosol hazards that may arise when the microscope chamber is pumped.
A complete simulation
SEM shell design built around JEOL JCM5700 ().
Through simulation, a comprehensive operation test of imaging and spectral collection was carried out by SEMup in place.
This allows the design to be adjusted before final manufacturing.
The operation of the use of electrical partitions was tested to ensure the correct electronic function, and adjustments were made to allow ergonomic positioning of the glove port and sample exchange, aperture alignment through the box
The cabinet is equipped with three HEPA filters for intake, exhaust and passage
By being the chamber of the gas gate that brings infectious specimens into the microscope (, ).
The filter can be easily removed while preventing any external contamination (
Is called "bag in/bag out" replacement).
After the port can be used to purify the use of the entire equipment, the gas phase hydrogen peroxide ().
The sensor provides a reading of the temperature and pressure inside the housing, which remains at a lower level than the outside, so when passing through
Insert the sample by opening the chamber and breathe in the air inward to protect the external environment from pollution ().
Close the equipment in the air
The tight box causes problems with the air cooling of the microscope, which is designed to work in a laboratory with adequate ventilation and temperature control systems.
Includes six Peltier refrigerator units to provide adequate cooling (, Blue arrow).
The design also has a back door through which the microscope can be pushed out on the ramp for repair (, ).
All controls of the microscope have external controls including electric stage motion, magnification selection, beam strength and focusing functions.
Electronic devices with external control are connected by electrical partitions (, ).
A common port (
Blue Circle, in)
Additional detectors or accessories are also included if required.
All fluid samples are filtered using a SPI hole pc track etching filter with a diameter of 13mm (
West Chester SPI Supplies, Pennsylvania, USA)
Hold 13mm Swinnex®Filter bracket (
Bianca, Massachusetts, United States of America)
Connect to the syringe with Luer-
Music®Joint to prevent leakage of samples.
In class II biosafety cabinets, bacterial suspended matter grows on agar plates.
About two cycles.
A large number of bacteria were suspended in 1 ml PBS.
If the suspension is too turbid, 10 times dilution is performed.
The filter is first wet through the device via 2 ml PBS. Then 0.
Apply 2 ml of bacterial suspension to the filter with 1 ml syringe, then use 2 ml syringe for 3 consecutive 2 ml PBS wash.
Finally, apply 2 ml 4% of propolis with a 2 ml syringe.
After waiting for an hour, wash the filter with 2 ml of 50% ethanol, 2 ml of 70% ethanol, 2 ml of 85% ethanol, 2 ml of 95% ethanol, and then with 2 ml of 100% ethanol
The syringe is either operated by hand or pumped with a Legato 200 syringe (
KD science, hollyston, United States of America).
Cut and install on SEM stub using double filter
Double-sided adhesive carbon sheet and silver glitter paint to create contact between the stub and the filter paper.
Q15r S using a legal quantity (
Quorum Technology, East Sussex, UK)containing a 0.
1mm gold target.
The sample was pumped down, washed with ar, and deposited 120sec sec with gold on the rotary table.
Working in Class II biosafety cabinets, powder samples are mounted directly in double
Spray a small amount of double-sided adhesive carbon disk attached to the metal sample stub using a spatula.
Then gently press the powder with a spatula to improve adhesion.
The stub is then dumped on the waste container and excavated to remove any excess loose particles.
When the case is open, the negative pressure alarm sounds until the case reaches operation 0.
5 "HO is below the environmental pressure that is usually achieved in less than a minute.
This confirms the operating status of the system and the function of the alarm system.
The internal negative pressure is displayed in red on the controller display screen ().
The temperature control is automatic, keeping the internal temperature of 20. 5°C. The SEM (
JEOL CarryScope Limited, Tokyo, Japan)
Is controlled by a computer and is equipped with X, Y and Z motor drives connected to the electrical head of the enclosure ().
The only difference in SEM operation is that at any time it is necessary to use SEM physical contact with the glove port or through the sample (, ).
Therefore, the operation includes opening SEM, opening and closing the sample insertion stage, tilting of the sample stage, in-
The flat rotation of the sample on the stage, the alignment of the condenser aperture, the repair of the electronic source and the adjustment of the condenser aperture all require the use of the glove port.
All other functions of SEM are electronically controlled externally (
Shift, focus, zoom in, Smudge, contrast and brightness).
SEM operates at 4 kv with a working distance of 7mm and a condenser aperture of 30 μm. Images (
2560x1920 pixels)
Secondary electron detector acquisition using a acquisition time of 160 seconds. X-
Collection of ray spectra using X-ray
Silicon Drift Detector max 80mm (SDD)
Micro-analysis software package with INCA (
Oxford Instruments, Abingdon, UK).
The entire detector is controlled by an external electron and connected to the computer through the electrical partition of the biosafety housing (). The X-
The Max SDD is cooled by the Peltier cooler, so no liquid nitrogen is required. For X-
X-ray microanalysis SEM works at 20kv with a working distance of 10 μm mm and a condenser aperture of 100 μm.
Collect maps and line scans in 512X352 pixels, 2048 X-
The light channel and 50 frames of each acquisition, the dwell time of each pixel is 100 μs: It takes 15 minutes for each acquisition.
The Zairian Ebola virus is transmitted in Vero E6 Cells and prepared as described earlier.
Sample Analysis with SDS-
Page and Western blot, and present non-
Fixed infection with more than 4% formaldehyde.
Remove the excess fixer by placing the fixed sample in the slideA-
Lyzer G2 cassette 0.
Capacity of 5 ml, 10,000 MWCO (
Thermal Science Pierce Protein Research Products in Rockford, Illinois, USA)
And then dialysis with PBS.
All with infectious Ebola virus (
Culture and purification of virus)
Conducted at biosafety level 4 laboratory, National Microbiology Laboratory, Canadian Public Health Agency, Winnipeg, Manitoba.
Hamster Kidney fiber cells (BHK-21: ATCC)
Growth in Dulbecco modified Eagle medium (Gibco)
Serum containing 10-Tal (FBS)(Gibco)
And twice the penicillin/L-glutamine (Gibco). BHK-
Ankara with improved acne (MVA)(
Please provide Dr.
Cao Jingxin, National Microbiology Laboratory)
Work at 37 °c for 1 hour
After washing with PBS, complete growth medium was added and cells were incubated for 48 hours at 37 °c.
MVA is the frozen harvest
Thawed cell culture 3X, in-
80 °c, room temperature.
After the final thaw, clear the supernatant by centrifuge for 3 minutes at 3000 x g to remove the cell fragments.
Culture on CAB plates and incubate for 24 hours at 37 °c.
1/10 Columbia broth 300 ml, including 0.
1mm MnSO, vaccinated in a circle, incubated on the orbital shaker for 96 hours at 37 °c.
The culture was centrifuge for 15 minutes at 4 °c at 4900g.
Pour the supernatant in sterile deionised water and wash the particles three times, then centrifuge at 4900 × g for 15 minutes at 4 °c.
The entire spore preparation was re-suspended in 40 ml of ethanol, transferred to a 50 ml tube, and then incubated at room temperature for 2 hours.
Then centrifuge at 4900g for 15 minutes at 4 °c and then decan the ethanol out.
Then wash twice with 40 ml of sterile deionized water and centrifuge at 49 °c for 15 minutes
The rest was washed away.
The final spore particles are suspended in 10 ml of sterile deionised water. Non-
Typhi is in one and a half overnight.
Solid agar plates at 35 °c
Growth in sugar phosphate Agar (TPA)
Exercise the tube at room temperature for two days and then divide
Culture on TPA plates at room temperature for another two days.
Fix the fluid sample for transmission electron microscopy inspection in 2% formaldehyde/1% formaldehyde.
The sample is adsorbed to the carbon emitted by the light-
Coating formvar film on 400-
Grid copper grid 1 min, negatively dyed with 2% methyl amine tungsten salt (Nano-W;
Nano probe, AP Hank, New York, USA).
Specimen observation of 200 kV transmission electron microscopy in the month of the scandal (
Hillsboro, a company in the United States)
Operate and enlarge the X25, 500 to X71, 000 in the instrument.
Use the AMT Advantage XR 12 CCD camera to obtain a digital image of the sample (
MA, Danvers, AMT, USA).