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To ensure the proper use and maintenance of the anaerobic incubator, follow these detailed guidelines:
1. Place the anaerobic incubator in an area with minimal temperature fluctuations and easy access for operation. Avoid direct sunlight and keep it away from heat sources. Ensure the unit is placed on a stable, level surface to prevent any instability or damage.
2. Securely position the gas cylinder and nitrogen bottle on a flat, stable platform. Install the pressure regulator (including the pressure gauge) separately and ensure it is properly aligned and positioned for easy access and monitoring.
3. Connect all gas lines carefully and check for leaks. If necessary, apply a sealant at each joint to prevent air leakage, ensuring airtight connections that maintain the anaerobic environment.
**Operating Room Anaerobic Environment Setup:**
1. Arrange all required accessories and equipment according to the user manual. Place two non-toxic plastic bags inside the operating room for gas exchange procedures.
2. Turn on the power supply and activate the lighting system. Set the temperature controller to the desired operational temperature and establish a safe temperature limit.
3. Insert 1000g of sealed palladium pellets and 500g of desiccant into the operating room. Also, place a sealed Meilan indicator inside for oxygen monitoring.
4. Close both the internal and external doors of the sampling chamber and perform a vacuum test to confirm airtightness.
5. First Gas Exchange (Nitrogen Replacement):
A. Insert the rubber tubing into the inlet port of the operating room and connect the other end to the plastic bag.
B. Open the nitrogen valve, allowing the plastic bags to fill with nitrogen. Once full, close the valve and secure the bag mouth.
C. Attach the latex gloves to the observation plate flange and tighten them securely.
D. Transfer the nitrogen-filled plastic bag into the operating room and release the gas completely.
6. Repeat the nitrogen replacement process for the second time. Begin by evacuating the sampling chamber, using the foot switch to control the exhaust as needed.
7. Third Gas Exchange (Mixed Gas Replacement):
A. Switch the gas path to open valve 3 and allow mixed gas (Nâ‚‚ 85%, Hâ‚‚ 10%, COâ‚‚ 5%) to enter. The sampling chamber should be evacuated first, and the exhaust should be controlled with the foot switch.
B. After filling the plastic bag with the mixed gas, close valve 3 and open valve 5. Adjust the flow meter to maintain a flow rate of approximately 10 ml/min.
C. Slowly release the mixed gas from the plastic bag into the operating room.
D. After three complete gas exchanges, the oxygen level inside the operating room will be significantly reduced.
8. Activate the deoxidizing agent and turn on the catalytic deoxygenation system. Monitor the Meilan indicator (Meilan bottle) for color change after one hour to verify the anaerobic condition.
9. Turn on the UV sterilization lamp to disinfect the interior. The duration of sterilization depends on the specific experimental requirements.
**Strain Implantation and Cultivation:**
1. Confirm that the sampling chamber door is closed properly.
2. Open the external door, place the strain into the sampling chamber, and then close the outer door.
3. Perform three nitrogen flushes in the sampling chamber:
A. Start the vacuum pump and evacuate the chamber to at least 500 mmHg (66 kPa).
B. Manually open valve 2 to introduce nitrogen, return the pressure gauge to zero, and close the valve. Repeat this process twice.
C. On the third cycle, stop the vacuum at 500 mmHg, open valve 4 to introduce the mixed gas, and return the gauge to zero before closing the valve again.
4. If the vacuum level is low, increase the number of nitrogen flushes accordingly.
5. Open and close the internal and external doors of the sampling chamber, then perform a low vacuum test of 100 mmHg (13 kPa) to ensure integrity.
**Long-Term Operation Requirements:**
A. Check the Meilan indicator daily. If it shows abnormal color changes, re-ventilate the chamber immediately.
B. Continuously supply a small amount of mixed gas at a flow rate of about 10 ml/min to maintain the anaerobic state. This helps absorb any trace oxygen present.
C. Replace the oxygen scavenger and desiccant daily during continuous culture.
**Temperature Control:**
The operating room can be set to any desired temperature and maintained accurately through the built-in controller.
**Gas Cylinder Pressure Adjustment:**
Adjust the pressure regulators so that the output pressure remains around 0.1 MPa for safe and consistent gas delivery.
**Precautions:**
1. Install the incubator in a clean, temperature-stable location to avoid environmental interference.
2. Before operation, thoroughly review the user manuals of all connected instruments and ensure correct usage.
3. Always ensure the culture is placed in a fully anaerobic environment.
4. In case of gas failure, the operating room can maintain anaerobic conditions for up to 12 hours. If longer than that, remove the cultures for further handling.
5. Regularly inspect the gas lines for leaks to prevent contamination.
6. When replacing gas cylinders, ensure all connections are tightly sealed to avoid introducing oxygen.
7. Follow the manufacturer’s guidelines for the vacuum pump, including regular oil checks and refills.
By following these steps and precautions, you can ensure a reliable and effective anaerobic environment for your experiments.