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The anaerobic incubator should be placed in a location with minimal temperature fluctuations and easy access for operation. Avoid direct sunlight and keep it away from heat sources to ensure stable conditions. Make sure the unit is placed on a flat, level surface to prevent any instability during use.
Gas cylinders, including nitrogen bottles, must be positioned securely and upright. Install pressure regulators (including pressure gauges) separately and ensure they are properly mounted in an accessible location. Always check that all connections are tight to avoid leaks.
Before starting, connect the gas lines carefully and perform a thorough leak test. If necessary, apply a small amount of sealing compound at each joint to prevent air infiltration. This step is crucial to maintain an effective anaerobic environment.
**Operating Room Preparation:**
1. Arrange all required tools and accessories according to the user manual. Place two non-toxic plastic bags inside the operating room.
2. Turn on the power supply and activate the temperature controller. Set the desired temperature and safety limit based on your experiment.
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 check to ensure proper sealing.
5. Begin the first nitrogen replacement:
- Connect one end of the rubber tubing to the operating room’s air inlet and the other to a plastic bag.
- Open the nitrogen valve and fill the plastic bags with nitrogen until full. Close the valve and seal the bags tightly.
- Attach the latex gloves to the observation plate flange and secure them firmly.
- Transfer the nitrogen-filled plastic bags into the operating room and release the gas completely.
6. Repeat the nitrogen replacement process for the second time. Before each refill, evacuate the sampling chamber using the foot switch to control exhaust.
7. For the third replacement, use a mixed gas consisting of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide:
- Switch the gas path to open intake valve 3. Vacuum the sampling chamber first, then use the foot switch to manage exhaust as needed.
- Once the mixed gas is in the plastic bag, close valve 3 and open valve 5. Adjust the flow meter to about 10 ml/min.
- Gradually transfer the mixed gas into the operating room. After three cycles, the oxygen level should be significantly reduced.
8. Open the deoxidizing agent and turn on the deoxygenation catalyst. Power on the Meilan indicator and monitor color change after one hour to confirm anaerobic conditions.
9. Activate the UV sterilization lamp to disinfect the interior. The duration depends on the specific experimental requirements.
**Strain Inoculation and Cultivation:**
1. Ensure the sampling chamber door is closed and sealed.
2. Open the outer door, place the strain inside, and close it immediately.
3. Perform three nitrogen flushes in the sampling chamber:
- Start by vacuuming the chamber to 500 mmHg (66 kPa) using the vacuum pump.
- Manually open nitrogen valve 2 to refill, return the pressure gauge to zero, and close the valve. Repeat this twice.
- On the third cycle, stop the vacuum at 500 mmHg, open valve 4 to introduce the mixed gas, and repeat the process.
4. If the initial vacuum is lower than expected, increase the number of nitrogen flushes accordingly.
5. Check the internal and external doors, then perform a low vacuum test of 100 mmHg (13 kPa).
6. For long-term operation, ensure the incubator runs continuously.
- Check the Meilan indicator daily. If it turns blue, re-ventilate the system.
- Maintain a slow flow of mixed gas (about 10 ml/min) to sustain anaerobic conditions.
- Replace the oxygen scavenger and desiccant every day to maintain optimal performance.
7. The temperature inside the operating room can be adjusted as needed.
8. Adjust the pressure regulators on the gas cylinders to maintain an output pressure of approximately 0.1 MPa.
**Precautions:**
1. Install the equipment in a clean, temperature-stable area.
2. Familiarize yourself with all connected instruments and their proper usage before operation.
3. Always ensure the culture is 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, remove the culture for further handling.
5. Regularly inspect the gas lines for leaks.
6. When replacing gas cylinders, make sure all connections are tightened to prevent oxygen contamination.
7. Follow the manufacturer’s guidelines for vacuum pump maintenance, including regular oil checks and refills.