I. Procedures for Establishing an Anaerobic Environment
1. Adjust the output pressure of the nitrogen cylinder or mixed-gas cylinder: Regulate the pressure-reducing valve to set the output pressure to approximately 0.1 MPa.
2. Switch on the main power supply, turn on the temperature controller, and set the desired temperature; the temperature within the incubator chamber located inside the operating chamber can be freely selected and controlled.
3. Place all necessary accessories and utensils into the operating chamber in accordance with operational requirements, and insert two non-toxic plastic bags into the chamber.
4. Place 1000g of dried palladium pellets (in a sealed container) and 500g of desiccant into the operating chamber, and also insert an anaerobic indicator (in a sealed container).
5. Tightly close both the inner and outer doors of the sampling chamber, and perform a vacuum check to verify the seal.
6. First Purge of the Operating Chamber (Nitrogen Purge):
(1) First, insert a rubber tube into the gas inlet port of the operating chamber, and insert the other end of the tube into one of the plastic bags.
(2) Connect the nitrogen gas supply line, open the nitrogen control valve, and allow nitrogen to fill the two plastic bags completely; then, tightly tie off the openings of the bags.
(3) Fit the latex gloves over the flanges of the observation window and secure them tightly.
(4) Gradually release the nitrogen contained within the plastic bags into the operating chamber until the bags are completely emptied.
7. Second Purge of the Operating Chamber (Nitrogen Purge): Repeat the nitrogen-filling process described in the first purge, ensuring to open and close the exhaust valve as needed during the procedure.
8. Third Purge of the Operating Chamber (Mixed-Gas Purge):
(The mixed-gas composition ratio is: N2 90%; H2 5%; CO2 5%).
(1) Switch the gas supply line to the mixed-gas channel, open the mixed-gas control valve to allow gas to flow in, and ensure the exhaust valve is opened and closed as needed during the filling process.
(2) Once the plastic bags are filled with the mixed gas, close the mixed-gas direct-flow valve (or three-way valve). (3) Gradually release the mixed gas contained within the plastic bag into the working chamber.
(4) After three cycles of gas exchange, the oxygen content within the working chamber will have reached trace levels.
9. Inside the working chamber, open the package of palladium-pellet oxygen scavenger and switch on the power supply for the catalytic oxygen remover to initiate catalytic oxygen removal. After one hour, check the anaerobic indicator strip to observe any color change; a change in color indicates that an anaerobic environment has been successfully established within the working chamber.
10. Switch on the UV sterilization lamp to sterilize the interior of the chamber. The duration of sterilization may be set as required.
II. Introduction and Cultivation of Microbial Strains
1. Inspect the inner door of the sampling chamber to ensure it is securely closed.
2. Open the outer door of the sampling chamber, place the microbial strains inside, and immediately close the outer door.
3. The nitrogen-purging process for the sampling chamber consists of three cycles: First, evacuate the chamber to a vacuum level of 500 mmHg (66 kPa) or higher, then stop; next, manually open the nitrogen inlet valve to introduce gas until the pressure gauge needle returns to the zero position, at which point the next cycle may begin.
4. If a lower vacuum level is selected for the evacuation step, the number of purging cycles must be increased accordingly.
5. The inner and outer doors of the sampling chamber are designed to withstand a low vacuum level of 100 mmHg (13 kPa) when closed; this feature facilitates leak testing and operational procedures.
6. Conditions for the long-term, continuous operation of the anaerobic incubator:
(1) Check the anaerobic indicator strip inside the working chamber daily; if the indicator appears normal, continuous operation may proceed. If the indicator appears abnormal, a complete gas exchange procedure must be performed.
(2) A trace amount of the mixed gas must be continuously supplied over the long term to ensure that the replenished hydrogen reacts with any trace oxygen present via catalytic absorption, thereby maintaining the anaerobic state within the chamber. The flow rate for the supplementary mixed gas should be set to approximately 10 mL per minute.
(3) For continuous cultivation runs, the oxygen scavenger and desiccant should be replaced periodically.
7. Operation of the Inoculation Loop Sterilizer:
(1) The nickel-chromium heating wire can be activated at any time to heat up to a glowing red temperature via an electrical short circuit, thereby sterilizing inoculation loops.
(2) The wax-melting port allows for the direct placement of a test tube; the wax-sealed opening of the test tube can be rotated against the heated surface to melt the wax seal.