I. Operation: Creating an Anaerobic Environment
1. Adjusting the output pressure of the mixed nitrogen and combined gas cylinders: Adjust the pressure reducing valve to approximately 0.1 MPa.
2. Turn on the power switch and the temperature controller, adjusting the desired temperature. The temperature inside the incubator in the operating chamber can be selected and controlled arbitrarily.
3. Place the necessary accessories and equipment according to the usage requirements, and put two non-toxic plastic bags into the operating chamber.
4. Place 1000g of dried palladium granules (sealed) and 500g of desiccant, along with an anaerobic indicator (sealed), into the operating chamber.
5. Close the inner and outer doors of the sampling chamber tightly and perform a vacuum check.
6. Secondary Nitrogen Replacement in the Operating Chamber:
(1) Insert a rubber tube into the air inlet of the operating chamber, and insert the other end into a plastic bag.
(2) Connect the nitrogen inlet, open the nitrogen control valve, fill both plastic bags with nitrogen, and then tie the bag openings tightly.
(3) Put latex gloves on the flange ring of the observation panel and tie them tightly.
(4) Gradually release the nitrogen from the plastic bags into the operating chamber until all the nitrogen is released.
7. Secondary Nitrogen Replacement in the Operating Chamber: Repeat the nitrogen filling process, and pay attention to opening and closing the exhaust valve at any time.
8. Tertiary Mixed Gas Replacement in the Operating Chamber:
(Mixed gas ratio: N2 90%; H2 5%; CO2 5%).
(1) Change the gas path, open the mixed gas inlet valve, and open and close the exhaust valve at any time during filling.
(2) After the mixed gas fills the plastic bags, close the mixed gas straight-through valve (three-way valve).
(3) Gradually release the mixed gas from the plastic bag into the operating chamber.
(4) After three air exchanges, the oxygen content in the operating chamber is reduced to trace amounts.
9. Turn on the palladium granule oxygen scavenger in the operating chamber and connect the power supply to the oxygen catalytic deoxygenation device. After one hour, observe the color change of the anaerobic indicator strip. A color change indicates that the operating chamber has reached an anaerobic environment.
10. Turn on the ultraviolet sterilization lamp to sterilize the chamber. The sterilization time is determined by the user.
II. Placement and Cultivation of Bacterial Strains
1. Check and close the door of the sampling chamber.
2. Open the outer door of the sampling chamber, place the bacterial strain into the sampling chamber, and then close the outer door.
3. Perform three nitrogen purging and replacement processes in the sampling chamber: first, evacuate to a vacuum level of 500 mmHg (66 kPa) or higher, then manually open the nitrogen valve to allow the pointer to return to zero before proceeding to the next operation.
4. If a lower vacuum level is selected, the number of replacement cycles needs to be increased.
5. After opening and closing the sampling room doors, a low vacuum of 100 mmHg (13 kPa) should be applied for inspection and assistance.
6. Conditions for long-term continuous use of the anaerobic incubator:
(1) Observe the anaerobic indicator bar in the operating room daily. If normal, it can continue to be used. If abnormal, the air must be replaced.
(2) A small amount of mixed gas should be continuously introduced to ensure the added hydrogen combines with trace amounts of oxygen for catalytic absorption, maintaining an anaerobic state. The flow rate of the mixed gas should be approximately 10 ml per minute.
(3) Replace the desiccant and desiccant every 3 days of continuous incubation.
7. Application of the inoculation rod sterilizer:
(1) Use a nickel-chromium heating wire to short-circuit and heat it to a red-hot state for sterilization (of the inoculation rod).
(2) The wax melting point can be placed directly on the inoculation rod, and the test tube sealing wax opening can be rotated to melt the wax.