Operating procedures for anaerobic incubators

Operating procedures of the anaerobic incubator (1) Anaerobic environment formation in the operating room: 1.
Place the necessary accessories and utensils according to the requirements of use; 2.
Turn on the lighting, turn on the temperature controller, adjust the required temperature; 3.
Put 1000g of palladium granules (closed) and 500g of desiccant in the operating room, and put in Meilan indicator paper (closed); 4.
Close the indoor and outdoor doors of sampling, and vacuum calibration; 5.
Initial replacement in the operating room (nitrogen displacement): A.
Put the latex glove on the observation plate flange ring and tie it tightly B.
Connect the nitrogen intake circuit, open the nitrogen control valve 1, make the glove bulge, close the valve 1, and then tighten the bag mouth; 6.
Operation room D secondary replacement (nitrogen displacement) Repeat the nitrogen filling process, the sampling room first evacuate, and pay attention to opening and closing the exhaust
gas with a foot switch at any time.

This is three consecutive nitrogen displacements
.

7.
Four displacements of operation chamber D (mixed gas displacement): (mixed gas ratio is N2 85%, H2 10%, CO2 5%) A.
The ventilation circuit opens the mixing valve 3 inlet, the sampling chamber is vacuumed first when inflating, and the exhaust is opened and closed at any time with a foot switch; B.
Turn off the gas mixture valve 3 and open the valve 5, so that the gas mixture passes through the flow meter, adjust the flow meter, the flow rate is about 10 ml per minute; C.
The mixture of gases is repeated 2-3 times, and after the above process, an anaerobic environment
is basically formed.

8.
Open the granule oxygen remover in the operation room, turn on the power supply of the deoxygenation catalytic catalyst for catalytic deoxygenation, open the Meilan indicator paper to observe its discoloration status after one hour, and do not change the color to achieve anaerobic environment in the operation room; 9.
Turn on the ultraviolet sterilization lamp, sterilize the room, and the sterilization time is customized
according to the specific experiment.

(2) Insertion and culture of strains: 1.
Check the sampling room door and close it tightly; 2.
Open the outside door of the sampling room, and close the outer door after placing the bacteria in the sampling room; 3.
The sampling chamber is filled with nitrogen and replaced three times: open the vacuum pump, first pump the vacuum degree of 500ml Hg (66kPa) above the stop, and then manually open the nitrogen valve 2 intake, so that the pointer returns to zero, turn off the valve 2
.

D Repeat the operation twice
.

D When operating three times, stop above the vacuum level of 500 ml Hg column (66 kPa), and then open the valve 4 to enter the mixture, so that the pointer returns to zero, and close the valve 4
.

End of the three-stage process of nitrogen filling replacement in the sampling chamber; 4.
If the selected vacuum is low, the number of substitutions needs to be increased; 5.
Open and close the outdoor door of the sampling room, and then pump the low vacuum 100 ml Hg (13 kPa) for inspection; 6.
Conditions for long-term continuous use of the anaerobic incubator: A.
Open the Meilan indicator paper in the operating room every day for observation, and re-ventilate if it is not normal; B.
To continuously input a trace amount of mixed gas for a long time, so that the combined nitrogen energy and the trace amount of oxygen can be absorbed by catalysis to ensure the anaerobic state of the room, and the flow rate of the supplementary gas mixture is selected to be about 10ml per minute; C.
Run continuously for one day, replacing the oxygen scavenger and desiccant
once.

D.
The operating room temperature can be arbitrarily selected and controlled
.

7.
Mixed gas cylinder, nitrogen cylinder output pressure adjustment: adjust the pressure reducing valve, so that the output pressure is about
0.
1Mpa.