Anaerobic culture method

Anaerobic bacteria cannot grow
under aerobic conditions.

To cultivate anaerobic bacteria, create an anaerobic environment
.

Usually, a reducing agent is added to the medium, or free oxygen in the environment is removed by physical or chemical methods to reduce the redox potential
.

Such as herpes culture medium, sodium thioacetate medium, bovine heart brain immersion medium and so on
.

There are many commonly used anaerobic culture methods, which can be selected according to
the actual situation.

1.
The anaerobic cylinder method inoculates the specimen of the plate or liquid medium test tube, which can be put into the anaerobic cylinder for culture, the anaerobic cylinder is an ordinary drying cylinder, and the anaerobic environment is caused by physical and chemical methods, so as to cultivate the anaerobic bacteria
.

2.
Anaerobic bag (Bio-bag) is to create an anaerobic environment in the plastic bag to cultivate anaerobic bacteria
.

The plastic bag is transparent and not breathable, and contains a gas generation tube (sodium bicarbonate solid with boron** sodium and ampoule 5% citrate), Meilan indicator tube, palladium catalyst tube, and desiccant
.

After placing the inoculated plates, try to squeeze out the air inside the bag, and then seal the mouth of the
bag.

First break the gas generation tube, then break the Meilan indicator tube, and the named bag will cause a gasless environment
within half an hour.

If there is no mutation, it means that the bag has reached an anaerobic state and can be incubated
.

3.
Anaerobie glove box (Anaerobie glove box) is an anaerobic incubator
.

It is a large closed metal box, the front of the box has a transparent panel made of plexiglass, and the board is equipped with two gloves, which can be operated in the box through gloves, hence the name
.

There is an exchange room on the side of the box, with two doors inside and outside, and the inner door is closed first inside the
box.

To put the object into the box, first open the outer door, put it in the exchange room, close the outer door for pumping and ventilation (H2, CO2, N2) to reach anaerobic state, and then reach the gloves to open the door of the exchange room, move the items into the box, and close the inner door
.

Maintaining an anaerobic state in the box is also the principle
of using the hydrogen in the aeration to catalyze palladium and the residual oxidation of the money in the box to synthesize water.

The box can adjust the temperature, itself is an incubator or incubator that is attached to it, but also into the dissecting microscope to facilitate the observation of anaerobic bacteria colonies, this anaerobic box is suitable for a large number of anaerobic bacteria culture studies, a large number of media can be put into the pre-reduction and anaerobic sterility test
.

The pumping, inflation, anaerobic environment and temperature of the metal hard-walled anaerobic box are automatically adjusted
.

4.
Anaerobic box: the principle is the same as the anaerobic bag, there are finished products for sale
.

5.
Biological oxygen consumption method: put organisms (mostly plants) in a closed container, consume oxygen, and produce carbon dioxide for bacterial growth
.

I haven't seen it
.

6.
Coke end-gallic acid method: spread gauze or filter paper on a clean slide, evenly sprinkle with scorched end-gallic acid, and then mix with NaHCO3 powder or NaOH solution, quickly inoculate the plate of bacteria upside down on it, and use melted ash to seal the edge, creating an enclosed space
.

Oxygen
consumption after the reaction of scorched end-palonic acid with alkali.

This method is used for the cultivation of anaerobic bacteria that are not strict with anaerobics, and is simple
.

If present, Clostridium
.

7.
Herpetic meat culture medium: itself is an anaerobic culture method
without special equipment.

The herpes and broth are packed into large test tubes, and the liquid level is sealed with vaseline, creating an anaerobic environment
.

Anaerobic environment formation in the operating room: place the necessary accessories and utensils according to the requirements of use, and put two non-toxic plastic bags
into the operating room.

Turn on the lights, turn on the thermometer, adjust the required temperature and safety temperature
.

Put 1000 g of palladium granules (blocked) and 500 g of desiccant in the operating room and put in Meilan indicator (blocked
).

Close the indoor and outdoor doors of the sampling and vacuum calibration
.

Secondary displacement in the operating chamber (nitrogen displacement): First insert the rubber tube into the air intake in the operating chamber, and insert the plastic bag
at the other end.

Turn on the nitrogen inlet path, open the nitrogen control valve, make the two plastic bags full of nitrogen, and then tighten the mouth of the
bag.

Put the latex gloves on the observation plate flange ring and tighten tightly
.

Gradually place the nitrogen in the plastic bag in the operating room until it is all released
.

Operation room D secondary replacement (nitrogen displacement) Repeat the nitrogen filling process, and pay attention to opening and closing the exhaust
gas with a foot switch at any time.

The operating chamber D triple displacement (mixed gas displacement) :( the mixed gas ratio is: N2↑ 85% H2 ↑ 10% CO2↑ 5 %) The ventilation circuit opens the mixed air channel through the valve intake, and the foot switch is opened and closed at any time when inflating
.

After the mixture is filled with a plastic bag, turn off the gas through the gas in-circuit valve (three-way valve), so that the gas mixture is operated through the flow meter input and adjust the flow meter, the flow rate is about
10 ml per minute.

Gradually discharge the mixture from the plastic bag in the operating room
.

After three air changes, the oxygen content of the gas in the operating room is already in a trace state
.

Open the palladium granular oxygenator in the operating room, turn on the power supply of the oxygenation catalyst for catalytic oxygen removal, and open the Meilan indicator (Meilan ampoule) to observe its discoloration after one hour, and the discoloration is to achieve an anaerobic environment
in the operation room.

Turn on the ultraviolet sterilization lamp and sterilize the room
.

The sterilization time is customized
.