CN105002126B - A kind of simple batch culture process of facultative aerobe - Google Patents

Abstract

The invention discloses a simple batch culture method for aerobic bacteria or facultative aerobic bacteria, which is cultivated by a multi-layer square plate solid plane fermentation device with a sealed box. The sealed box and the square plate are made of heat-resistant sheet material that is not easy to deform. The size of the sealed box is based on the size that can only accommodate a specified number of stacked square plates. There are two opposite corners of the sealed cover. Inoculation hole. It adopts one-time injection of culture medium, one-time sterilization, one-time formation of multi-layer culture surface, one-time inoculation, one-time washing and harvesting. Its equipment is simple, low in cost, easy to operate, not easy to pollute, and high in production efficiency. Compared with the traditional fermenter liquid fermentation culture method, its culture efficiency can be increased by more than 50 times, and the bacterial concentration can be as high as 150-200 billion/ml, reducing The production cost is more than 50%, which solves the common bottleneck problems such as low production efficiency, difficult concentration of bacterial solution, excessive sewage discharge or high production cost in the mass production and preparation of aerobic bacteria or facultative aerobic bacteria.

Description

A Simple Batch Cultivation Method of Facultative Aerobic Bacteria

technical field

The invention belongs to the field of microbial fermentation, and in particular relates to a simple batch culture method for facultative aerobic bacteria.

Background technique

Fermentation refers to the process in which people use the life activities of microorganisms under aerobic or anaerobic conditions to prepare microbial cells themselves, or direct metabolites or secondary metabolites (Qiu Liyou, Wang Mingdao editor-in-chief. Microbiology: Chemical Industry Press, 2012: 343). With the continuous development of biotechnology, the demand for pure culture of facultative aerobic bacteria in the field of microbial fermentation is increasing. At present, laboratory culture of facultative aerobic bacteria is a common, simple and mature technology, such as liquid, solid Cultivation; test tube, plate, eggplant-shaped bottle culture, etc., but the preparation of mass production is difficult. In the field of pure culture and fermentation of large quantities of facultative aerobic bacteria, traditional fermentation tank liquid aeration culture methods are mostly used. This method generally has the disadvantages of easy pollution in the production process, low production efficiency, difficulty in concentrating the bacterial liquid, excessive sewage discharge and production costs. Advanced bottleneck.

The multi-layer solid fermentation culture method in a sealed box has simple equipment, low cost, convenient operation, no pollution, and high production efficiency; the application of this technology can significantly reduce the amount of medium used, save product concentration costs, reduce energy consumption, and reduce sewage emission. Compared with the traditional fermenter liquid fermentation culture method, the culture efficiency can be increased by more than 50 times, the bacterial concentration can be as high as 150-200 billion/ml, and the production cost can be reduced by more than 50%.

After searching words such as "solid plane fermentation", "solid plane culture" and "multi-layer square plate fermentation culture" through Baidu search engine and VIP Chinese science and technology journal database, no identical or similar content was found. The current microbial solid fermentation culture is a traditional technology and method widely used in this field. Its process is characterized by mixed bacteria fermentation culture, that is, microorganisms and solid medium particles are mixed together. The multi-layer solid culture method in a sealed box provided by this patent technology is characterized by concentrated pure culture, that is, the purified culture of a single bacterium. The batch purification and cultivation of bacteria has a wide range of technical requirements in many biotechnology fields such as strain production, vaccine production, probiotics and biological enzyme production.

Contents of the invention

The purpose of the present invention is to provide a simple batch method for facultative aerobic bacteria in view of the common bottleneck problems such as low production efficiency, difficulty in concentrating bacterial solution, excessive sewage discharge or high production cost in the batch production and preparation process of facultative aerobic bacteria. The cultivation method has simple equipment, low cost, convenient operation, no pollution, and high production efficiency. Compared with the traditional fermentation tank liquid fermentation cultivation method, the cultivation efficiency can be increased by more than 50 times, and the bacterial concentration can be as high as 150 to 200 billion pcs/ml, reducing the production cost by more than 50%.

In order to realize the above-mentioned purpose of the invention, the present invention adopts following technical scheme:

A simple batch culture method for facultative aerobic bacteria, which adopts a sealed box multi-layer square plate solid plane fermentation device for cultivation; the sealed box and square plate are made of heat-resistant thin plate material that is not easily deformed, and the square plate The quantity is 10~20, the size of the sealed box is subject to the specified number of stacked square plates, and two inoculation holes with a diameter of 22mm are set at the opposite corners of the sealing cover, and the inoculation holes are sealed with silicone plugs; The opening of the sealed box body is provided with a sealing cover for closing the opening, and a sealing rubber pad is provided between the sealing cover and the opening edge of the box body, and the sealing cover, the sealing rubber pad and the box body are locked through the three. The connection is fixed by screws; the locking screws are set at the four corners of the sealing cover.

The material of the non-deformable heat-resistant thin plate is stainless steel.

The size of each square plate is set according to production requirements, such as: length 45cm~60cm; width 30cm~45cm; height 1.5cm~2.5cm.

The simple batch culture method of described facultative aerobic bacteria comprises the following steps:

1) Through the inoculation hole reserved on the sealing cover, inject the agar medium suspension that can form a gel with a thickness of 3mm~5mm according to the inner surface area of the square plate, seal the inoculation hole with a silica gel plug, and place it in a high-temperature sterilization equipment for routine use. Sterilize, wait for the temperature of the box to drop to 50°C~60°C, shake the box to mix the culture medium, erect the box based on the position of the square plate, let the agar flow into the square plate from the gap for a while, and wait for the agar solution The surface is balanced and stable, then quickly place the box horizontally and make the opening of the square plate face up, so that the melted agar is evenly distributed in each square plate;

2) After the agar cools down and solidifies, inoculate the liquid strains according to 1%~2% of the volume of the box. The inoculation adopts an aseptic method, and injects or drops the strains into the inoculation hole with the help of an ultra-clean workbench or flame closure. Keep the concentration above 500 million/ml. After inoculation, erect the box with the position of the square plate as the reference, let the bacteria liquid flow into the square plate from the gap for a while, wait for the liquid level of the bacteria to be balanced and stable, then quickly place the box horizontally and make it With the opening of the square plate facing upwards, gently tilt the box and vibrate the box back and forth, so that the bacteria are fully coated on the surface of the agar medium, and cultured at a suitable temperature for 24 h to 72 h to form a bacterial lawn;

3) When the culture is harvested, 15%~20% of the box volume is injected with washing liquid, the box is shaken to fully wash the bacterial lawn, and then the box is tilted, and the washing liquid is exported through the inoculation hole to obtain concentrated pure culture liquid.

In the simple batch culture method of facultative aerobic bacteria, the bacteria are fermented and cultivated on a multi-layer square plate solid medium, and the culture medium is injected into the culture medium in one time, sterilized in one time, a multi-layer culture surface is formed in one time, and inoculated in one time. , One-time washing harvest.

In order to prevent product contamination, through aseptic operation, the method of injecting needles, rubber hose connections and pressurization in the filtered air box is used to introduce the concentrated bacterial liquid into the container to obtain a non-polluting concentrated pure culture bacterial liquid.

The medium remaining in the box is sterilized by heating or high temperature, poured out through the inoculation hole before the medium cools down and solidifies, rinses with water, turns the inoculation hole upside down, and dries for later use.

The invention effectively amplifies the agar plate culture method of bacteria in the laboratory. The method of cultivating bacteria on the agar plate of the plate or eggplant-shaped bottle commonly used in the laboratory is only applicable to small batches of bacterial culture because of its small effective culture area, and is not suitable for the mass production and preparation of bacteria. The effective culture area of a multi-layer square plate with a sealed box is about 30000 cm ² , which is equivalent to the culture area of 200 eggplant-shaped bottles with a specification of 500ml or the culture area of 468 plates with a diameter of 9 cm. The multi-layer square plate culture technology in a sealed box has significant application advantages in the production and preparation of large quantities of facultative aerobic bacteria.

The beneficial effects of the present invention are: the equipment of the present invention is simple, low in cost, easy to operate, not easy to pollute, and high in production efficiency. Compared with the traditional fermenter liquid fermentation culture method, its culture efficiency can be increased by more than 50 times, and the bacterial concentration can be as high as 150-200 billion/ml, reducing the production cost by more than 50%; solving the common bottleneck problems in the mass production and preparation of facultative aerobic bacteria, such as low production efficiency, difficult concentration of bacterial liquid, excessive sewage discharge or high production cost.

Description of drawings

Fig. 1 is the structural representation of square plate;

Fig. 2 is a schematic diagram of a partially decomposed structure of a sealed incubator;

Fig. 3 is a schematic diagram of the decomposition structure of the overall structure of the sealed incubator;

In the figure, 10—square plate, 20—box body, 30—sealing rubber pad, 40—sealing cover, 410—locking screw, 50—inoculation hole.

detailed description

The present invention further illustrates the present invention with following examples, but protection scope of the present invention is not limited to following examples.

An animal micro-ecological preparation factory in Fujian used a liquid fermenter aerated fermentation method to produce Escherichia coli C600. The production process is relatively complicated. The manual labor in the centrifugal concentration process is heavy, it is easily contaminated by bacteria, and the production efficiency is low. Also used eggplant-shaped bottle nutrient agar plane culture, which significantly reduced the consumption of raw materials, because of its small effective culture area, heavy manual labor, cumbersome operation links, high probability of pollution, and limited production efficiency. Applying the sealing box multi-layer square plate solid plane fermentation culture technology of the present invention, the equipment is simple, the investment is small, the production process is simplified, the chance of product pollution is reduced, the production cost is reduced, the sewage discharge is reduced, the production efficiency is significantly improved, and the continuous production of liquid There are more than 200 tons of finished microecological preparations, and good benefits have been achieved.

Taking the production of 10 L of Escherichia coli (C600) concentrated bacterial liquid with a concentration of 150 billion/ml as an example, the above three cultivation methods are compared as follows:

Embodiment 1 Liquid fermenter aerated culture fermentation

1) Equipment

500 L fermentation tank; ultra-clean bench (1400×690×520mm); large-capacity refrigerated centrifuge (DL-4000B, 500 ml×6).

2) Culture medium preparation

Ordinary broth medium: peptone 5 kg, beef extract 1.5 kg, sodium chloride 2.5 kg, distilled water 500 L (beef extract and peptone were produced by Beijing Aoboxing Biological Company).

After the medium was mixed, the pH was adjusted to 7.4 with 10% sodium hydroxide, and the fermenter was autoclaved at 121 °C for 15 min, and then cooled for later use (sodium chloride was a commercially available analytical reagent).

3) Inoculation and culture

Insert 100 ml of Escherichia coli C600 strain through aseptic technique, the strain concentration is 500 million/ml, pass sterile ultra-filtered air into the bottom of the fermenter, and open the sterile ultra-filter exhaust on the top of the fermenter Wells were incubated at 37°C for 36 h with aeration, and the bacterial concentration reached 3 billion/ml.

4) Bacterial liquid concentration

The subpackage of the bacterial culture solution is carried out on the ultra-clean workbench, and the culture solution is exported to plastic centrifuge bottles several times. The centrifuge capacity is 3000 ml (500 ml×6) each time. After sealing the centrifuge bottle, Place them in a centrifugal cup and balance them in a diagonal centrifugal cup. Centrifuge at 4000 rpm for 10 min, discard the supernatant, take 60 ml of sterilized normal saline to wash the sediment in 6 centrifuge bottles in turn, and obtain about 60 ml of concentrated bacterial solution with a concentration of 150 billion cells/ml. 500 L of fermentation culture liquid was centrifuged for a total of 167 times, and the washing liquid was collected to obtain about 10 L of concentrated bacterial liquid with a concentration of 150 billion cells/ml.

5) Major equipment investment and production cost

Equipment: 102,000 yuan (fermentation tank 48,000 yuan; ultra-clean workbench 12,000 yuan; large-capacity freezer

scheming 42,000).

Medium: 1480 yuan (peptone 5 kg×200 yuan; beef extract 1.5 kg×300 yuan; sodium chloride

2.5 kg x 12 yuan).

Water and electricity fee: 591 yuan (sterilization 15 KV×3 h×1 yuan; culture 1 KV×36/2 h×1 yuan;

Centrifuge 1 KV×167/6 h×1 yuan; distilled water 500 L×1 yuan).

Staff salary: 800 yuan (2 people x 2 d x 200 yuan).

6) Waste discharge

500 L of discarded culture medium; 200 L of water for washing equipment.

Embodiment 2 Nutrient agar plane culture in eggplant-shaped bottle

1) Equipment and equipment

Horizontal rectangular pressure steam sterilizer (specification: 30L, 1018×500×600mm); constant temperature incubator (specification: 610×530×1360 mm); ultra-clean workbench (specification: 1400×690×520mm); large capacity Shaker; eggplant-shaped bottle (specification: 500ml).

2) Culture medium preparation

Nutrient agar medium: peptone 0.1 kg; beef extract 0.03 kg; sodium chloride 0.05 kg; agar 0.15 kg; distilled water 10 L.

Mix the culture medium, adjust to pH 7.4 with 10% sodium hydroxide, distribute to 200 eggplant-shaped bottles, 50 ml per bottle, seal the bottle mouth with a silica gel stopper, place at 121 ℃ for 15 min, and wait for temperature Lower the temperature to 50°C~60°C, shake the eggplant-shaped bottle to mix the medium, place it horizontally, wait for the agar to cool down and solidify, and form a nutrient agar gel with a thickness of 3mm~5mm for later use.

3) Inoculation culture

With the help of an ultra-clean workbench, through aseptic procedures, 3 ml of Escherichia coli C600 strains were inserted into each eggplant-shaped bottle, with a strain concentration of 500 million/ml, and a silicone plug sealed the mouth of the bottle. Tilt and shake the eggplant-shaped bottle so that the bacteria are fully coated on the surface of the agar medium, and cultured at 37 °C for 36 h, until the bacteria grow and form a lawn.

4) Bacterial liquid collection

Inject 50ml of sterilized physiological saline into each eggplant-shaped bottle through aseptic technique, seal the bottle mouth, stabilize the eggplant-shaped bottle on the shaker, shake at 50 times/min for 15 minutes, fully wash the bacterial lawn, and export the washing liquid to a sterile container , to obtain about 10 L of concentrated bacterial liquid with a concentration of 150 billion cells/ml.

5) Major equipment investment and production cost

Equipment and equipment: 67,000 yuan (28,000 yuan for steam sterilizer; 20,000 yuan for constant temperature incubator; 12,000 yuan for ultra-clean workbench; 3,000 yuan for large-capacity oscillator; 200×20 yuan for eggplant-shaped bottles)

Medium: 44.6 yuan (peptone 0.1 kg×200 yuan; beef extract 0.03 kg×300 yuan; chlorinated

Sodium 0.05 kg×12 yuan; agar 0.15 kg×100 yuan).

Water and electricity fee: 103 yuan (sterilization 5 KV×3 h×5 times×1 yuan; culture 1 KV×36/2 h×1 yuan;

10 L of distilled water × 1 yuan; 1000 L of tap water for washing equipment).

Staff salary: 1200 yuan (2 people x 3 d x 200 yuan).

6) Waste discharge

10 L of discarded culture medium; 1000 L of water for washing equipment.

Example 3 Culture on Solid Plane with Multi-layer Square Plate in Sealed Box

1) Equipment

Horizontal rectangular pressure steam sterilizer (specification: 30L, 1018×500×600 mm); constant temperature incubator (specification: 750×600×600 mm); ultra-clean workbench (specification: 1400×690×520 mm); Large-capacity oscillator; stainless steel sealed box multi-layer square plate, including: 12 stainless steel square plates, length, width and height 60×45×2.0㎝; 1 stainless steel sealed box, length, width and height 60×45×24㎝; sealed 1 cover, length and width 49×28㎝, two inoculation holes that can be sealed with silicone plugs are set at the opposite corners of the sealing cover surface, and the sealing cover is connected with the box body with the help of silicone pads, and the sealing is sealed with bolts (note: sealing The size of the box is subject to the ability to accommodate 12 stacked stainless steel square plates, see attached picture).

2) Culture medium preparation

Nutrient agar medium: peptone 0.1 kg; beef extract 0.03 kg; sodium chloride 0.05 kg; agar 0.15 kg; distilled water 10 L.

Mix the medium evenly, adjust the pH to 7.4 with 10% sodium hydroxide, inject the medium into the sealed box through the inoculation hole of the sealed box, seal the inoculated hole with a silica gel plug, and put it at 121 ℃ for 15 minutes to autoclave, and wait for the box to Lower the temperature to 50°C-60°C, shake the box to mix the culture medium, stand the box upright based on the position of the square plate and let the agar flow into the square plate from the gap, then quickly place the box horizontally and make the square plate Facing upward, distribute the melted nutrient agar evenly in each square plate, wait for the agar to cool down and solidify, and form a nutrient agar gel with a thickness of 3 mm to 5 mm for later use.

3) Inoculation culture

With the help of an ultra-clean workbench, insert 1000 ml of Escherichia coli C600 strains through the inoculation hole of the sealed box through aseptic surgery, with a strain concentration of 500 million/ml, and seal the inoculation hole with a silica gel plug. Take the position of the square plate as the reference to erect the box and let it stand for a while to allow the bacteria liquid to flow into the square plate from the gap. After the liquid level of the bacteria is balanced and stable, quickly place the box horizontally with the square plate facing upwards, gently tilt and vibrate the box back and forth The liquid strains were fully spread on the surface of the agar medium, and cultured at 37 °C for 36 h to allow the strains to grow and form a lawn.

4) Bacterial liquid collection

Inject 10 L of sterilized physiological saline through the inoculation hole of the sealed box through the aseptic method, seal the inoculation hole, place the box on a shaker, shake at 50 times/min for 15 minutes, fully wash the bacterial lawn, and export the washing liquid to aseptic container to obtain about 10 L of concentrated bacterial liquid with a concentration of 150 billion cells/ml.

5) Major equipment investment and production cost

Equipment and equipment: 55,000 yuan (28,000 yuan for a steam sterilizer; 10,000 yuan for a constant temperature incubator; 12,000 yuan for an ultra-clean workbench; 3,000 yuan for a large-capacity oscillator; 2,000 yuan for a sealed multi-layer square plate)

Medium: 44.6 yuan (peptone 0.1 kg×200 yuan; beef extract 0.03 kg×300 yuan; chlorinated

Sodium 0.05 kg×12 yuan; agar 0.15 kg×100 yuan).

Utilities: 34 yuan (sterilization 5KV×3 h×1 yuan; culture 0.5 KV×36/2 h×1 yuan; distillation

10 L of water x 1 yuan; 200 L of tap water for equipment washing).

Staff salary: 200 yuan (1 person x 1 d x 200 yuan).

6) Waste discharge

10 L of discarded culture medium; 200 L of tap water for equipment washing.

Three kinds of fermentation culture methods of embodiment 1, embodiment 2, embodiment 3 are compared

The fermentation tank liquid culture method requires high equipment investment (102,000 yuan), simple fermentation culture process, high raw material cost, cumbersome bacterial liquid concentration process, high labor intensity, easy pollution of products, and high material consumption cost.

The eggplant-shaped bottle nutrient agar planar culture method requires high equipment investment (67,000 yuan), cumbersome fermentation culture and harvesting processes, high labor intensity, easy pollution of products, and low material consumption costs.

The multi-layer square plate solid plane culture method has low equipment investment (55,000 yuan), simple fermentation culture and harvesting process, low labor intensity of personnel, low probability of product pollution, and low material consumption cost. See Table 1 for details.

Table 1 Comparison of main costs of producing 10 L concentrated bacterial liquid by three fermentation methods

Example 4

A large pig farm in Tongliao City cultivated a large number of Escherichia coli k88 and k99 genetically engineered bacteria due to production needs, using liquid culture methods and eggplant-shaped bottle nutrient agar plane culture methods, but the products were more polluted and the yield of the cultures was low. Can not meet production needs. After applying the multi-layer square plate solid plane culture method, the labor intensity of personnel and the probability of product contamination have been reduced, and the yield of culture products has been significantly improved. The accumulative production of Escherichia coli k88 and k99 genetically engineered bacteria concentrates several tons, and good production benefit. The required equipment and specific operation methods for preparing Escherichia coli k88 and k99 genetically engineered bacteria by using multi-layer square plate solid plane culture are as follows:

1) Equipment

Horizontal rectangular pressure steam sterilizer, constant temperature incubator, ultra-clean workbench, oscillator, stainless steel sealed box and multi-layer square plate (including: 12 stainless steel square plates, length, width and height 60×45×2.0㎝; stainless steel seal 1 box, length, width and height 60×45×24㎝; 1 sealing cover, length and width 49×28㎝), two inoculation holes that can be sealed with silicone plugs are set at the opposite corners of the sealing cover surface, and the operation is performed with the help of silicone pads Connect the sealing cover and the box body, and lock and seal with bolts.

2) Culture medium preparation

Nutrient agar medium: peptone 0.1 kg; beef extract 0.03 kg; sodium chloride 0.05 kg; agar 0.15 kg; distilled water 10 L.

Mix the components of the culture medium evenly, adjust the pH to 7.4 with 10% sodium hydroxide, inject the culture medium into the sealed box through the inoculation hole of the sealed box, seal the inoculated hole with a silica gel plug, and place it at 121 °C for 15 minutes to autoclave. When the temperature of the box drops to 50 ℃ ~ 60 ℃, shake the box to mix the culture medium, stand the box upright based on the position of the square plate for a while, let the agar flow into the square plate from the gap, and then quickly place the box horizontally and Make the square plates face up, and distribute the melted nutrient agar evenly in each square plate, wait for the agar to cool down and solidify, and form a nutrient agar gel with a thickness of 3 mm to 5 mm for later use.

3) Inoculation culture

With the help of an ultra-clean workbench, 1000 ml of Escherichia coli k88 and k99 genetically engineered strains were inserted through the inoculation hole of the sealed box through aseptic surgery. The concentration of the strain was kept above 500 million/ml, and the inoculation hole was sealed with a silica gel plug. Take the position of the square plate as the reference to erect the box and let it stand for a while to allow the bacteria liquid to flow into the square plate from the gap. After the liquid level of the bacteria is balanced and stable, quickly place the box horizontally with the square plate facing upwards, gently tilt and vibrate the box back and forth The liquid strains were fully spread on the surface of the agar medium, and cultured at 37 °C for 48 h to allow the strains to grow and form a lawn.

4) Bacterial liquid collection

Inject 10 L of sterilized physiological saline through the inoculation hole of the sealed box through the aseptic method, seal the inoculation hole, place the box on a shaker, shake at 50 times/min for 15 minutes, fully wash the bacterial lawn, and export the washing liquid to aseptic container to obtain 10 L of concentrated bacterial liquid with a concentration of about 150 billion cells/ml.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (5)

1. A simple batch culture method of facultative aerobic bacteria is characterized in that: the multilayer square plate solid plane fermentation device of a sealed box is used to cultivate; The number of square plates is 10-20, and the size of the sealed box is based on the size that can only accommodate a specified number of stacked square plates; the opening of the sealed box is provided with a sealing cover that covers the opening. There is a sealing rubber pad between the cover and the opening edge of the box body, and the sealing cover, the sealing rubber pad and the box body are fixedly connected by locking screws passing through the three; the locking screws are set at the four corners of the sealing cover, and the opposite corners of the sealing cover Two inoculation holes with a diameter of 22 mm were arranged at each place, and the inoculation holes were sealed with silica gel plugs. 2. The simple batch culture method of facultative aerobic bacteria according to claim 1, characterized in that: the material of the heat-resistant thin plate that is not easily deformed is stainless steel. 3. The simple batch culture method of facultative aerobic bacteria according to claim 1, characterized in that: the size of each square plate is set according to production requirements. 4. the simple and easy batch culture method of facultative aerobic bacteria according to claim 1, is characterized in that: comprise the following steps: 1) Through the inoculation hole reserved on the sealing cover, inject the agar medium suspension that can form a gel with a thickness of 3mm~5mm according to the inner surface area of the square plate, seal the inoculation hole with a silica gel plug, and place it in a high-temperature sterilization equipment for routine use. Sterilize, wait for the temperature of the box to drop to 50°C~60°C, shake the box to mix the culture medium, erect the box based on the position of the square plate, let the agar flow into the square plate from the gap for a while, and wait for the agar solution The surface is balanced and stable, quickly place the box horizontally and make the opening of the square plate face up, so that the melted agar is evenly distributed in each square plate; 2) After the agar cools down and solidifies, inoculate the liquid strains according to 1%~2% of the volume of the box. The inoculation adopts an aseptic method, and injects or drops the strains into the inoculation hole with the help of an ultra-clean workbench or flame closure. Keep the concentration above 500 million/ml. After inoculation, erect the box with the position of the square plate as the reference, and let the bacteria liquid flow into the square plate from the gap for a while. With the opening of the plate facing upwards, gently tilt and vibrate the box back and forth so that the bacteria are fully coated on the surface of the agar medium, and cultured to form a lawn; 3) When the culture is harvested, 15%~20% of the box volume is injected with washing liquid, the box is shaken to fully wash the bacterial lawn, and then the box is tilted, and the washing liquid is exported through the inoculation hole to obtain concentrated pure culture liquid. 5. the simple and easy batch culture method of facultative aerobic bacteria according to claim 4, is characterized in that: bacterium is fermented and cultivated on multi-layer square plate solid medium, adopts disposable injection medium, disposable sterilization, once It can form a multi-layer culture surface, one-time inoculation, one-time washing and harvesting.