AU2020103917A4

AU2020103917A4 - Method for preparing a 3d printing gradient antibacterial film, product and application thereof

Info

Publication number: AU2020103917A4
Application number: AU2020103917A
Authority: AU
Inventors: Jingman Li; Shanshan Li; Siying Li; Yan Li; Xinyi WU; Shengkui Yi
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-12-10
Filing date: 2020-12-07
Publication date: 2021-02-11
Anticipated expiration: 2028-12-07
Also published as: CN111138723A; CN111138723B

Abstract

The application relates to a method for preparing a 3D printing gradient antibacterial film, a product and application thereof, which belongs to the field of microbes. The method comprises the following steps: taking alginic acid and pectin, adding glycerol 5 and distilled water, and performing uniform stirring to obtain several groups of mixed solution I; taking and dissolving chitosan into acetic acid solution to obtain several groups of mixed solution II with gradient concentration; correspondingly adding the several groups of mixed solution II with gradient concentration into the several groups of mixed solution I one to one, and performing uniform stirring to form a colloidal 10 state to obtain several groups of 3D printing raw materials; respectively performing 3D printing by using the several groups of 3D printing raw materials to obtain several films, and sequentially splicing the several films together seamlessly according to the mass fraction of chitosan; soaking the film in CaCl 2, and performing drying to obtain a 3D printing gradient antibacterial film. The 3D printing gradient antibacterial film 15 provided by the application can be used to observe the movement trajectories of microbial strains. 1/1 FIG.1 FIG.2 FIG.3

Description

1/1

FIG.1

FIG.2

FIG.3

METHOD FOR PREPARING A 3D PRINTING GRADIENT ANTIBACTERIAL FILM,

PRODUCT AND APPLICATION THEREOF

Technical Field

The application belongs to the field of microbes, in particular to a method for preparing a 3D

printing gradient antibacterial film, a product and application thereof.

Background Art

It is one of the most basic skills of living beings to perceive the external environment and

guide their movement. More and more experimental data show that microbial strains can

actively respond to external stimulation, such as light, electricity, compound and oxygen.

Their migration along the direction of the gradient of the concentration of a compound is

called as chemotaxis.

In the past, the study on the movement of microbial strains realized the swimming mode of

the microbial strains in the bacterial suspension by means of biological convection, or

migration along the direction of the gradient of the concentration of the compound. The

movement of the microbial strains is driven by the power produced through the rotation of

one flagellum or a bundle of flagellums with length of about 10um. In a liquid culture

medium, the microbial strains will accumulate on the surface of the culture medium. Usually,

the specific gravity of the microbial strains is slightly larger than that of water, resulting in a

density distribution of "heavy top and light bottom". When the instability accumulates to a

certain extent, the upper fluid will suddenly fall down at a certain point, and absorb the nearby

upper fluid to form a downward flow channel, thus causing biological convection. However,

the movement of the microbial strains caused by biological convection is caused by the

influence of the gravity gradient of the microbial strains, which can only be observed by

macroscopic phenomena in the liquid culture medium. When the microbial strains encounter a

compound that is harmful to them, they will stay away from it. Therefore, in the prior art,

compounds with gradient concentration in liquid can only be used to explore the tactic movement of the microbial strains in the liquid culture medium. Both the movement of the microbial strains and the fluidization of the liquid culture medium will make it difficult to observe the movement states of the microbial strains or make the observation effect not good.

In conclusion, in view of the problems of the prior art, an antibacterial film which facilitates

the observation of the movement states of the microbial strains needs to be studied.

Summary

The purpose of the application is to provide a method for preparing a 3D printing gradient

antibacterial film, a product and application thereof, so as to improve the observability of the

movement states of microbial strains.

The application adopts the following technical solution: a method for preparing a 3D printing

gradient antibacterial film includes the following steps:

(1) taking alginic acid and pectin, adding glycerol and distilled water, and performing uniform

stirring to obtain several groups of mixed solution I with the same concentration;

(2) taking and dissolving chitosan into acetic acid solution to obtain several groups of mixed

solution II with gradient concentration of chitosan;

(3) correspondingly adding the several groups of mixed solution II into the several groups of

mixed solution I one to one, and performing uniform stirring to form a colloidal state to

obtain several groups of 3D printing raw materials with gradient concentration of chitosan;

(4) respectively performing 3D printing by using the several groups of 3D printing raw

materials to obtain several films, sequentially splicing the several films together seamlessly

according to the gradient concentration of chitosan from low to high, soaking the film in

CaCl2 solution, taking out the film, and then performing drying to obtain a 3D printing

gradient antibacterial film.

Preferably, in step (1), the mass ratio of alginic acid to pectin to glycerol to distilled water is

1-2:1-3:0.2-0.4:90-100.

Preferably, in step (2), the mass fraction of the acetic acid solution is 1-2%, the range of the gradient mass fraction of chitosan in the mixed solution II is 0-20%, and a difference between the mass fractions of chitosan in the mixed solution II with adjacent gradient concentration is

4-5%. The mass fraction of chitosan may be 0, since the antibacterial film is formed by

splicing several groups of films with gradient concentration of chitosan. Therefore, when the

mass fraction of one group of chitosan is zero, it does not affect the property that the whole

antibacterial film has gradient concentration.

Preferably, in step (3), the 3D printing parameters are as follows: the printing speed

30-50mm/s, the nozzle aperture is 0.5-0.7mm, the filling rate 80-100%, the wall thickness

1.0-1.4mm. The specific 3D printing parameters may be adjusted adaptively based on the

thickness and size of the required film according to the prior art.

Preferably, in step (4), the mass fraction of the CaCl2 solution is 1-3%, the time of soaking is

3-5min, the temperature of drying is 30-40°C, and the time of drying was 2-3h. The properties

of the film are more stable by soaking in the CaCl2 solution.

Preferably, application of the 3D printing gradient antibacterial film to observation of

movement states of microbial strains includes the following steps:

(1) taking and dissolving a culture of to-be-detected bacteria in NaCl solution with mass

fraction of 0.7-0.9% to obtain a concentrated bacterial suspension with turbidity of

10-20MCF;

(2) weighing and dissolving agar powder in NaCl solution with mass fraction of 0.7-0.9% to

obtain a semi-solid culture medium;

(3) performing high-temperature sterilization to the semi-solid culture medium for 15-20min

at 120-125°C, and then performing ultraviolet sterilization for 20-30min;

(4) under an aseptic operation condition, cooling the semi-solid culture medium prepared in

step (3) to 50-60°C, pouring the semi-solid culture medium into a culture dish, then

inoculating the concentrated bacterial suspension, and performing uniform mixing;

(5) putting the 3D printing gradient antibacterial film into the culture dish prepared in step (4)

to obtain an experimental group, and repeating steps (1)-(4) to obtain a blank group without the 3D printing gradient antibacterial film;

(6) placing and culturing the experimental group and the blank group prepared in step (5) at

35-37°C, and observing the turbidity change of the culture medium every 3-4h;

(7) observing the diameter of turbidity change areas in the experimental group and the blank

group by using a phase contrast microscope, and observing movement forms of microbial

strain cells with the blank group as reference.

Preferably, in step (2), the mass fraction of the agar powder in the semi-solid culture medium

is 0.2-0.7%.

Preferably, in step (4), the volume ratio of the semi-solid culture medium to the concentrated

bacterial suspension is 200-400:1-3.

The prior art does not use the 3D printing technology to explore the movement behavior of

the microbial strains. In the fields of 3D printing and microbes, the past research on the tactic

behavior of the microbial strains mainly includes chemotaxis, phototaxis, geotaxis,

magnetotaxis, oxytaxis, etc. What is adopted in the application is glycotaxis. The chemotaxis

of the microbial strains to amino acids and saccharides are regulated by receptor proteins

located on cell surfaces, and signals are transmitted by intracellular molecules, which

ultimately affect the movement of the microbial strains. The output signals of tactic behaviors

are the movement direction of the microbial strains, which indicates that the determinant of

the movement states is the propeller, i.e., flagellum of the microbial strains.

In the application, the semi-solid culture medium is used and a gradient film is prepared to

study the movement behavior of the microbial strains, which abandons the traditional method

of studying the tactic movement of the microbial strains by using the compound with gradient

concentration only in the liquid culture medium. Since chitosan with polycations attracts the

negative charges on the cell membranes of the microbial strains, which will lead to the

leakage of intracellular protease, we use chitosan as an antibacterial gradient film to study the

taxis of the microbial strains, which is not available in the prior art. The microbial strains will

instinctively stay far away from the substances harmful to them. By adding the gradient film

unfavorable to the microbial strains into the semi-solid culture medium, the movement behavior of the microbial strains can be observed.

The thickness, size and shape of the film can be accurately controlled through 3D printing,

which is incomparable with traditional tape casting and electrospinning. 3D printing uses a

melt extrusion technology to print the prepared printing materials according to the original

path through preset model parameters, which can realize the uniformity of quantity, size and

shape, is highly efficient and can save time. The gradient of the film can be realized by

replacing different gradient printing materials and printing paths. If the same materials are

used to prepare the film by adopting tape casting, firstly, the process of film preparation is

time-consuming and the size and shape of the film cannot be controlled; secondly, the shape is

single and unchanged; finally, the gradient of the film cannot be realized. In the application,

sodium alginate and pectin may be used as printing substrates, and the antibacterial gradient

of the printing substrates can be realized by adding chitosan with different gradient

concentration. Chitosan has polycations, which will attract the negative charges on the cell

membranes of the microbial strains, but the microbial strains will instinctively stay far away

from the compounds that are harmful to them. The gradient film prepared by adopting 3D

printing can be used to study the influence on the movement behavior of the microbial strains,

and thus the movement states of the microbial strains can be observed.

The application has the following beneficial effects:

(1) The 3D printing antibacterial film with gradient concentration prepared in the application,

based on the response principle of the microbes to external environment stimulation, can be

used to observe the movement states of the strains through the chemotactic response of the

strains to chitosan with different concentration.

(2) The polycations of chitosan used in the application interact with the negative charges on

the cell membranes of the microbial strains, which causes the leakage of protease and other

components in the cells, thus achieving the antibacterial and bactericidal effects. Furthermore,

the higher the gradient concentration of chitosan is, the more the microbial strains adsorbed

by the cations, the less the microbial strains escape, the movement of the microbial strains is

inhibited, the turbidity is clearer microscopically, and thus the movement trajectories of the microbial strains can be observed.

(3) In the application, the 3D printing gradient antibacterial film is used to explore the

movement behavior of the microbial strains. By studying the glycotaxis of the microbial

strains, the chemotaxis of the microbial strains to amino acids and saccharide compounds is

regulated by receptor proteins located on cell surfaces, and signals are transmitted by

intracellular molecules, which ultimately affect the movement of the microbial strains.

Chitosan with an antibacterial effect is used to explore the taxis of the microbial strains, such

that the movement states of the microbial strains can be observed.

Description of the Drawings

FIG1 illustrates a 3D printing gradient antibacterial film.

FIG 2 illustrates turbidity diameter changes of Escherichia coli in a semi-solid culture

medium in an experimental group and a blank group.

FIG 3 illustrates turbidity diameter changes of Saccharomycetes in a semi-solid culture

medium in an experimental group and a blank group.

Description of the Embodiments

The technical solution of the application will be further described below in detail. However,

the scope of protection of the application is not limited to what described below.

Example 1

(1) Alginic acid and pectin were taken, glycerin and distilled water were added, and uniform

stirring was performed to obtain five groups of mixed solution I. The mass ratio of alginic

acid to pectin to glycerol to distilled water was 1:3:0.4:300.

(2) Chitosan was taken and dissolved in acetic acid solution with mass fraction of 2% to

obtain several groups of mixed solution II with gradient mass fraction. The range of the

gradient mass fraction of chitosan in the several groups of mixed solution II was 0-20%, a

difference between the mass fractions of chitosan in the mixed solution II with adjacent

gradient mass fraction was 5%, and the mass fractions were specifically 0, 5%, 10%, 15%, and 20%.

(3) The several groups of mixed solution II prepared in step (2) were correspondingly added

into the several groups of mixed solution I one to one, and uniform stirring was performed to

form a colloidal state to obtain several groups of 3D printing raw materials.

(4) 3D printing was respectively performed by using the several groups of 3D printing raw

materials to obtain several films, the several films were sequentially spliced together

seamlessly according to the gradient mass fraction of chitosan from low to high, the spliced

gradient antibacterial film was soaked for crosslinking for 5min in 3% CaCl2 solution, and

then drying was performed to obtain a 3D printing gradient antibacterial film for observing

the movement states of bacteria.

Setting of 3D printing parameters: a small cube model was selected, a model with parameters

of 1.0*1.5*0.2cm was set on cura software, the printing speed was 50mm/s, the nozzle

aperture was 0.7mm, the filling rate was 100%, and the wall thickness was 1.0-1.4mm.

The specific printing steps were as follows: the 3D printing material prepared in step (3) was

put into a barrel, and an aluminum cover was screwed tightly to complete the filling work;

one end of an air pipe was inserted into a quick plug, and the other end was connected with an

outlet end of a pressure regulating valve; an air valve was opened, and at this time, the

material entered a large sleeve part (aluminum block) of an extruder under the pushing force

of air pressure. A motor was controlled to rotate through a preset program, and the motor

drove a screw to rotate through a coupling and a bearing to accurately control the extrusion of

the material; the model parameters set in advance in step (3) were selected, a "PRINT" button

was clicked, and a 3D printer (ZD-2000A, manufactured by Shenzhen Zhandong Industry Co.,

Ltd.) completed the printing process according to the preset model path.

Method of application:

(1) A culture of Escherichia coli was taken and dissolved in NaCl solution with mass fraction

of 0.9% to obtain a concentrated bacterial suspension with turbidity of 20MCF.

(2) Agar powder was weighed and dissolved in NaCl solution with mass fraction of 0.9% to obtain a semi-solid culture medium. The mass fraction of the agar powder in the semi-solid culture medium was 0.7%.

(3) High-temperature sterilization was performed to the semi-solid culture medium for 20min

at 135°C, and then ultraviolet sterilization was performed for 30min.

(4) Under an aseptic operation condition, the semi-solid culture medium prepared in step (3)

was cooled to 60°C, the semi-solid culture medium was poured into a culture dish, then 100ul

of the concentrated bacterial suspension prepared in step (1) were inoculated, and uniform

mixing was performed. The volume ratio of the semi-solid culture medium to the

concentrated bacterial suspension was 400:3.

(5) The 3D printing gradient antibacterial film was put into the culture dish prepared in step (4)

to obtain an experimental group, and steps (1)-(4) were repeated to obtain a blank group

without the 3D printing gradient antibacterial film.

(6) The experimental group and the blank group prepared in step (5) were placed and cultured

at 37C, and the turbidity change of the film with gradient concentration in the culture

medium was observed every 4h. The diameter of bacterial turbidity change in the gradient

film was 10mm, and the diameter of change in the blank group without the gradient film was

4mm.

(7) The diameter of turbidity change areas in the experimental group and the blank group was

observed by using a phase contrast microscope, and movement forms of bacterial cells were

observed with the blank group as reference.

Referring to FIG. 2, the upper part of the picture represents the range of diffusion of

Escherichia coli in the gradient film. Since Escherichia coli are Gram-negative bacteria, a

peptidoglycan layer in a cell wall is thin and is a two-dimensional plane with loose structure,

when contacting with the chitosan film with gradient concentration, the signals received on

the Escherichia coli film tend to be far away from the gradient film. When observed on the

semi-solid culture medium by using a microscope, it shows that larger turbidity diameter and

faster change speed. The lower part of the picture represents the movement states of

Escherichia coli in the absence of the gradient film. Since the glycotaxis of the microbial strains has a tendency of diffusion to the surrounding nutrients, but the diffusion speed is slow, it shows slow speed of turbidity diameter change and smaller diameter.

Example 2

stirring was performed to obtain six groups of mixed solution I. The mass ratio of alginic acid

to pectin to glycerol to distilled water was 1:1:0.2:300.

gradient mass fraction was 4%, and the mass fractions were specifically 0, 4%, 8%, 12%,

16%, and 20%.

form a colloidal state to obtain several groups of 3D printing raw materials.

seamlessly according to the mass fraction of chitosan from low to high, the spliced gradient

antibacterial film was soaked for crosslinking for 3min in 1% CaCl2 solution, and then drying

was performed to obtain a 3D printing gradient antibacterial film for observing the movement

states of bacteria.

of 1.0*1.5*0.2cm was set on cura software, the printing speed was 30mm/s, the nozzle

aperture was 0.5mm, the filling rate was 80%, and the wall thickness was 1.0mm.

The specific printing steps were the same as that in example 1.

Method of application:

(1) A culture of Staphylococcus aureus was taken and dissolved in NaCl solution with mass fraction of 0.7% to obtain a concentrated bacterial suspension with turbidity of10MCF.

(2) Agar powder was weighed and dissolved in NaCl solution with mass fraction of 0.7% to

obtain a semi-solid culture medium. The mass fraction of the agar powder in the semi-solid

culture medium was 0.2%.

(3) High-temperature sterilization was performed to the semi-solid culture medium for 15min

at 121°C, and then ultraviolet sterilization was performed for 20min.

was cooled to 50°C, the semi-solid culture medium was poured into a culture dish, then 50ul

mixing was performed. The volume ratio of the semi-solid culture medium to the

concentrated bacterial suspension was 200:1.

without the 3D printing gradient antibacterial film.

at 35°C, and the turbidity change of the film with gradient concentration in the culture

film was 5mm, and the diameter of change in the blank group without the gradient film was

2mm.

observed with the blank group as reference.

The turbidity diameter change of Staphylococcus aureus on the gradient film is smaller than

that of Escherichia coli. Staphylococcus aureus are Gram-positive bacteria, and a

peptidoglycan layer on a cell wall is thick and is a solid three-dimensional network structure.

The signals that the chemical receptors on the cell membrane surfaces receive the gradient

chitosan stimulation are weak, so the flagella of the microbial strains move slowly, and the range of movement toward the surround is small. Under a microscope, the turbidity diameter is small and the change speed is slow.

Example 3

acid to pectin to glycerol to distilled water was 1:2:0.3:300.

(2) Chitosan was taken and dissolved in acetic acid solution with mass fraction of 1.4% to

gradient mass fraction was 5%, and the mass fractions were specifically 0, 5%, 10%, 15%,

and 20%.

form a colloidal state to obtain several groups of 3D printing raw materials.

gradient antibacterial film was soaked for crosslinking for 4min in 3% CaCl2 solution, and

the movement states of bacteria.

of 1.0*1.5*0.2cm was set on cura software, the printing speed was 35mm/s, the nozzle

aperture was 0.6mm, the filling rate was 90%, and the wall thickness was 1.2mm.

The specific printing steps were the same as that in example 1.

Method of application:

(1) A culture of Saccharomycetes was taken and dissolved in NaCl solution with mass fraction of 0.8% to obtain a concentrated bacterial suspension with turbidity of 15MCF.

(2) Agar powder was weighed and dissolved in NaCl solution with mass fraction of 0.8% to

culture medium was 0.5%.

(3) High-temperature sterilization was performed to the semi-solid culture medium for 17min

at 128°C, and then ultraviolet sterilization was performed for 25min.

was cooled to 56°C, the semi-solid culture medium was poured into a culture dish, then 70ul

mixing was performed. The volume ratio of the semi-solid culture medium to the

concentrated bacterial suspension was 150:1.

without the 3D printing gradient antibacterial film.

at 36°C, and the turbidity change of the film with gradient concentration in the culture

medium was observed every 3h. The diameter of Saccharomycetes turbidity change in the

gradient film was 6mm, and the diameter of change in the blank group without the gradient

film was 3mm.

observed with the blank group as reference.

Referring to FIG. 3, the turbidity diameter change of Saccharomycetes in the gradient film is

greater than that in the blank group. Saccharomycetes are eukaryotic microbes. After

receiving the stimulation of gradient chitosan, the specific chemical receptors on the fungal

cell membranes do not respond as violently as Escherichia coli and diffuse rapidly around.

After receiving the stimulation signal of chitosan, information is transmitted to the cytoproteins in the cells. The intracellular proteins control the movement of flagella, such that the microbial strains diffuse around. The transmission speed of the stimulation signals from the outside of the cells to the flagella is slower than the transmission speed of the microbial strains. Therefore, the turbidity diameter is smaller than that of Escherichia coli, and the change speed is relatively slower.

In the application, by adopting the method of combining 3D printing of the antibacterial film

with gradient concentration and the response of microbes to external environment stimulation,

the chemotactic response of different strains (Escherichia coli, Staphylococcus aureus and

Saccharomycetes) to chitosan with different concentration is studied. Escherichia coli and

Staphylococcus aureus are respectively the representatives of Gram-negative bacteria and

Gram-positive bacteria, Saccharomycetes are the representative of eukaryotic microbes, and

Saccharomycete can be used to compare the different influence of the 3D printed gradient

film on the movement behavior of bacteria and fungi.

According to the chemotactic response of Escherichia coli, Staphylococcus aureus and

Saccharomycete to chitosan with different concentration, the results show that the movement

trajectory of Escherichia coli is more obvious than that of the other two kinds of bacteria.

Since the cell wall of Gram-negative bacteria is a two-dimensional plane with loose structure,

the stimulation response to chitosan with polycations is fiercer, the tendency that the bacteria

escape from the gradient film is more obvious, and the movement trajectory is larger and

more obvious.

What are described above are just preferred implementation modes of the application. It

should be understood that the application is not limited to the modes disclosed herein, and

should not be regarded as excluding other embodiments, but may be used for various other

combinations, modifications and environments, and may be modified through the

above-mentioned teaching or technology or knowledge in the related art within the scope of

the concept described herein. However, any modifications and changes made by those skilled

in the art without departing from the spirit and scope of the application shall fall within the

scope of protection defined by the attached claims of the application.

Claims

1. A method for preparing a 3D printing gradient antibacterial film, wherein the

method comprises the following steps:

(1) taking alginic acid and pectin, adding glycerol and distilled water, and performing

uniform stirring to obtain several groups of mixed solution I with the same

concentration;

(2) taking and dissolving chitosan into acetic acid solution to obtain several groups of

mixed solution II with gradient concentration of chitosan;

(3) correspondingly adding the several groups of mixed solution II into the several

groups of mixed solution I one to one, and performing uniform stirring to form a

colloidal state to obtain several groups of 3D printing raw materials with gradient

concentration of chitosan;

(4) respectively performing 3D printing by using the several groups of 3D printing

raw materials to obtain several films, sequentially splicing the several films together

seamlessly according to the gradient concentration of chitosan from low to high,

soaking the film in CaCl 2 solution, taking out the film, and then performing drying to

obtain a 3D printing gradient antibacterial film.

2. The method according to claim 1, wherein in step (1), the mass ratio of alginic

acid to pectin to glycerol to distilled water is 1-2:1-3:0.2-0.4:90-100;

wherein in step (2), the mass fraction of the acetic acid solution is 1-2%, the range of

the gradient mass fraction of chitosan in the mixed solution II is 0-20%, and a

difference between the mass fractions of chitosan in the mixed solution II with

adjacent gradient concentration is 4-5%;

wherein in step (4), the mass fraction of the CaCl 2 solution is 1-3%, the time of

soaking is 3-5min, the temperature of drying is 30-40°C, and the time of drying was

2-3h.

3. A 3D printing gradient antibacterial film prepared by adopting the method

according to any one of claims 1-2.

4. Application of the 3D printing gradient antibacterial film according to claim 3 to

observation of movement states of microbial strains.

5. The application of the 3D printing gradient antibacterial film to observation of

movement states of microbial strains according to claim 4, wherein the application

comprises the following steps:

(1) taking and dissolving a culture of to-be-detected bacteria in NaCl solution with

mass fraction of 0.7-0.9% to obtain a concentrated bacterial suspension with turbidity

of 10-20MCF;

(2) weighing and dissolving agar powder in NaCl solution with mass fraction of

0.7-0.9% to obtain a semi-solid culture medium;

(3) performing high-temperature sterilization to the semi-solid culture medium for

-20min at 120-125°C, and then performing ultraviolet sterilization for 20-30min;

(4) under an aseptic operation condition, cooling the semi-solid culture medium

prepared in step (3) to 50-60°C, pouring the semi-solid culture medium into a culture

dish, then inoculating the concentrated bacterial suspension, and performing uniform

mixing;

(5) putting the 3D printing gradient antibacterial film into the culture dish prepared in

step (4) to obtain an experimental group, and repeating steps (1)-(4) to obtain a blank

group without the 3D printing gradient antibacterial film;

(6) placing and culturing the experimental group and the blank group prepared in step

(5) at 35-37°C, and observing the turbidity change of the culture medium every 3-4h;

(7) observing the diameter of turbidity change areas in the experimental group and the

blank group by using a phase contrast microscope, and observing movement forms of

microbial strain cells with the blank group as reference; wherein in step (2), the mass fraction of the agar powder in the semi-solid culture medium is 0.2-0.7%; wherein in step (4), the volume ratio of the semi-solid culture medium to the concentrated bacterial suspension is 200-400:1-3.