CN102703308A - Bacteria filtering cup and application thereof to bacteriostatic activity detection of biocontrol bacteria metabolin - Google Patents

Abstract

The invention relates to a bacterial filter cup and its application in the detection of antibacterial activity of bacterial metabolites for biocontrol. The invention is based on the fact that bacterial cells cannot penetrate the water agar medium, but the bacterial metabolites can diffuse in the water agar medium Based on the principle that the bacterial fermentation broth can penetrate the water agar medium, a bacterial filter cup with a simple structure was designed and applied to the detection of antibacterial activity of metabolites in the biocontrol bacterial fermentation broth, suitable for biopesticides in the process of biopesticide development. Determination of antibacterial activity of antibacterial fermentation broth, screening of bacterial strain culture formula, optimization of bacterial agent production process and stability detection of antibacterial active substances of biopesticide products.

Description

Bacterial filter cup and its application in detection of antibacterial activity of biocontrol bacterial metabolites

technical field

The invention relates to a simple bacterial filter cup in the development of biological pesticides and its application in the detection of biological control bacterial metabolites and biological pesticide antibacterial activity.

Background technique

In recent years, beneficial microorganisms and their metabolites have been widely used at home and abroad to inhibit the survival and activities of pathogens, which has led to the rapid development of biological control, among which biocontrol bacteria have played a very important role in the control of plant diseases. Antagonism is the main mechanism of action of bio-control bacteria, which refers to the antibacterial substances that bio-control bacteria can produce and secrete into the environment during their metabolism. Generally, at low concentrations, they can inhibit the growth and metabolism of pathogenic bacteria, and even kill them. dead pathogens. Such antibacterial substances mainly include antibiotics, bacteriocins (bactericidal proteins or polypeptides), extracellular lyases, lysozymes, proteases, defective phages, lipopeptides, exopolysaccharides, and some initial metabolic pathways such as ammonia and organic acids by-products and other secondary metabolites. Bacillus bacteria are widely distributed in nature and can tolerate a variety of adverse environments. Some Bacillus bacteria can produce antibacterial substances that inhibit plant pathogens, including lipopeptides, peptides, proteins, etc., and are widely used in plant diseases of biological control.

Under experimental conditions, the plate confrontation method is usually used for preliminary screening of biocontrol bacteria based on the width of the inhibition zone between the bacteria to be tested and the pathogenic bacteria. As for the detection and evaluation of the antibacterial activity of the metabolites of the fermentation broth of bio-control bacteria, the drug-containing plate method is often used to determine the effect of the sterile fermentation broth on the growth of the bacterial colony, or the Oxford cup method is used to determine the antibacterial activity according to the diameter of the inhibition zone. The effect of bacterial fermentation broth on germination of pathogenic spores.

At present, the preparation methods of aseptic fermented liquid generally include bacterial filter sterilization, high temperature sterilization and organic solvent extraction. (1) Bacterial filter sterilization method: After high-speed centrifugation of the bio-control bacterial fermentation liquid, take the supernatant and filter it with a bacterial filter. It can be used only after the sterility test, which is inconvenient to operate and the cost of the bacterial filter is high. , when the antibacterial activity of the fermented liquid is detected by the drug-containing plate method, more sterile filtrate is often required, and the sterile filtrate Oxford cup method is used. Although the amount of sample added is small, it is affected by the bottom and wall of the tube. It may affect the diffusion direction and diffusion amount of the biocontrol bacterial fermentation liquid. The antibacterial plaques formed during the bioassay are not necessarily regular, and it is impossible to quantitatively distinguish the fermentation liquid with the same inhibition zone size and different clarity; (2) high temperature Sterilization method: It may lead to the inactivation of some antibacterial metabolites in the fermentation liquid that are not resistant to high temperature, which will affect the reliability and authenticity of the test results; One-polar antibacterial active substances, the operation process is relatively complicated, usually need to use a large amount of fermentation broth and organic solvents for extraction.

Contents of the invention

Aiming at the above problems, the present invention proposes a simple bacterial filter cup, which has simple structure, low cost and convenient application.

The technical solution of the present invention is: a bacterial filter cup, which is characterized in that it includes a cylindrical cup body, a round hole is opened at the bottom of the cup body, and a layer of filter net is arranged on the round hole. A water agar layer is arranged on the filter net, the upper surface of the water agar layer is provided with a groove for placing the fermentation liquid of bio-control bacteria, and the outer peripheral wall of the bottom of the cup is provided with supporting feet.

Another object of the present invention is to use the bacterial filter cup to detect the antibacterial activity of the biocontrol bacterial metabolites. The detection method has good operability and the detection result of the antibacterial activity of the biocontrol bacterial metabolites is accurate.

Another technical solution of the present invention is: a preparation method of a bacterial filter cup and its application in the detection of antibacterial activity of biocontrol bacterial metabolites, characterized in that: the following steps are followed:

(1) Take a sterilized large glass plate, and make a 1.8% water agar plate in it, and the thickness of the water agar plate is 3mm;

(2) Take a cylindrical stainless steel cup body with a filter net at the bottom, the bottom of the cylindrical stainless steel cup body is provided with a circle of support feet with a height of 3mm, and place the cylindrical stainless steel cup body in the water of step (1). On the agar plate, press slightly so that the support feet at the bottom of the cup touch the bottom of the large glass plate, where the diameter of the large glass plate is larger than the diameter of the cylindrical stainless steel cup body;

(3) Inject water agar at a temperature of 50-60°C into the cylindrical stainless steel cup in step (2), so that the height of the water agar in the cup is 6-7mm, and then immediately put it into a sterilized small glass dish to make It floats in the middle of the water agar, and after the water agar solidifies, take out the small glass plate, so that a water agar groove with a depth of 3~4mm is formed in the cylindrical stainless steel cup, and the diameter of the cylindrical stainless steel cup is larger than that of the small glass plate. diameter;

(4) Remove the water agar plate in step (1), and scrape off the water agar adhered to the bottom of the filter with a sterile blade, and the bacterial filter cup is completed;

(5) Take another sterilized small glass plate, and make a PSA medium plate in it, and the thickness of the PSA medium plate is 2~3mm;

(6) Take another sterilized large glass plate, and put the lid of the sterilized small glass plate into the large glass plate;

(7) Use a sterilized medium puncher to punch a hole in the PSA medium described in step (5), take out a piece of PSA medium disc, and place the PSA medium disc in step (6). on the lid of a small glass petri dish;

(8) Place the bacterial filter cup prepared in steps (1) to (4) above the PSA medium disk in step (7), and make the filter at the bottom of the bacterial filter cup directly contact with the PSA medium disk ;

(9) Use a pipette gun to inject the bacterial fermentation broth to be tested into the groove on the surface of the water agar layer in the bacterial filter cup described in step (8), and let it stand on the ultra-clean workbench or under sterile conditions After more than 15 hours, the metabolites of the bacterial fermentation liquid in the cup are filtered through water agar to filter out the bacteria, and then fully and naturally penetrate into the PSA medium disc. The outer edge flows into the large glass petri dish;

(10) Remove the bacterial filter cup, and move the PSA medium disc infiltrated by the fermentation broth of bacteria and its metabolites to a sterile glass plate;

(11) Insert the mycelium block of the indicator pathogen into the PSA medium disk described in step (10), culture it in a dark environment at 28°C for 72 hours, measure the colony growth diameter of the mycelium block of the indicator pathogen, and at the same time in the step ( 9) In the other PSA medium disk, sterile water was used instead of bacterial fermentation broth as a blank control.

Description of drawings

Fig. 1 is a schematic structural view of the bacterial filter cup of the present invention.

Among them: 1 is the cylindrical cup body, 2 is the round hole, 3 is the filter screen, 4 is the water agar layer, 5 is the groove on the water agar layer, 6 is the supporting foot of the cylindrical cup body, 7 is the large glass plate , 8 small glass petri dish lids, 9 for the PSA medium disk.

Detailed ways

A bacterial filter cup, characterized in that it includes a cylindrical cup body 1, a round hole 2 is provided at the bottom of the cylindrical cup body, a layer of filter screen 3 is provided on the round hole, and a filter screen is provided on the filter screen. A water agar layer 4, the upper surface of the water agar layer is provided with a groove 5 for placing the bio-control bacterial fermentation liquid, and the outer peripheral wall of the bottom of the cup is provided with a supporting foot 6.

The cylindrical cup body is a stainless steel cylindrical cup body, the filter screen is a stainless steel filter screen, the height of the supporting feet of the cylindrical cup body is 3 mm, the aperture of the filter screen is 1 mm, and the depth of the groove is 3 ~ 4mm, the thickness of the water agar between the bottom of the groove and the filter is 3~4mm.

A method for preparing a bacterial filter cup and its application in the detection of antibacterial activity of bio-control bacterial metabolites, characterized in that: the operation is performed according to the following steps:

(1) Take a sterilized large glass plate with a diameter of 89 mm, and make a 1.8% water agar plate in it, and the thickness of the water agar plate is 3 mm;

(2) Take a cylindrical stainless steel cup body with a filter net at the bottom, the bottom of the cylindrical stainless steel cup body is provided with a circle of support feet with a height of 3mm, and place the cylindrical stainless steel cup body in the water of step (1). On the agar plate, press slightly so that the support feet at the bottom of the cup touch the bottom of the large glass plate, where the diameter of the large glass plate is larger than the diameter of the cylindrical stainless steel cup body;

(3) Inject water agar at a temperature of 50-60°C into the cylindrical stainless steel cup in step (2), so that the height of the water agar in the cup is 6-7mm, and then immediately put a sterilized small glass with a diameter of 59mm Make it float in the middle of the water agar. After the water agar is solidified, take out the small glass plate to form a water agar groove with a depth of 3~4mm in the cylindrical stainless steel cup. The diameter of the cylindrical stainless steel cup is larger than that of the small glass. The diameter of the glass dish;

(4) Remove the water agar plate in step (1), and scrape off the water agar adhered to the bottom of the filter with a sterile blade, and the bacterial filter cup is completed;

(5) Take another sterilized small glass plate, and make a PSA medium plate in it, and the thickness of the PSA medium plate is 2~3mm;

(6) Take another sterilized large glass petri dish 7, and put the lid 8 of the sterilized small glass petri dish into the large glass petri dish;

(7) Use a sterilized medium puncher to punch holes in the PSA medium described in step (5), take out a piece of PSA medium disc 9, and place the PSA medium disc in step (6) on the lid of a small glass petri dish;

(8) Place the bacterial filter cup prepared in steps (1) to (4) above the PSA medium disk in step (7), and make the filter at the bottom of the bacterial filter cup directly contact with the PSA medium disk ;

(9) Use a pipette gun to inject the bacterial fermentation broth to be tested into the groove on the surface of the water agar layer in the bacterial filter cup described in step (8), and let it stand on the ultra-clean workbench or under sterile conditions After more than 15 hours, the metabolites of the bacterial fermentation liquid in the cup are filtered through water agar to filter out the bacteria, and then fully and naturally penetrate into the PSA medium disc. The outer edge flows into the large glass petri dish;

(10) Remove the bacterial filter cup, and move the PSA medium disc infiltrated by the fermentation broth of bacteria and its metabolites to a sterile glass plate;

(11) Insert the mycelium block of the indicator pathogen into the PSA medium disk described in step (10), culture it in a dark environment at 28°C for 72 hours, measure the colony growth diameter of the mycelium block of the indicator pathogen, and at the same time in the step ( 9) In the other PSA medium disk, sterile water was used instead of bacterial fermentation broth as a blank control.

First, the ink tracking color development experiment in Example 1 shows that the bacterial fermentation liquid in the present invention can pass through the water agar layer in the bacterial filter cup and fully diffuse into the PSA medium.

Example 1: Ink tracking and color development observation

1) Materials and methods:

Take a large glass plate with a diameter of 89mm to make a PSA medium plate, and use a medium puncher to punch a round hole with a diameter of 50mm on the PSA medium plate, remove the medium in the hole and inject 2.5ml of 15% ink into the hole , at room temperature, after 4h, 6h, 15h, and 24h, observe the diffusion of the ink in the PSA medium plate at fixed points, and measure the ink diffusion distance;

Simultaneously adopt the bacterial filter cup detection method of the present invention, the size of the bacterial filter cup is as follows: the diameter of the cylindrical cup body is 75mm, and the bottom of the cup of the cylindrical cup body has a diameter of 55mm round hole, and the round hole is provided with a diameter of 55mm. There is a layer of filter screen, the screen aperture of the filter screen is 1mm, the filter screen is provided with a water agar layer, the thickness of the water agar layer is 6 ~ 7mm, and the upper surface of the water agar layer is provided with a shelf for placing the bio-control bacterial fermentation liquid. Groove, the depth of the groove is 3 ~ 4mm, the thickness of the water agar between the bottom of the groove and the filter screen is 3 ~ 4mm, the peripheral wall at the bottom of the cup is provided with supporting feet, and the height of the supporting feet is 3mm; Add 3ml of 15% ink to the water agar groove, and observe the coloring degree of the PSA medium disk at the bottom of the filter cup at different times after infiltration treatment for 4h, 5h, 6h, 8h and 15h at room temperature;

At the same time, the Oxford cup method was adopted in the PSA medium plate with a diameter of 89mm, that is, the Oxford cup was used to punch holes, and the PSA disk at the bottom of the hole was removed, and 10 μl of 15% ink was added to each Oxford cup, and the ink diffusion and its formation were observed after 15 hours. Color circle condition.

2) Results and analysis:

When 15% ink was processed in the PSA flat panel for several hours, the results shown in Table 1 were obtained. From Table 1, after 15% ink was processed in the PSA flat panel for 4h, 6h, 15h and 24h respectively, the width values of the formed black bands were respectively It is 5.0mm, 6.9mm, 11.0mm and 11.9mm, and there are significant differences between the treatments, indicating that the ink can spread laterally in the PSA plate, and its black substance can penetrate into the medium, and the width of the formed black band increases with the ink treatment time And increase.

Table 1 The rate of lateral diffusion of 10% ink in PSA flat plate

                                                 

After the 15% ink permeates downward for 4 hours, 5 hours, 6 hours, 8 hours and 15 hours using the bacterial filter cup method, the PSA medium disc under the filter at the bottom of the cup can be colored black, and as the ink penetration time increases, the PSA medium The darker the coloring is; the ink penetrates downward for 6-8 hours, and the PSA medium is unevenly colored, and after 15 hours of treatment, the PSA medium is uniformly colored and darker; it shows that the ink can diffuse longitudinally in the water agar, and the bacterial filter cup The black substance in the ink can seep down into the PSA medium disk

The Oxford cup method was used to remove the PSA disc with the bottom hole, and the ink was treated for 15 hours. It was found that the ink color circle formed by the PSA medium plate was not very regular, and the diameter of the color circle was different.

3) Conclusion:

15% ink is treated in the PSA plate for 15 hours, the width of its lateral diffusion can reach 11.0mm, the bottom thickness of the water agar groove in the bacterial filter cup is 3~4mm, and the PSA medium under the gauze at the bottom of the bacterial filter cup is 2~3mm , the maximum total thickness is 7mm. Therefore, it is theoretically speculated that the ink in the bacterial filter cup can penetrate into the PSA medium under the gauze at the bottom of the filter cup; The chromogenic substance of the ink in the cup can fully diffuse into the PSA medium disk, and the biocontrol bacterial metabolites with inhibitory activity can diffuse and penetrate in the agar medium. It is speculated that the biocontrol bacterial fermentation liquid is infiltrated in the bacterial filter cup. After 15 hours, its metabolites with inhibitory activity can also penetrate into the PSA medium disk.

Example 2: Antibacterial activity detection of metabolites of Bacillus subtilis

1) Materials and methods

a. Tested strains and medium

Bacillus subtilis (Bacillus subtilis) T122F was selected as the biocontrol bacterium for the test, and Fusarium oxysporum f.sp.cubense (FO) was used as the indicator bacteria for the detection of antibacterial activity of bacterial metabolites. The width of the inhibition zone formed by confrontation culture in the PSA plate can reach 12mm.

First, liquid culture of Bacillus subtilis T122F was carried out in various mediums, and the medium used in this experiment was as follows:

NB medium: glucose 2.5g, beef extract 3g, peptone 5g, pH=7, water 1000ml;

Soybean meal medium: glucose 5g, soybean meal 8g, pH=7, water 1000ml;

Maltose medium: 5g maltose, 3g beef extract, 5g peptone, pH=7, 1000ml water;

Corn flour medium: corn flour 5g, beef extract 3g, peptone 5g, pH=7, water 1000ml;

Glucose medium: glucose 5g, beef extract 3g, peptone 5g, pH=7, water 1000ml.

The culture medium required for plate culture of No. 4 physiological race of Fusarium wilt of banana is PSA medium: 200 g of potatoes, 20 g of sucrose, 18 g of agar, and 1000 ml of water.

b. Antibacterial activity of biocontrol bacteria Bacillus subtilis T122F fermentation broth metabolites with different penetration times:

Dip a small ring of Bacillus subtilis T122F bacterial lawn with an inoculation loop, transfer it into a 300 ml Erlenmeyer flask filled with 100 ml NB culture solution, cultivate it at 30°C and 180 rpm for 2 days, then use it as the inoculation mother solution, and draw 1ml of the mother bacteria The solution was transferred to a 300 ml Erlenmeyer flask filled with 100 ml NB culture solution, and cultured under the same conditions for 2 days before use.

Using the bacterial filter cup and detection method thereof of the present invention, 2.5ml of the fermented liquid of the biocontrol bacterium Bacillus subtilis T122F is moved into the water agar groove of the bacterial filter cup, after infiltrating 4h, 6h, 15h and 24h respectively, the filter cup Move the PSA medium disc under the bottom filter to a sterile large glass plate, and insert the activated hyphae of Fusarium wilt (d=5mm) into the PSA medium disc, and at the same time use sterile The treatment of water infiltration was the blank control, and each group was treated with 4 repeated samples. After culturing in a dark environment at 28°C for 3 days, the diameter of the colony on the plate was measured by the cross method, and the antibacterial activity of the fermentation broth at different infiltration times was compared.

c. Detect the antibacterial activity of different types of biocontrol bacterial metabolites:

The antibacterial activity of different types of biocontrol bacterial metabolites was detected by the bacterial filter cup method. The 1:100 dilution of Bacillus subtilis biopesticides and sterile water and the NB fermentation broth of Bacillus subtilis T122F were mixed with sterile water. 2.5ml of the dilutions of 1:2 and 2:1 were transferred into the water agar groove of the bacterial filter cup, and each group was treated with 4 repeated samples, and the infiltration time was 15h.

d. Comparison of the test results between the bacterial filter cup method and the drug-containing plate test method:

Prepare Bacillus subtilis NB, soybean meal, maltose, corn flour and glucose liquid medium, the bottling volume of each medium, the initial inoculum volume of Bacillus subtilis T122F strain, and the culture conditions are the same as above, and the bacteria filter cup and drug-containing plate are used to detect respectively The antibacterial activity of the metabolites produced by the fermentation of the Bacillus subtilis T122F strain in different culture media was detected by the method, and the penetration time of the bacterial filter cup detection method was 15 hours. After centrifugation at 16000rpm for 5min, the supernatant was filtered and sterilized with a bacterial filter (Φ=0.22μm), and the obtained sterile filtrate was mixed with PSA medium at 50°C in a ratio of 1:5. , pour it into a petri dish with a diameter of 59mm to make a plate, insert the activated mycelium block of Fusarium wilt fungus (d=5mm) in the center of the plate after condensation, and add the same amount of sterile water as a control, each group of treatment After 4 repeated samples were cultured in the dark at 28°C for 3 days, the diameter of the bacterial colony on the flat plate was measured by the cross method, and the detection results of the bacterial filter cup method and the drug-containing plate were compared.

2) Results and analysis

a. Test results of antibacterial activity of biocontrol bacteria Bacillus subtilis T122F fermentation broth metabolites with different penetration times

Table 2 shows the impact of infiltration time on the detection of antibacterial activity of metabolites in the Bacillus subtilis T122F fermentation broth. From the results in Table 2, it can be seen that the biocontrol bacteria Bacillus subtilis T122F fermentation broth permeates downward in the bacterial filter cup for 6h, 15h and 24h. , the growth of the pathogen in the PSA medium disc was significantly inhibited, and the inhibitory effect was better when the fermentation broth was infiltrated for 15h and 24h, indicating that the biocontrol bacteria Bacillus subtilis T122F fermentation broth contains FO that is effective against banana Fusarium wilt No. 4 physiological race The metabolites of the inhibitory effect can penetrate into the PSA medium disc under the gauze at the bottom of the cup through the water agar in the bacterial filter cup. Considering the convenience of actual operation, it is advisable to choose to infiltrate for 15 hours.

Table 2 The effect of penetration time on the detection of antibacterial activity of metabolites in T122F fermentation broth

b. Test results of antibacterial activity of different types of biocontrol bacterial metabolites

As can be seen from Table 3, the bacteria filter cup detection method of the present invention can be used to detect the antibacterial activity of bacterial metabolites in the biopesticide Bacillus subtilis biocontrol agent, and the antibacterial activity of the NB fermentation liquid of the biocontrol bacteria Bacillus subtilis T122F The activity is related to its different dilution ratios, the higher the dilution ratio, the lower its antibacterial activity.

Table 3 Detection of antibacterial activity of different types of bacterial metabolites

c. Comparison of the results of the antibacterial activity of bacterial metabolites in biocontrol with bacterial filter cup and drug-containing plate detection method

As can be seen from Table 4, adopt the bacterial filter cup detection method of the present invention, compared with the contrast of aseptic water treatment, the metabolites of different culture medium fermentation liquids of Bacillus subtilis T122F bacterial strain have antibacterial activity in different degrees, wherein with biocontrol The bacterial Bacillus subtilis T122F soybean meal fermentation broth had the best antibacterial activity, while the glucose fermentation broth had the worst antibacterial activity. The order of antibacterial activity of the five bacterial fermentation broths tested was 1>2>3>5 >4; while using the drug-containing plate detection method, compared with the sterile water-treated control, the metabolites of different formulations of Bacillus subtilis T122F strains have different degrees of antibacterial activity, and the order of antibacterial activity is from high to low It is Ⅰ>Ⅱ>Ⅲ>Ⅴ>Ⅳ.

Table 4 Test results of antibacterial activity of metabolites in different culture media of Bacillus subtilis T122F strain

3) Conclusion

Illustrate adopting bacterial filter cup of the present invention, can make antibacterial active substance in the fermented liquid of biocontrol bacterium Bacillus subtilis T122F penetrate in the PSA culture medium disc under the filter screen at the bottom of the cup through the water agar in the filter cup, the present invention relates to The detection method obtained has good application value in the detection and evaluation of the antibacterial activity of the metabolites of the fermentation broth of different culture media of biocontrol strains and the stability of antibacterial active substances in biopesticide products, and has good operability, especially in the case of bacteria-free In the case of a filter, the bacterial filter cup of the present invention and its detection method can be considered as a simple and effective alternative method. The consumable material water agar involved in the detection process is cheap and has a convenient source. In the implementation step (1) can be reused in .

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. bacterium filtering cup; It is characterized in that: comprise the tubular cup; The cup end of said tubular cup, offer a circular hole, and said circular hole is provided with one deck filtering net, and said filtering net is provided with a water agar layer; The upper surface of said water agar layer is provided with the groove that is used to place the biocontrol bacteria fermented liquid, and said cup end periphery wall is provided with feet.

2. bacterium filtering cup according to claim 1 is characterized in that: said tubular cup is a stainless steel tubular cup, and said filtering net is the stainless steel filtering net.

3. bacterium filtering cup according to claim 1 is characterized in that: the feet height of said tubular cup is 3mm, and the aperture of filtering net is 1mm.

4. bacterium filtering cup according to claim 1 is characterized in that: said depth of groove is 3 ~ 4mm, and the water agar thickness between bottom portion of groove and the filtering net is 3 ~ 4mm.

5. the preparation method of a bacterium filtering cup and the application in biocontrol bacteria metabolite bacteriostatic activity detects thereof is characterized in that: operate according to the following steps:

(1) get one the sterilization big glass dish, make 1.8% water agar plate within it, wherein the thickness of water agar plate is 3mm;

(2) get the tubular stainless steel cup that a bottom has filtering net; The bottom of said tubular stainless steel cup is provided with a circle and highly is the feet of 3mm; Tubular stainless steel cup is placed on the water agar plate of step (1); Press the feet at the bottom of making glass to contact at the bottom of the ware of big glass dish slightly, wherein the diameter of big glass dish is greater than the diameter of tubular stainless steel cup;

(3) implantation temperature is 50 ~ 60 ℃ a water agar in the tubular stainless steel cup of step (2); Make that the height of water in the cup agar is 6 ~ 7mm, put into the little glass dish of a sterilization then immediately, it is swum in the middle of the water agar; After treating that water agar solidifies; Take out little glass dish, make to form the water agar groove that the degree of depth is 3 ~ 4mm in the tubular stainless steel cup, wherein the diameter of tubular stainless steel cup is greater than the diameter of little glass dish;

(4) the water agar plate in the step (1) is removed, and strike off the water agar that sticks to the filtering net bottom with sterile razor blade, the bacterium filtering cup promptly completes;

(5) get in addition one the sterilization little glass dish, make the PSA culture medium flat plate within it, wherein the thickness of PSA culture medium flat plate is 2 ~ 3mm;

(6) get the big glass dish of a sterilization in addition, the ware lid of the little glass dish of sterilization is put into big glass dish;

(7) the substratum punch tool with sterilization punches in the described PSA substratum of step (5), takes out a slice PSA substratum disk, and the ware that PSA substratum disk is displaced in the little glass dish of step (6) is covered;

(8) the bacterium filtering cup that makes in step (1) ~ (4) is placed on the PSA substratum disk top of step (7), and the filtering net of bacterium filtering cup bottom is directly contacted with PSA substratum disk;

(9) inject ferment product to be measured with liquid-transfering gun in the water agar groove in the described bacterium filtering cup of step (8); Leaving standstill more than the 15h on the Bechtop or under aseptic condition; The metabolite that makes the ferment product in the cup is behind water agar filtering thalline; Fully and naturally be penetrated in the PSA substratum disk, unnecessary no thalline fermented liquid can flow in the big glass dish along the outer of little glass dish ware lid;

(10) the bacterium filtering cup is removed, will by the PSA substratum disk of the fermented liquid of no thalline and metabolite thereof infiltration move to an aseptic glass dish in;

(11) in the described PSA substratum of step (10) disk, insert indication pathogenic bacteria mycelia piece; In 28 ℃ dark surrounds, cultivate 72h; Measure the colony growth diameter of indication pathogenic bacteria mycelia piece, in described another PSA substratum disk of step (9), replace ferment product as blank simultaneously with sterilized water.

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