CN109091685A - A kind of pair of keratitis extracts the ablation method of bacterial pathogens - Google Patents
A kind of pair of keratitis extracts the ablation method of bacterial pathogens Download PDFInfo
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- CN109091685A CN109091685A CN201810906621.4A CN201810906621A CN109091685A CN 109091685 A CN109091685 A CN 109091685A CN 201810906621 A CN201810906621 A CN 201810906621A CN 109091685 A CN109091685 A CN 109091685A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
- A61L2/08—Radiation
- A61L2/085—Infrared radiation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
- A61L2/08—Radiation
- A61L2/084—Visible light
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
Present invention relates particularly to the ablation methods that a kind of pair of keratitis extracts bacterial pathogens.The ablation method of bacterial pathogens provided by the invention is one to be inactivated bacterial pathogens using photodynamic therapy;The process of the photodynamic therapy is, photosensitizer is excited using the light irradiation of specific wavelength, and the photosensitizer of excitation state energy transmission to the oxygen of surrounding, generate highly active singlet oxygen, oxidation reaction occurs for singlet oxygen and adjacent bacterial pathogens, cytotoxic effect is generated, and then leads to cell damage or even death.Currently, when treating eye pathogenic infection, it is main still by being treated using antibiotic;However, the excessive use of antibiotic is easy that bacterium is made to generate drug resistance, and influence treatment time and effect, eye bacterial pathogens purpose is killed by both can achieve using photodynamic therapy, the use that antibiotic can be reduced again, avoids the generation of bacterial drug resistance, is conducive to the health of patient.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of pair of keratitis extracts the ablation method of pathogen.
Background technique
Photodynamic therapy (PDT) is the treatment method of a kind for the treatment of cancer and other diseases, has been obtained in many countries
Supervision department approval.It swashs PS molecule using the visible light for the non-toxic dye and appropriate wavelength for being referred to as photosensitizer (PS)
It is dealt into excited singlet, the system under this excitation state enters the triplet of long-life, it can react production with molecular oxygen
Raw cytotoxic substance, such as singlet oxygen, superoxides and free radical.These active oxygens can aoxidize many biomolecule, such as egg
White matter, nucleic acid and lipid, lead to cell death.PDT can inactivate or kill a series of microbial pathogens, especially several resistance to
Medicine microbial pathogens, such as Vancomycin-resistant Enterococcus, methicillin-resistant staphylococcus aureus and mycobacterium tuberculosis.This
Outside, it has been reported that treating the infection and some clinical tests of certain animal models using PDT, it is blue to reduce leather by PDT as the result is shown
The activity of the virulence factor of family name's negative bacterium.
Infected keratitis is a kind of disease that vision threatens, and may be caused by bacterium, virus, fungi, helminth etc..?
, there are 3,000,000 corneal blindness patients in China, and infectiousness corneal blindness patient increases by 100,000 every year.Bacterial keratitis is most common
One of ocular infection, progress are rapidly and serious.Staphylococcus epidermis and staphylococcus aureus are to cause bacterial keratitis most
Common microorganism.Current antibacterial therapy method usually requires a large amount of antibiotic dosage.However, with broad-spectrum antibiotic
It is widely used, nearest researches show that more and more evidences to show that microorganism has drug resistance to antibacterials.Although using wide
Spectrum antibiotic and some more serious consequences, antibiotic resistance may cause the continuing advances of disease process.Therefore, it is badly in need of out
The effective substitution antimicrobial technology of one kind is sent out to treat ocular bacterial infection.
Summary of the invention
For current treatment bacterial keratitis, clinically or by being treated using antibiotic, other therapies phase
To being more short of, treatment means also by larger limitation, exist and are easy to produce resistance problems, and it is an object of that present invention to provide one
Kind keratitis extracts the ablation method of bacterial pathogens.
The present invention is by treating bacterial keratitis using photodynamic therapy;This method is by utilizing visible light pair
Photosensitizer is excited, and is at excitation state and is carried out reacting cellulation toxicant with oxygen molecule, these toxicants
Again in conjunction with biomolecule, achieve the purpose that kill bacteria pathogeny body cell;It is treated using photodynamic therapy, without using
Antibiotic avoids the generation of antibiotic resistant, and can play therapeutic effect, and patient is made to restore health, more preferable to meet
The demand of clinical treatment bacterial keratitis.
To achieve the above object, the present invention is achieved by the following technical programs:
A kind of pair of keratitis extracts the ablation method of bacterial pathogens, and the ablation method of the bacterial pathogens is to pass through
The bacterial pathogens of eye surface are inactivated under photodynamic action and mediation substance collective effect using photodynamic therapy.
Preferably, the light that the photodynamic action uses is that bandwidth is 20nm, and wavelength is the visible of 575nm~670nm
Light.It is highly preferred that it is 20nm that the light that the photodynamic action uses, which is bandwidth, wavelength is the visible light of 600nm~650nm.
Preferably, the mediation substance is the hydrophilic cations photosensitizer with film destruction.
It is highly preferred that the hydrophilic cationic photosensitizer with film destruction is blutene.
Preferably, the bacterial pathogens of the eye surface are staphylococcus epidermis or staphylococcus aureus one kind or two
Kind;The microbes cause bacterial keratitis.
Preferably, the visible light is one of red visible or white visible light.
It is highly preferred that the visible light is red visible.
Preferably, the optical power density of the visible light is 0.550~7.500mW/cm2, radiated time be 20~
45min。
It is highly preferred that the optical power density of the visible light is 5.750~7.350mW/cm2, radiated time be 25~
40min。
Preferably, the concentration of the blutene is 45~80 μM.
Preferably, the ablation method for the bacterial pathogens that the keratitis is extracted comprising the steps of:
S1. above-mentioned 100~200 μ L 15~25min of incubated at room temperature of bacterial suspension is taken, is placed it in 96 orifice plates, is made thin
Strain density maintains 107CFU/mL or so, and 5~80 μM of blutene are added in the drip hole of part thereto;
It S2. is 0.50~8.50J/cm by being exposed to added with the hole of blutene as illumination flow after the completion of cultivating2, light
It is 0.550~7.500mW/cm according to density2Under conditions of, 20~45min of radiated time;
S3. it cultivates 48 hours under the conditions of 37 DEG C, after the completion of culture, bacteria living is commented using colony counting method
Estimate.
It is treated currently, clinical treatment bacterial keratitis mainly passes through using antibiotic, but therapeutic process
In largely there is the risk that develops drug resistance using antibiotic, be unfavorable for patient body health;
It is treated for present clinical treatment bacterial keratitis, it is excessive that there are antibiotic dosages, easily leads to bacterium
It develops drug resistance, is unfavorable for the risk of patient body health and recovery, the object of the invention is degermed by providing a kind of keratitis
The ablation method of pathogen, is treated by this method, can be had to avoid the problem of antibiotic treatment develops drug resistance is taken
The few advantage of toxic side effect is more advantageous to patient body health.
Detailed description of the invention
Fig. 1 is that different disposal group radiates aureus growth situation after preceding and radiation.
Fig. 2 is different experiments group survival Microflora.
Specific embodiment
The present invention is further elaborated combined with specific embodiments below, the embodiment is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.Test method as used in the following examples is routine unless otherwise specified
Method;Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
The separation and culture of bacterial strain
This research selects 12 staphylococcus epidermidis and 12 plants of staphylococcus aureus separation strains, these bacterial strains are by Beijing
All bacterial strains that bacterial keratitis provided by institute of ophthalmology's microbe research group is isolated.It will be stored in glycerol tube
Bacteria culture is inoculated in blood culture vessel, and makes its 2 times of logarithmic growth phase of recovering.Then flora is struck off with oese, and
It puts it into bacterium diluent, is mixed and made into 1*108~2*108The bacterial suspension of CFU/ml concentration.
Experimental light sources and photosensitizer
Light source is made of High Power LED array, wavelength 625nm, bandwidth 20nm.LED light source is by small-sized straight
Galvanic electricity source (MCH-k305d) power supply.The size of LED array can cover a part of 96 orifice plates, and have power-adjustable density.
Optical power output is measured using light power meter (VLP-2000).This experiment uses blutene as photosensitizer.
Embodiment 1
A kind of pair of keratitis extracts the ablation method of bacterial pathogens comprising the steps of:
S1. above-mentioned 130 μ L incubated at room temperature 18min of bacterial suspension is taken, places it in 96 orifice plates, makes sample bacterial density
Maintain 107CFU/mL or so, and 50 μM of blutene are added in part of hole inward;
It after the completion of S2 culture, will be exposed under feux rouges environment added with the hole of blutene, illumination flow is 8.50J/cm2,
Illumination density is 5.750mW/cm2, radiated time 20min;
S3. it cultivates 48 hours under the conditions of 37 DEG C, after the completion of culture, bacteria living is commented using colony counting method
Estimate.
Embodiment 2
A kind of pair of keratitis extracts the ablation method of bacterial pathogens comprising the steps of:
S1. above-mentioned 150 μ L incubated at room temperature 20min of bacterial suspension is taken, places it in 96 orifice plates, makes sample bacterial density
Maintain 107CFU/ml or so, and 65 μM of blutene are added in part of hole inward;
S2. it after the completion of cultivating, will be exposed under feux rouges environment added with the hole of blutene, illumination flow is 5.25J/
cm2, illumination density is 6.700mW/cm2, radiated time 30min;
S3. it cultivates 48 hours under the conditions of 37 DEG C, after the completion of culture, bacteria living is commented using colony counting method
Estimate.
Embodiment 3
A kind of pair of keratitis extracts the ablation method of bacterial pathogens comprising the steps of:
S1. above-mentioned 150 μ L incubated at room temperature 25min of bacterial suspension is taken, places it in 96 orifice plates, makes sample bacterial density
Maintain 107CFU/mL or so, and 75 μM of blutene are added in part of hole inward;
S2. it after the completion of cultivating, will be exposed under feux rouges environment added with the hole of blutene, illumination flow is 7.50J/
cm2, illumination density is 7.450mW/cm2, radiated time 40min;
S3. it cultivates 48 hours under the conditions of 37 DEG C, after the completion of culture, bacteria living is commented using colony counting method
Estimate.
1. photosensitizer factor and illumination factor are to staphylococcus epidermis and the pathogen inactivated effect of S. aureus bacterium
The influence of fruit
Photosensitizer blutene (TBO) and illumination factor are studied to the shadow of staphylococcus epidermis and staphylococcus aureus
It rings.By bacterial suspension (108CFU/mL it) is placed in 96 drip hole plates and is trained with the blutene of various concentration (60 μM)
It supports.The drip hole of control group 1 is cultivated with 60 μM of PBS.Standing cultivates 20min, the population of laboratory sample at room temperature
Density is maintained at 107CFU/mL.Illumination is carried out after incubation.Illumination flow is 5.25J/cm2.With 6.150mW/cm2Irradiation
Degree.The irradiation time of all samples is 20min;The drip hole of control group 2 with 60 μM of TBO at room temperature, in dark surrounds
Carry out culture 20min;Population density is maintained at 107CFU/ml。
After the completion of above-mentioned culture, data are counted with statistics software SPSS software, use descruotuve statu statistical method
Data in Multi-way analysis are summarized.The result shows that this method is effective.Statistical significance between group uses variance
(variance analysis) is analyzed to determine.P < 0.05 thinks statistically significant, and related data is as shown in table 1.
1 photosensitizer factor of table and illumination factor are to staphylococcus epidermis and the pathogen inactivated effect of S. aureus bacterium
The influence of fruit
Group | Bacterial number variation | To bacteria pathogeny Cytotoxicity |
TBO (60 μM)+red light irradiation | Bacterial number significantly reduces | With stronger cytotoxicity |
TBO (60 μM)+dark | Bacterial number is without significant change | Do not show cytotoxicity |
PBS (60 μM)+red light irradiation | Bacterial number is without significant change | Do not show cytotoxicity |
PBS (60 μM)+dark | Bacterial number is without significant change | Do not show cytotoxicity |
Aureus growth situation is as shown in Figure 1, wherein Fig. 1 different disposal group before the radiation of different disposal group and after radiation
The staphylococcus epidermal growth situation of (e, f, g, h) after (a, b, c, d) and irradiation in 48 hours.The PDT group (h) that TBO is mediated is in spoke
Apparent growth inhibition is shown according to after.(a, e) control group, (b, f) independent TBO group, (c, g) independent light irradiation group, (d, h)
The PDT group that TBO is mediated.Different experiments group survival Microflora (T:TBO;L: feux rouges) as shown in Figure 2.
It is thin that above-mentioned experimental data shows that photosensitizer does not have photosensitizer all in the dark or in light environment and cannot make
Bacterium pathogen quantity is reduced;Only in the case where photosensitizer TBO and illumination condition are concured, it can just play and kill epidermis grape ball
The effect of bacterium and S. aureus bacterium pathogen.
2. influence of the photosensitizer concentration factor to bacterial pathogens inactivating efficacy
Study influence of photosensitizer blutene (TBO) concentration to staphylococcus epidermis and staphylococcus aureus.It will be thin
Bacterium suspension (108CFU/ml) with the blutene of various concentration (20 μM, 40 μM, 60 μM, 80 μM) be placed in 96 orifice plates into
Row culture.Standing cultivates 20min at room temperature, and the population density of laboratory sample is maintained at 107CFU/mL.It is training
Illumination is carried out after supporting, illumination flow is 5.25J/cm2.With 6.150mW/cm2Irradiation level.The irradiation time of all samples is
20min。
After the completion of above-mentioned culture, data are counted with statistics software SPSS software, use descruotuve statu statistical method
Data in Multi-way analysis are summarized.The result shows that this method is effective.Statistical significance between group uses variance
(variance analysis) is analyzed to determine.P < 0.05 thinks statistically significant, and related data is as shown in table 2.
Shadow of the 2 photosensitizer concentration factor of table to staphylococcus epidermis and the pathogen inactivated effect of S. aureus bacterium
It rings
Group | Bacterial number variation | To bacteria pathogeny Cytotoxicity |
TBO (20 μM) illumination (feux rouges) | Bacterial number is reduced | There is certain cytotoxicity |
TBO (40 μM) illumination (feux rouges) | Bacterial number reduction increases | Cytotoxicity further enhances |
TBO (60 μM) illumination (feux rouges) | Bacterium reduces more | Cytotoxicity further enhances |
TBO (80 μM) illumination (feux rouges) | Bacterium reduces quantity and increased | Cytotoxicity further enhances |
Shown by the experiment digital display in table 2, under same illumination condition, with the increase of photosensitizer TBO concentration, for epidermis
The killing effect of staphylococcus and S. aureus bacterium pathogen is constantly enhancing, the epidermis that TBO is killed at 80 μM
Staphylococcus and S. aureus bacterium pathogen quantity are most, and the ability for inactivating bacterial pathogens is most strong, but it is killed
The increased number of bacterial pathogens of going out and ability growth rate obviously weaken, this may with bacterial pathogens sum with
The concentration of TBO has largely reduced during increasing, and makes to subsequent deactivation to increase to be restricted;Meanwhile for thin
The enhancing of the deactivation of bacterium pathogen is also influenced by illumination factor, is the coefficient result of the two;Thus above-mentioned
In under bacteria pathogeny bulk concentration and illumination condition, the sterilization effect of 60 μM of TBO concentration is preferable.
3. influence of the illumination dose to bacterial pathogens inactivating efficacy
Have studied influence of the light exposure dose to staphylococcus epidermis and staphylococcus aureus.Bacterial suspension is used respectively
Concentration is that 60 μM of blutenes are cultivated as described above.Some bacterial suspensions are cultivated on 96 orifice plates, use light energy
Respectively 0.81J/cm2、1.28J/cm2、2.97J/cm2、6.33J/cm2And 8.76J/cm2Red light irradiation 20min.Experiment knot
Shu Hou calculates bacterial clump quantity, and has carried out the calculating of Survival probability of bacteria.
After the completion of above-mentioned culture, data are counted with statistics software SPSS software, use descruotuve statu statistical method
Data in Multi-way analysis are summarized.The result shows that this method is effective.Statistical significance between group uses variance
(variance analysis) is analyzed to determine.P < 0.05 thinks statistically significant, and corresponding situation is as shown in table 3.
Influence of 3 illumination dose of table to bacterial pathogens inactivating efficacy
Light exposure dose be also studied to the Different Effects of staphylococcus epidermis and staphylococcus aureus.With luminous energy
Increase, the survival rate of staphylococcus epidermis and staphylococcus aureus gradually decreases.However, under given optical power, observation
To being remarkably decreased for staphylococcus epidermis.Low light dosage (1.28J/cm2), the cell survival rate of staphylococcus epidermis is almost than gold
Staphylococcus aureus is 10 times low.Difference is statistically significant (P < 0.05).
Deactivation of the intensity of illumination (being indicated with optical power density) to bacterium is had studied simultaneously, light irradiation time is respectively provided with
For 20min, change the power density of irradiation light, respectively 0.675mW/cm2,1.065mW/cm2,2.474mW/cm2,
5.273mW/cm2and 7.299mW/cm2, the survival rate of bacterium after statistics light irradiation.The result shows that mentioning with intensity of illumination
Height, Survival probability of bacteria reduce rapidly, and then further increase intensity of illumination, and Survival probability of bacteria can also further decrease.
Deactivation of the irradiation time to bacterium is further studied, and optical power density is maintained at 5.273mW/cm2。
Statistics is respectively with the survival rate of staphylococcus epidermis and staphylococcus aureus after red light irradiation 5,10,20,30,40min.When
When TBO concentration is 60 μM, even if the only irradiation of 5min, the survival rate of staphylococcus epidermis is also reduced to 30% hereinafter, and golden
The CNN surviving fraction of staphylococcus aureus is reduced to 50%.The result shows that the dead of most of bacteriums nearly all occurs in initial light
According to the stage.This quick inactivating effect may be of great significance to the treatment of staphylococcic ocular infection, because this
Bacterium has very strong invasion.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected
The limitation of range is protected, although explaining in detail referring to preferred embodiment to the present invention, those skilled in the art are answered
Work as understanding, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the reality of technical solution of the present invention
Matter and range.
Claims (9)
1. the ablation method that a kind of pair of keratitis extracts bacterial pathogens, which is characterized in that the inactivation of the bacterial pathogens
Method is by utilizing photodynamic therapy under photodynamic action and mediation substance collective effect to the bacterial pathogens of eye surface
It is inactivated.
2. the ablation method that a kind of pair of keratitis according to claim 1 extracts bacterial pathogens, which is characterized in that described
The light that uses of photodynamic action be bandwidth be 20nm, wavelength is the visible light of 575nm~670nm.
3. the ablation method that a kind of pair of keratitis according to claim 1 extracts bacterial pathogens, which is characterized in that described
Mediation substance be the hydrophilic cations photosensitizer with film destruction.
4. the ablation method that a kind of pair of keratitis according to claim 3 extracts bacterial pathogens, which is characterized in that described
Hydrophilic cationic photosensitizer be blutene.
5. the ablation method that a kind of pair of keratitis according to claim 1 extracts bacterial pathogens, which is characterized in that described
The bacterial pathogens of eye surface be that staphylococcus epidermis or staphylococcus aureus are one or two kinds of;The microbes cause
Bacterial keratitis.
6. the ablation method that a kind of pair of keratitis according to claim 2 extracts microbial pathogens, which is characterized in that institute
The visible light stated is one of red visible or white visible light.
7. the ablation method that a kind of pair of keratitis according to claim 6 extracts microbial pathogens, which is characterized in that institute
The optical power density for the visible light stated is 0.550~7.500mW/cm2, radiated time is 20~45min.
8. the ablation method that a kind of pair of keratitis according to claim 4 extracts microbial pathogens, which is characterized in that institute
The concentration for the blutene stated is 45~80 μM.
9. the ablation method that a kind of pair of keratitis according to claim 1 extracts microbial pathogens, which is characterized in that packet
Include following steps:
S1. above-mentioned 100~200mL of bacterial suspension, 15~25min of incubated at room temperature is taken, places it in 96 orifice plates, keeps bacterium close
Degree maintains 107CFU/mL or so, and 5~80 μM of blutene are added in the drip hole of part thereto;
It S2. is 0.50~8.50J/cm by being exposed to added with the hole of blutene as illumination dose after the completion of cultivating2, optical power
Density is 0.550~7.500mW/cm2Under conditions of, 20~45min;
S3. it cultivates 48 hours under the conditions of 37 DEG C, after the completion of culture, bacteria living is assessed using colony counting method.
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