CN101619339B - Production method of bacterial exotoxin - Google Patents
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- CN101619339B CN101619339B CN2009100751847A CN200910075184A CN101619339B CN 101619339 B CN101619339 B CN 101619339B CN 2009100751847 A CN2009100751847 A CN 2009100751847A CN 200910075184 A CN200910075184 A CN 200910075184A CN 101619339 B CN101619339 B CN 101619339B
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- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 title claims abstract description 29
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a production method of bacterial exotoxin, comprising the procedures of bacterial toxin production culture, bacterial exotoxin extraction, bacterial exotoxin purification and bacterial exotoxin preservation, wherein the bacterial toxin production culture is carried out in a microorganism dialysis incubator which comprises a culture medium chamber and a culture chamber, a dialysis membrane and a dialysis membrane supporting and protecting device are arranged between the culture medium chamber and the culture chamber, the ratio of the volume of the culture medium chamber to the total volume of the culture chamber is selected between 3:1 and 1:6 according to a microorganism culture time, and facilities needed by microorganism growth are arranged in the culture medium chamber. The production method comprises the procedures of toxin production culture, sterilization, filtration, concentration, precipitation, percrystallization and preservation. The production method of bacterial exotoxin is adopted to produce toxin, optimizes bacterial growth environment, simplifies the technologies of concentration and purification of bacterial exotoxin, increases the yield to 90 percent from 17-33 percent, and shortens the purification time to about 12-24 hours from the original natural dialysis of 12-15 days.
Description
Technical field
The present invention relates to a kind of microorganism preparation method, refer in particular to a kind of bacterial exotoxin cultivation, extraction, concentrate, the processing method of purifying.
Background technology
Bacterial exotoxin is a kind of high molecular weight protein that comes out from the thalline intracellular metabolite in the bacteria growth process.These high molecular weight proteins have various virulence.Bacterial exotoxin is applied in many fields such as medical science, the evils of going out at present, has been applied to the preventive medicine field as C type Clostridium botulinum vaccine, toxoid, and 1989 years U.S. FDA official approval botulinum toxin type As are gone into operation as new drug and gone on the market.Botulinum toxin type A has been applied to clinical medicine and beauty treatment smoothing wrinkle field at present, as being used for the myodystonia disease in fields such as neurology department, ophthalmology---the treatment of blepharospasm, mimic convulsion, stravismus and spasmodic torticollis etc., Type B, E BOTULINUM TOXIN TYPE A A have also entered clinical trial, C type D BOTULINUM TOXIN TYPE A A can be used for killing mouse, and the Su Jin cloud bacillus fly mosquito aspect of going out is on probation.Begun the application practice of tetanus bacillus toxin on the recent international.Bacterial exotoxin can also be applied to national defense construction.
The method for cultivation of bacteria of present popular use is traditional fermentation method.It is with the bacterial classification direct inoculation in substratum, the bacterium procreation of in substratum, growing, the extracellular toxin of substratum, bacterium and bacterium mixes during cultivation, bacterium grows in having nutrition, excremental hybird environment.Simple if desired extracellular toxin just must adopt isolating technique means that bacterial exotoxin is separated from mixture.Such cultivation and method of purification not only bacterium and ectotoxicly yield poorly, yield rate is little, and purifying technique complexity.Because the growing environment of bacterium is bad, so the quality of bacterium is not high yet.The technology that the present U.S. prepares botulinum toxin type A is to comprise: Clostridium botulinum is cultivated Acid precipitation entirely---extraction of toxin phosphate buffered saline buffer---, and ammonium sulfate concentrates steps such as reaching spontaneous nucleation.This method exists certain problem at aspects such as process time, consumption, yields.Chinese patent 97118838.6 also discloses a kind of production technique of A type meat poison crystalline toxin and has produced the required frozen-dried protective liquid of this toxin, and the technology of this invention is: dilution, the freeze-drying of the refining purification-toxin of toxin producing medium-product poison cultivation-Sterile Filtration-toxin.Its main technical schemes comprises: with the process that casein, pancreas enzyme powder, yeast dialyzate, distilled water, glucose are made substratum, produce malicious cultivation, Sterile Filtration, the refining purification of toxin, the operations such as dilution freeze-drying of toxin.Its venom of collecting is the mixed solution of substratum and bacterial exotoxin, must comprise steps such as enzymeization, dialysis, chromatography in its processing step, could separate bacterial exotoxin from venom.
Russian Mei Niqi scolded the novel method that the husband has invented culturing bacterium in 1896---the dialysis culture method.Dialysis culture is with dialysis membrane substratum and culture to be separated, the part at bacterium, culture place is culturing room, the part at substratum (being nutrient) place is Culture Medium Laboratory, culture is by the nutrient of the absorption of the dialysis membrane between culturing room and Culture Medium Laboratory substratum, and the small molecules movement of culture enters Culture Medium Laboratory by dialysis membrane.Therefore purified the growing environment of bacterium.But dialysis method is laid aside and neglected always, just in the laboratory, use, can't suitability for industrialized production, major cause is the dialyzer that dialysis method is used bag type paste always, the capsule bag of will dialysing is soaked in the liquid medium, dialysis capsule bag is in free state, and owing to reasons such as the dialysis capsule bag of depositing culture soak in substratum, moves, the capsule bag breaks easily.And its to require the shared volume of substratum be 10 times of culture place volume, that is to say that the dialysis ratio of general dialysis culture method is 10: 1.And that the volume of dialysis capsule bag can not be done is too big, otherwise easier breakage.Though so dialysis method can obtain high-quality bacterium of high reactivity and derivative thereof, because cost height, success ratio are low, few people use.Though the dialysis culture method had been passed through partly and to have been improved afterwards, but still to continue to use substratum be 10: 1 dialysis culture ratio than culture.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of method of the high-quality bacterial exotoxin of production that can be efficient, stable.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
A kind of production method of bacterial exotoxin comprises bacterium and produces poison cultivation, the extraction of bacterial exotoxin, purifying, preservation operation; Its production method comprises the steps:
A, product poison are cultivated
It is to carry out in the microbial dialysis incubator that the product poison of bacterium is cultivated, the microbial dialysis incubator comprises Culture Medium Laboratory and culturing room, be provided with the device of a dialysis membrane and an overfill protection dialysis membrane between Culture Medium Laboratory and the culturing room, Culture Medium Laboratory is one, on culturing room's assembling Culture Medium Laboratory, the quantity of culturing room is the multiple of Culture Medium Laboratory, the beguine of the volume of Culture Medium Laboratory and the cubic capacity of culturing room is selected between 3: 1 to 1: 6 according to the length of the incubation time of microorganism, the needed facility of microbial growth is arranged in the Culture Medium Laboratory, provide growth conditions by Culture Medium Laboratory for microorganism, with the growing environment of guaranteeing that culturing room is good; The shape of microbial dialysis incubator should adopt in the solid geometry principle the big and relatively little shape of volume of surface-area; Its culturing step comprises:
A1. modulate nutrient solution and toxin producing medium,
A2. select, assemble corresponding dialysis culture device according to the size and the incubation time of turnout, substratum and nutrient solution are filled into respectively in the Culture Medium Laboratory and culturing room of dialysis culture device, high-temperature sterilization postcooling to 35 ℃～40 ℃, the ratio of substratum and nutrient solution are by kind of bacterium and microorganism and growth cycle decision; High-temperature sterilization can be sterilized 30 minutes under 113 ℃ of conditions;
A3. will be inoculated in the culturing room through the cultivation bacterium bacterial classification of suitably cultivating, inoculum size is produced poison by the incubation time of cultivating bacterium and culture temperature and is cultivated for by cultivating 3%～5% of liquid measure in the culturing room; The output of dialysis culture is directly proportional with the dialysis area.
B, Sterile Filtration
After microscopy is pure culture, collect cultured cultivation bacterium bacterium liquid from dialyzer culturing room with liquid collecting tube, centrifugal treating under≤0 ℃ environment is got supernatant liquor then, with Φ 0.22mm degerming filter plate or there is not asbestos degerming membrane filtration, promptly gets the extracellular toxin venom; Centrifugal treating is preferably on the low-temperature and high-speed whizzer with 6000 rev/mins speed centrifugal 10 minutes.
C, precipitation concentrate
With venom and-70 ℃ dehydrated alcohol mixed by 1: 2, pour mixed solution into tophan pot, the environment (as refrigerator-freezer) of putting into-20 ℃ left standstill 8～12 hours, under≤0 ℃ environment centrifugal 25～30 minutes then with 12000 rev/mins speed, remove supernatant liquor, last solid promptly is the bacterial exotoxin of sex change.
D, percrystallization
Centrifugal back gained solids is dissolved and thorough mixing with an amount of ammoniumsulphate soln, pour in the dialysis tubing then, the macromole sorbent material will be adjacent to around the dialysis tubing, use the macromole sorbent material with the ammoniumsulphate soln sucking-off in the dialysis tubing in≤5 ℃ of environment, last solids is crystalline toxin in the dialysis tubing.Can repeat this step, till the OD260/OD280 value is less than 0.6.
E, preservation, with purity reach the OD260/OD280 value less than 0.6, crystallisate that virulence is qualified puts into≤15 ℃ protective material dilutes, and by proteinic freeze-drying curve freeze-drying, refrigeration then.
The further concrete scheme of method for culturing bacteria of the present invention is: the relation between the volume of Culture Medium Laboratory and the cubic capacity of culturing room is that the volume of the long more Culture Medium Laboratory of incubation time is big more with the ratio of the cubic capacity of culturing room, otherwise it is also anti-, incubation time is during greater than 120 hours, and the volume of Culture Medium Laboratory is 3: 1 with the ratio of the cubic capacity of culturing room; When incubation time is 96～120 hours, the volume of Culture Medium Laboratory is 2.5: 1 with the ratio of the cubic capacity of culturing room, when incubation time is 72～96 hours, the volume of Culture Medium Laboratory is 2: 1 with the ratio of the cubic capacity of culturing room, incubation time is 48～72 hours, the volume of Culture Medium Laboratory is 1.5: 1 with the ratio of the cubic capacity of culturing room, when incubation time is 24～48 hours, the volume of Culture Medium Laboratory is 1: 1～2 with the ratio of the cubic capacity of culturing room, incubation time is during less than 24 hours, and the volume of Culture Medium Laboratory is 1: 3～6 with the ratio of the cubic capacity of culturing room.
Being further defined to of nutrient solution of the present invention: nutrient solution is a 0.6%NaCl solution.
Being further defined to of substratum of the present invention: described toxin producing medium is selected according to the particular case of bacterial classification, and adds the ratio adding yeast extract of 200 ± 50g yeast extract according to the 1000ml substratum.Substratum is selected the digestion type as far as possible.
Protectant composition used in the preservation operation of the present invention is: protective material is formulated by gelatin, sucrose, N.F,USP MANNITOL, distilled water.
Protectant ratio used in the preservation operation of the present invention is: the proportioning of gelatin, sucrose, N.F,USP MANNITOL, distilled water is 3/5/5/100 in the protective material.
Protectant protective material compound method used in the preservation operation of the present invention is: take by weighing gelatin, sucrose, N.F,USP MANNITOL respectively by configuration amount; earlier gelatin is added distilled water; be heated to till the gelatin off-bottom; add sucrose and N.F,USP MANNITOL again; fully after the dissolving; filter bulb filters, and sterilizes through 30 minutes under 113 ℃ of conditions.
Owing to adopted technique scheme, the present invention to obtain following technical progress:
Adopt method of the present invention to produce toxin, optimize the bacterial growth environment, widen the breeding space of bacterium, thereby the cost of bacterial exotoxin goods, price are descended significantly, there is a sharp increase in output, and every milliliter of bacterium number of dialysis culture method can reach 10
10～10
11, than 10 of every milliliter of bacterium number of fermentation method
8～10
9Improved 100 times, its yield rate can bring up to 90% by 17%～33% of original technology.Adopt method of the present invention to carry out the cultivation of bacterium and derivative thereof, concentrated, the purifying technique of bacterial exotoxin have been simplified greatly, exempted loaded down with trivial details separation circuit, the purification time is shortened to about 12～24 hours by original 12～15 days of dialysing naturally, shortened the production cycle greatly, reduced staff and recruitment time, improved production efficiency greatly, reduce the consume of goods in the making processes to greatest extent, improved output, saved time and starting material.Solid basic substance is provided for the utilization of bacterial exotoxin, has huge economic benefit and social benefit.
Use method of the present invention, the microbial growth environment is optimized, its biological activity maximizes the retention, its Products Quality and the raising thereupon of tiring.The production of bacterial exotoxin of the present invention does not all pollute environment with use.All pass through high temperature, autoclaving sterilization before and after the production unit of bacterial exotoxin, container also is like this.Therefore, no poisonous, the gas, waste water, the waste discharge that are harmful to, have bacterium.In use, bacterial exotoxin is a protein, very easily degrades at occurring in nature, does not have residually, can not cause savings to poison and secondary poisoning, is the technology and the product of environmental protection.
The venom of gained of the present invention only is bacterium and extracellular toxin, can not comprise substratum, adopt high speed centrifugation processing under the low temperature environment in the Sterile Filtration process, can remove most bacterium thalline rapidly, therefore omit existing fermentation method and cultivated steps such as enzymeization in the ectotoxic separation of produced method, dialysis, chromatography, can save filtration time, reduce labor intensity, reduce the energy and materials consumption.Adopt pressure dialysis technologies such as high speed centrifugation processing, can shorten crystallization time, the time of natural percrystallization was generally 12～15 days, forced the crystallization time of dialysis then to shorten to about 12 hours.
Adopt organic solvent ethanol that the bacterial exotoxin sex change from venom in the venom is precipitated out in the precipitation enrichment process; and reach isolating purpose; crossing cold organic solvent, to concentrate quick-frozen be loss of activity in order to make slow decline that extracellular toxin can Yin Wendu; protect the biological activity of toxin to greatest extent, improve the quality.
Adopt dialysis tubing and sorbent material to carry out crystallization treatment, can the absorption of the damping fluid in the bag is clean, the impurity small-molecule substance of conduct simultaneously (as the metabolite of remaining nutritive medium, bacterium, and NaCl solution etc.) clean with the damping fluid dialysis, promptly reach the purpose of purifying.
Protective material proportioning of the present invention is simple, and raw material is easy to get, and is widely used material in the medicament, the advantage that have safely, has no adverse reaction.Add N.F,USP MANNITOL in the protective material and can subdue the untoward reaction of detoxifying function in human body, improve the recovery of the function at human injection position, in addition, N.F,USP MANNITOL is a kind of vehicle, and the protective material that adds N.F,USP MANNITOL is after making the extracellular toxin goods, and the product phase outward appearance of goods is good.
Embodiment
Below by a concrete experimentation of producing A type novain goods the present invention is described in further details, and the two kind A type novains of experimental result with the U.S., the production of Chinese Lanzhou is contrasted:
1, microbial dialysis incubator
It is to carry out in the microbial dialysis incubator that the product poison of bacterium of the present invention is cultivated, the microbial dialysis incubator comprises Culture Medium Laboratory and the culturing room that fits together, be provided with the device of a dialysis membrane and an overfill protection dialysis membrane between Culture Medium Laboratory and the culturing room, Culture Medium Laboratory is one, the quantity of culturing room is the multiple of Culture Medium Laboratory, on culturing room's assembling Culture Medium Laboratory, the needed facility of the growth of substratum and bacterium is arranged in the Culture Medium Laboratory, provides food and other growth conditions by Culture Medium Laboratory for bacterium; The shape of microbial dialysis incubator should adopt in the solid geometry principle the big and relatively little shape of volume of surface-area.The beguine of the volume of Culture Medium Laboratory and the cubic capacity of culturing room is selected between 3: 1 to 1: 6 according to the length of the incubation time of microorganism, and the relation between the volume of Culture Medium Laboratory and the cubic capacity of culturing room is that the volume of the long more Culture Medium Laboratory of incubation time is big more with the ratio of the volume of culturing room.
2, bacterial classification, nutrient solution and substratum
Employed bacterial classification is the bacterial strain of isolating a kind of general quality in northwest, Sichuan University biotechnology research place Guyuan, the Ruoergai area soil in the experimentation, and tentatively cultivates.Nutrient solution is a 0.6%NaCl solution.Toxin producing medium is selected commercially available common anaerobic bacteria culture base, and in substratum, add yeast extract, the ratio that adds is that every 1000ml substratum adds the 200g yeast extract, selecting the volume ratio of Culture Medium Laboratory and culturing room is 2.5: 1, and Culture Medium Laboratory and culturing room are fitted together formation dialysis culture device.Deployed substratum and nutrient solution are filled into respectively in Culture Medium Laboratory and the culturing room.Under 113 ℃ of conditions,, be cooled to 35 ℃～40 ℃ then through sterilization in 30 minutes.
3, producing poison cultivates
Bacterial classification by cultivating 3%～5% of liquid measure in the culturing room, is inoculated into culturing room, produces poison in suitable culture temperature and cultivate, incubation time is 110 hours.
4, Sterile Filtration
Carry out the mirror mirror to cultivating bacterium, after microscopy is pure culture, cultured cultivation bacterium bacterium liquid is collected by liquid collecting tube from dialyzer culturing room, centrifugal through low-temperature and high-speed whizzer (＜0 ℃, 6000 rev/mins) 10 minutes then.Get supernatant liquor, with the degerming filter plate of Φ 0.22mm or there is not asbestos degerming membrane filtration, promptly get venom.
5, concentrating and precipitating
Mix with venom to about-70 ℃ ratios dehydrated alcohol is freezing, mixed solution is put into-20 ℃ of refrigerator-freezers and left standstill 8～12 hours with 2: 1, centrifugal through low-temperature and high-speed whizzer (＜0 ℃, 12000 rev/mins) 25～30 minutes then.Get supernatant liquor, last solid promptly is the bacterial exotoxin of sex change.
6, percrystallization
Centrifugal back gained solids is added an amount of saturated ammonium sulphate solution, pour in the dialysis tubing behind the thorough mixing, place＜5 ℃ refrigerator-freezer and being adjacent to the macromole sorbent material, with the ammoniumsulphate soln in the dialysis tubing together with as the impurity small-molecule substance (as the metabolite of remaining nutritive medium, bacterium, and NaCl solution etc.) sucking-off, last solids is crystalline toxin in the dialysis tubing.
Can repeat this step, till the OD260/OD280 value is less than 0.6.
7, detect virulence
With the crystallisate of 1 milligram of electronic scales weighing, with using small white mouse to measure virulence after the damping fluid renaturation.
8, preserve preservation
Protective material is cooled to≤add toxin after 15 ℃, by the service requirements can, by proteinic freeze-drying curve freeze-drying.Toxin after the freeze-drying is measured virulence with small white mouse once more, carries out aseptic detection and water content and detects, the refrigeration of qualified back.
Wherein protectant prescription is: gelatin 3g, sucrose 5g, N.F,USP MANNITOL 5g, distilled water 100ml.
The protective material compound method is: take by weighing gelatin, sucrose, N.F,USP MANNITOL respectively by configuration amount, earlier gelatin is added distilled water, be heated to till the gelatin off-bottom; add sucrose and N.F,USP MANNITOL again; fully after the dissolving, filter bulb filters, and sterilizes through 30 minutes under 113 ℃ of conditions.
The performance comparison table of A type novain goods
The bacterial classification that the contrast experiment uses is the isolating high toxic bacterial strain of HALL strain U.S. state university food test.This is the very good bacterial classification of a kind of quality.
Bacterial classification used in the present invention is an isolating general bacterial strain in northwest, Sichuan University biotechnology research place Guyuan, the Ruoergai area soil.
Consumption when tiring described in the table is the onset of each injection point during with smoothing wrinkle is a standard, is to be the biological unit of each injection point onset of standard with the wrinkle improvement effect, and U represents biological unit.Onset consumption during A type novain smoothing wrinkle of the present invention has only 0.7U to get final product onset, and the smoothing wrinkle validity period is 6～9 months, is longer than the A type novain that other two kinds of methods are produced.
If use other bacterial classification beyond this embodiment, perhaps culture condition changes and when incubation time is changed, should use the microbial dialysis incubator of other dialysis ratio, just the ratio of the Culture Medium Laboratory of microbial dialysis incubator and culturing room also should change with incubation time.
Claims (2)
1. the production method of a bacterium A type novain comprises the cultivation of Production by Bacteria poison, the extraction of bacterial exotoxin, purifying, preservation operation, it is characterized in that: comprise the steps
A, product poison are cultivated
It is to carry out in the microbial dialysis incubator that the product poison of bacterium is cultivated, the microbial dialysis incubator comprises Culture Medium Laboratory and culturing room, be provided with the device of a dialysis membrane and an overfill protection dialysis membrane between Culture Medium Laboratory and the culturing room, Culture Medium Laboratory is one, on culturing room's assembling Culture Medium Laboratory, the quantity of culturing room is the multiple of Culture Medium Laboratory, the shape of microbial dialysis incubator should adopt in the solid geometry principle the big and relatively little shape of volume of surface-area, the beguine of the volume of Culture Medium Laboratory and the cubic capacity of culturing room is selected between 3: 1 to 1: 6 according to the length of the incubation time of microorganism, and the needed facility of microbial growth is arranged in the Culture Medium Laboratory; Its culturing step comprises
A1. modulate nutrient solution and toxin producing medium, described nutrient solution is a 0.6%NaCl solution, and described toxin producing medium is a digestion type substratum, and adds yeast extract according to the ratio of 1000ml substratum adding 200 ± 50g yeast extract,
A2. according to the size of turnout and incubation time select, the corresponding dialysis culture device of assembling, substratum and nutrient solution are filled into respectively in dialysis culture basal cell and the culturing room, 113 ℃ of following high-temperature sterilization postcooling to 35 ℃～40 ℃,
A3. will be inoculated in the culturing room through the cultivation bacterium bacterial classification of suitably cultivating, inoculum size is produced poison by the incubation time of cultivating bacterium and culture temperature and is cultivated for by cultivating 3%～5% of liquid measure in the culturing room;
B, Sterile Filtration
Collect cultured cultivation bacterium bacterium liquid, then under≤0 ℃ environment with 6000 rev/mins speed centrifugal treating, get supernatant liquor, filter, promptly get the extracellular toxin venom;
C, concentrating and precipitating
With venom and-70 ℃ dehydrated alcohol mixed by 1: 2, mixed solution being put into-20 ℃ of environment left standstill 8～12 hours, under≤0 ℃ environment centrifugal 25～30 minutes then with 12000 rev/mins speed, remove supernatant liquor, last solid promptly is the bacterial exotoxin of sex change;
D, percrystallization
Centrifugal back gained solids is dissolved and thorough mixing with an amount of ammoniumsulphate soln, pour in the dialysis tubing then, in≤5 ℃ of environment, use the macromole sorbent material with the ammoniumsulphate soln sucking-off in the dialysis tubing, repeat this step, till the OD260/OD280 value was less than 0.6, last solids was crystalline toxin in the dialysis tubing;
E, preservation
With purity reach the OD260/OD280 value less than 0.6, crystallisate that virulence is qualified puts into≤15 ℃ protective material dilutes, and by proteinic freeze-drying curve freeze-drying, refrigeration then; Described protective material is formulated in 3/5/5/100 ratio by gelatin, sucrose, N.F,USP MANNITOL, distilled water.
2. the production method of a kind of bacterium A type novain according to claim 1 is characterized in that: described incubation time is during greater than 120 hours, and the volume of Culture Medium Laboratory is 3: 1 with the ratio of the cubic capacity of culturing room; When incubation time is 96～120 hours, the volume of Culture Medium Laboratory is 2.5: 1 with the ratio of the cubic capacity of culturing room, when incubation time is 72～96 hours, the volume of Culture Medium Laboratory is 2: 1 with the ratio of the cubic capacity of culturing room, when incubation time is 48～72 hours, the volume of Culture Medium Laboratory is 1.5: 1 with the ratio of the cubic capacity of culturing room, when incubation time is 24～48 hours, the volume of Culture Medium Laboratory is 1: 1～2 with the ratio of the cubic capacity of culturing room, incubation time is during less than 24 hours, and the volume of Culture Medium Laboratory is 1: 3～6 with the ratio of the cubic capacity of culturing room.
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