CN105238718A - Multistage cultural method of Escherichia coli - Google Patents
Multistage cultural method of Escherichia coli Download PDFInfo
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- CN105238718A CN105238718A CN201510708159.3A CN201510708159A CN105238718A CN 105238718 A CN105238718 A CN 105238718A CN 201510708159 A CN201510708159 A CN 201510708159A CN 105238718 A CN105238718 A CN 105238718A
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Abstract
The invention relates to the field of a biology technology, and concretely relates to a multistage cultural method of Escherichia coli. The method comprises the following steps: 1) inoculating bacterial strains of Escherichia coli to a first medium for cultivation, and carrying out a streak inoculation on an agar plate; 2) inoculating a first level cultivation seed to a second medium for cultivation; 3) inoculating a second level cultivation seed to a third medium for cultivation and preservation; 4) inoculating the third level cultivation seed to a bacteria liquid, introducing the bacteria liquid into a centrifuge tube, adding 9-16% agar, placing the centrifuge tube on a shaking table for a night, adding a solution of calcium chloride, suspending the bacteria liquid, carrying out a centrifugation, removing a clear liquid on a upper level to obtain the product. The multistage cultural method of Escherichia coli can stabilize cultivation of Escherichia coli, and simultaneously simplify cultivation conditions of the microbial fermentation; the cultivation process is practical, and ideal live bacteria of high concentration can be obtained for laboratory.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of colibacillary multistage cultural method.
Background technology
Intestinal bacteria are bacterioids very close with our daily life relation, and formal name used at school is called " escherichia coli ", belong to the one in the large class of intestinal bacilli.It colonizes in a kind of unicellular organism harmless in human body large intestine, and structure is simple, and rapidly, cultivate easily, it is biologically important experiment material in breeding.In the preparation of various animal large intestine bacillus inactivated vaccine, primary link is exactly colibacillary cultivation.In order to obtain desirable viable bacteria concentration, industry has carried out large quantity research to intestinal bacteria culture process.Affect intestinal bacteria and cultivate a lot of because have of bacteria concentration, as substratum composition, incubation time, strain inoculation amount and environment of bacteria growth etc. generally, productive rate and the Escherichia coli fermentation density of bio-pharmaceutical are closely related, and colibacillary yield is directly connected to the productive rate of bio-pharmaceutical.So when thalline expressing protein efficiency is suitable, colibacillary fermentation density directly has influence on the production cost of bio-pharmaceutical.Particularly under same purification technique condition.
Summary of the invention
For solving the deficiencies in the prior art, the invention provides a kind of colibacillary multistage cultural method.
A kind of colibacillary multistage cultural method, comprises the following steps:
1) strain Escherichia coli is inoculated in the first substratum, cultivates 24 ~ 32 hours under 36.5 ~ 37.3 DEG C of conditions, streak inoculation on agar plate, cultivate 20 ~ 24 hours under 37.4 ~ 37.8 DEG C of conditions, obtain one-level and cultivate seed.
Concrete, step 1) in, the nutritive ingredient in the first described substratum comprises: glucose, peptone, potassium primary phosphate, magnesium sulfate heptahydrate, yeast extract, glycerine, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2 6H2O and distilled water.
2) by step 1) one-level that obtains cultivates seed and is inoculated in the second substratum, cultivate 15 ~ 18 hours under 34.5 ~ 35.5 DEG C of conditions, obtain secondary and cultivate seed.
Concrete, step 2) in, the nutritive ingredient in the second described substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, magnesium sulfate, magnesium, yeast extract, Iron trichloride hexahydrate, boric acid, Zinc Sulphate Heptahydrate, vitamin H, manganous sulfate, Sodium Glutamate and distilled water.
3) by step 2) secondary that obtains cultivates seed and is inoculated in the 3rd substratum, cultivate 16 ~ 18 hours, obtain third stage culture seed, preserve as under 1 ~ 6 DEG C of condition under 37.8 ~ 38.5 DEG C of conditions.
Concrete, step 3) in, the nutritive ingredient in the 3rd described substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, sulfuric acid, rose-bengal solution, agar, distilled water, Deoxycholic Acid sodium solution, Streptomycin Solution and Calcium dichloride dihydrate.
4) by step 3) the third stage culture seed that obtains is inoculated in bacterium liquid, and is directed in centrifuge tube, add the agar of 9 ~ 16%, incubator overnight, then add calcium chloride solution, suspendible bacterium liquid, centrifugation, removed by supernatant liquid, to obtain final product.
Preferably, the rotating speed of described shaking table is 120 ~ 160 revs/min.
Preferably, the temperature of described calcium chloride solution is 3 ~ 6 DEG C.
Stable the cultivating intestinal bacteria of colibacillary multistage cultural method energy provided by the present invention, simplify fermentable culture condition, culture process is practical, can obtain desirable use for laboratory high viable bacteria concentration simultaneously.
Embodiment
Be described principle of the present invention and feature below, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1
A kind of colibacillary multistage cultural method, comprises the following steps:
1) strain Escherichia coli is inoculated in the first substratum, cultivates 28 hours under 36.8 DEG C of conditions, streak inoculation on agar plate, cultivate 22 hours under 37.6 DEG C of conditions, obtain one-level and cultivate seed.
Wherein, the nutritive ingredient in the first substratum comprises: glucose, peptone, potassium primary phosphate, magnesium sulfate heptahydrate, yeast extract, glycerine, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2 6H2O and distilled water.
2) by step 1) one-level that obtains cultivates seed and is inoculated in the second substratum, cultivate 16 hours under 35.0 DEG C of conditions, obtain secondary and cultivate seed.
Wherein, the nutritive ingredient in the second substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, magnesium sulfate, magnesium, yeast extract, Iron trichloride hexahydrate, boric acid, Zinc Sulphate Heptahydrate, vitamin H, manganous sulfate, Sodium Glutamate and distilled water.
3) by step 2) secondary that obtains cultivates seed and is inoculated in the 3rd substratum, cultivate 17 hours, obtain third stage culture seed, preserve as under 4 DEG C of conditions under 38.2 DEG C of conditions.
Wherein, the nutritive ingredient in the 3rd substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, sulfuric acid, rose-bengal solution, agar, distilled water, Deoxycholic Acid sodium solution, Streptomycin Solution and Calcium dichloride dihydrate.
4) by step 3) the third stage culture seed that obtains is inoculated in bacterium liquid, and is directed in centrifuge tube, add the agar of 13%, incubator overnight, then add calcium chloride solution, suspendible bacterium liquid, centrifugation, removed by supernatant liquid, to obtain final product.
Wherein, the rotating speed of shaking table is 150 revs/min.The temperature of calcium chloride solution is 3.5 DEG C.
Gained viable bacteria concentration is 9.5 × 10
9cFU/ml.
Embodiment 2
A kind of colibacillary multistage cultural method, comprises the following steps:
1) strain Escherichia coli is inoculated in the first substratum, cultivates 24 hours under 37.3 DEG C of conditions, streak inoculation on agar plate, cultivate 20 hours under 37.8 DEG C of conditions, obtain one-level and cultivate seed.
Wherein, the nutritive ingredient in the first substratum comprises: glucose, peptone, potassium primary phosphate, magnesium sulfate heptahydrate, yeast extract, glycerine, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2 6H2O and distilled water.
2) by step 1) one-level that obtains cultivates seed and is inoculated in the second substratum, cultivate 15 hours under 35.5 DEG C of conditions, obtain secondary and cultivate seed.
Wherein, the nutritive ingredient in the second substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, magnesium sulfate, magnesium, yeast extract, Iron trichloride hexahydrate, boric acid, Zinc Sulphate Heptahydrate, vitamin H, manganous sulfate, Sodium Glutamate and distilled water.
3) by step 2) secondary that obtains cultivates seed and is inoculated in the 3rd substratum, cultivate 16 hours, obtain third stage culture seed, preserve as under 6 DEG C of conditions under 38.5 DEG C of conditions.
Wherein, the nutritive ingredient in the 3rd substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, sulfuric acid, rose-bengal solution, agar, distilled water, Deoxycholic Acid sodium solution, Streptomycin Solution and Calcium dichloride dihydrate.
4) by step 3) the third stage culture seed that obtains is inoculated in bacterium liquid, and is directed in centrifuge tube, add the agar of 9 ~ 16%, incubator overnight, then add calcium chloride solution, suspendible bacterium liquid, centrifugation, removed by supernatant liquid, to obtain final product.
Wherein, the rotating speed of shaking table is 120 revs/min.The temperature of calcium chloride solution is 5 DEG C.
Gained viable bacteria concentration is 2.9 × 10
10cFU/ml.
Embodiment 3
A kind of colibacillary multistage cultural method, comprises the following steps:
1) strain Escherichia coli is inoculated in the first substratum, cultivates 32 hours under 36.5 DEG C of conditions, streak inoculation on agar plate, cultivate 24 hours under 37.4 DEG C of conditions, obtain one-level and cultivate seed.
Wherein, the nutritive ingredient in the first substratum comprises: glucose, peptone, potassium primary phosphate, magnesium sulfate heptahydrate, yeast extract, glycerine, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2 6H2O and distilled water.
2) by step 1) one-level that obtains cultivates seed and is inoculated in the second substratum, cultivate 18 hours under 34.5 DEG C of conditions, obtain secondary and cultivate seed.
Wherein, the nutritive ingredient in the second substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, magnesium sulfate, magnesium, yeast extract, Iron trichloride hexahydrate, boric acid, Zinc Sulphate Heptahydrate, vitamin H, manganous sulfate, Sodium Glutamate and distilled water.
3) by step 2) secondary that obtains cultivates seed and is inoculated in the 3rd substratum, cultivate 18 hours, obtain third stage culture seed, preserve as under 1 DEG C of condition under 37.8 DEG C of conditions.
Wherein, the nutritive ingredient in the 3rd substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, sulfuric acid, rose-bengal solution, agar, distilled water, Deoxycholic Acid sodium solution, Streptomycin Solution and Calcium dichloride dihydrate.
4) by step 3) the third stage culture seed that obtains is inoculated in bacterium liquid, and is directed in centrifuge tube, add the agar of 16%, incubator overnight, then add calcium chloride solution, suspendible bacterium liquid, centrifugation, removed by supernatant liquid, to obtain final product.
Wherein, the rotating speed of shaking table is 120 revs/min.The temperature of calcium chloride solution is 5 DEG C.
Gained viable bacteria concentration is 1.8 × 10
10cFU/ml.
The foregoing is only better embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. a colibacillary multistage cultural method, is characterized in that, comprise the following steps:
1) strain Escherichia coli is inoculated in the first substratum, cultivates 24 ~ 32 hours under 36.5 ~ 37.3 DEG C of conditions, streak inoculation on agar plate, cultivate 20 ~ 24 hours under 37.4 ~ 37.8 DEG C of conditions, obtain one-level and cultivate seed;
2) by step 1) one-level that obtains cultivates seed and is inoculated in the second substratum, cultivate 15 ~ 18 hours under 34.5 ~ 35.5 DEG C of conditions, obtain secondary and cultivate seed;
3) by step 2) secondary that obtains cultivates seed and is inoculated in the 3rd substratum, cultivate 16 ~ 18 hours, obtain third stage culture seed, preserve as under 1 ~ 6 DEG C of condition under 37.8 ~ 38.5 DEG C of conditions;
4) by step 3) the third stage culture seed that obtains is inoculated in bacterium liquid, and is directed in centrifuge tube, add the agar of 9 ~ 16%, incubator overnight, then add calcium chloride solution, suspendible bacterium liquid, centrifugation, removed by supernatant liquid, to obtain final product.
2. colibacillary multistage cultural method according to claim 1, it is characterized in that: step 1) in, the nutritive ingredient in the first described substratum comprises: glucose, peptone, potassium primary phosphate, magnesium sulfate heptahydrate, yeast extract, glycerine, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2 6H2O and distilled water.
3. colibacillary multistage cultural method according to claim 2, it is characterized in that: step 2) in, the nutritive ingredient in the second described substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, magnesium sulfate, magnesium, yeast extract, Iron trichloride hexahydrate, boric acid, Zinc Sulphate Heptahydrate, vitamin H, manganous sulfate, Sodium Glutamate and distilled water.
4. colibacillary multistage cultural method according to claim 3, it is characterized in that: step 3) in, the nutritive ingredient in the 3rd described substratum comprises: ammonium sulfate, Sodium phosphate dibasic, potassium primary phosphate, citric acid, sulfuric acid, rose-bengal solution, agar, distilled water, Deoxycholic Acid sodium solution, Streptomycin Solution and Calcium dichloride dihydrate.
5., according to the arbitrary described colibacillary multistage cultural method of Claims 1-4, it is characterized in that: step 4) in, the rotating speed of described shaking table is 120 ~ 180 revs/min.
6., according to the arbitrary described colibacillary multistage cultural method of Claims 1-4, it is characterized in that: step 4) in, the temperature of described calcium chloride solution is 2 ~ 5 DEG C.
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Citations (5)
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|---|---|---|---|---|
| CN104293668A (en) * | 2013-07-17 | 2015-01-21 | 南京朗恩生物科技有限公司 | Recombinant escherichia coli high-density fermentation method |
| CN104498391A (en) * | 2014-11-27 | 2015-04-08 | 苏州嘉禧萝生物科技有限公司 | Escherichia coli and culture method of culture medium thereof |
| CN104593306A (en) * | 2015-02-04 | 2015-05-06 | 烟台恒源生物股份有限公司 | High-density culture method of escherichia coli strains HY-05C |
| CN104877939A (en) * | 2015-05-27 | 2015-09-02 | 成都易创思生物科技有限公司 | Culture medium and cultural method applied to escherichia coli |
| CN105176887A (en) * | 2015-10-22 | 2015-12-23 | 江苏大学 | Method for culturing escherichia coli |
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- 2015-10-27 CN CN201510708159.3A patent/CN105238718A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293668A (en) * | 2013-07-17 | 2015-01-21 | 南京朗恩生物科技有限公司 | Recombinant escherichia coli high-density fermentation method |
| CN104498391A (en) * | 2014-11-27 | 2015-04-08 | 苏州嘉禧萝生物科技有限公司 | Escherichia coli and culture method of culture medium thereof |
| CN104593306A (en) * | 2015-02-04 | 2015-05-06 | 烟台恒源生物股份有限公司 | High-density culture method of escherichia coli strains HY-05C |
| CN104877939A (en) * | 2015-05-27 | 2015-09-02 | 成都易创思生物科技有限公司 | Culture medium and cultural method applied to escherichia coli |
| CN105176887A (en) * | 2015-10-22 | 2015-12-23 | 江苏大学 | Method for culturing escherichia coli |
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Application publication date: 20160113 |