CN107099481B - It is a kind of suitable for culture medium of clostridium difficile and preparation method thereof - Google Patents
It is a kind of suitable for culture medium of clostridium difficile and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of suitable for culture medium of clostridium difficile and preparation method thereof, belong to field of microbial culture technology.In order to overcome the bad technical deficiency of culture effect of the existing culture medium for clostridium difficile, it is a kind of suitable for culture medium of clostridium difficile and preparation method thereof present invention aims at providing, not only include the compounding ingredients such as peptone and dehydration beef heart infusion in the culture medium, further include the supplemental components such as glycine and laminine, test result indicates that, the culture medium is good for the culture effect of clostridium difficile, this is for studying its biological characteristics and the treatment of infection for seeking its initiation has great importance.
Description
Technical field
The present invention relates to a kind of suitable for culture medium of clostridium difficile and preparation method thereof, belong to microbial culture technique neck
Domain.
Background technology
Clostridium difficile (Clostridium difficile) is a kind of obligate anaerobe of fusobacterium, very sensitive to oxygen,
It is difficult to be separately cultured, therefore gain the name.Nineteen thirty-five is found in, but this bacterium and some antibiotic of clinical long-time service were found until 1977
Pseudomembranous enteritis caused by (ampicillin, cephalosporin, erythromycin, lindamycin etc.) is related, is just taken seriously.Difficult shuttle
Bacterium is distributed widely in natural habitat, as soil, hay, sand, some larger animals (ox, donkey and horse) excrement, and dog, cat,
Rodent and the excrement of people, are in addition also largely present in water and the enteron aisle of animal in the excrement of baby often containing difficult
Clostridium, is normal flora in neonate's enteron aisle, has clostridium difficile, more than 2 years old youngster in the enteron aisle of about 50% 12 month infants
Virgin bacterial bearing rate is about 3%, but this bacterium frequency of occurrences in health adult is relatively low, and the asymptomatic adult that carries disease germs is in Sweden
1.9%, it is 15.4% in Japan.
Clostridium difficile can trigger following disease：1. pseudomembranous enteritis (pseudomenbranous colitis, PMC)：
Clinical manifestation abruptly starts to for diarrhea, abdominal pain, with systemic toxicity profiles symptom, symptom, and low with blood pressure, can be lethal when serious.
Usually also with fever, leukocytosis, can cause death afterwards, be very serious a kind of disease.2. antibiotic associated abdomen
Rush down (antibiotic-associated diarrhoea)：Incubation period in vivo is 5-10 days, causes substantial amounts of brown afterwards
Or watery diarrhea, left and right for 1 week.In addition to above-mentioned disease, clostridium difficile still causes pyelonephritis, meningitis, abdominal cavity and the moon
Road infection, bacteremia and emphysematous gangrene etc..The bacterium is as one of pathogen of inside-hospital infection in recent years, increasingly by people institute
Pay attention to.How the suitable medium culture bacterial strain is selected for studying its biological characteristics and for seeking its sense triggered
The treatment of dye has great importance.
The prior art shows that this bacterium is stringent obligate anaerobe, is not easy to grow with conventional methods,anaerobic.Growth temperature
Spend for 25 DEG C~45 DEG C, and optimum temperature is 30 DEG C~37 DEG C.In the tablets such as blood agar, cattle heart brain infusion agar and CCFA, warp
48 it is small when culture after, 3~5mm of diameter of bacterium colony is circular, slightly raised, white or faint yellow, opaque, edge is irregular, surface
It is coarse.The not haemolysis on blood plate, does not form milkiness ring on egg yolk agar tablet.The bacterium colony grown on CCFA tablets is ultraviolet
Visible yellow-green fluorescence under line irradiation.For this bacterium through meat soup culture more than 2 days, thalline dissolved phenomenon.
The content of the invention
A kind of suitable for culture medium of clostridium difficile and preparation method thereof present invention aims at providing, it is for studying it
The treatment of biological characteristics and the infection for seeking its initiation has great importance.
The present invention is achieved through the following technical solutions above-mentioned technique effect：
A kind of culture medium suitable for clostridium difficile, it is prepared by the component of following parts by weight：Peptone 10-15
Part, small 6-8 parts of powder of bovine brain leaching is dehydrated, is dehydrated 10-15 parts of beef heart infusion, 1.0-1.5 parts of glucose, 0.1-0.5 parts of magnesium sulfate, elder brother
1.5-2.5 parts of cloth propylhomoserin, 0.01-0.03 parts of natamycin, 0.5-0.9 parts of sodium dihydrogen phosphate, 0.5-0.9 parts of glycine, dimension life
B10.3-0.5 parts plain, 800-1000 parts of purified water, pH is adjusted to 7.2-7.4.
As a kind of embodiment preferred for this invention, the component of the following parts by weight of the culture medium is prepared：
12.5 parts of peptone, is dehydrated small 7 parts of powder of bovine brain leaching, is dehydrated 12.5 parts of beef heart infusion, 1.25 parts of glucose, 0.3 part of magnesium sulfate, elder brother
2 parts of cloth propylhomoserin, 0.02 part of natamycin, 0.7 part of sodium dihydrogen phosphate, 0.7 part of glycine .4 parts of vitaminB10, purified water 900
Part, pH is adjusted to 7.3.
The preparation method of the above-mentioned culture medium of the present invention is：The purified water of recipe quantity half amount is measured, is added thereto
Peptone, dehydration small bovine brain leaching powder, dehydration beef heart infusion and glucose, stir to dissolve, add surplus purified water, Xiang Qi
Middle addition magnesium sulfate and sodium dihydrogen phosphate, adjust its pH to 7.2-7.4,121 DEG C of sterilizing 20min, after culture medium is cooled to room temperature
Glycine, laminine and vitamin thereto, up to the culture medium of clostridium difficile is suitable for.
The above-mentioned culture medium of the present invention can be used for the preservation of its bacterial strain as the culture medium of clostridium difficile, activate and
Separation.During for preservation, the agar of 1.5%-2% is further included in its culture medium, the addition time of agar adds before sterilizing.
Beneficial effect is the present invention compared with prior art：
The present invention overcomes the technical deficiency that clostridium difficile in the prior art is difficult to cultivate, the culture medium is very suitable for
The growth of clostridium difficile, in being suitable as storage medium or being screened for dominant strain.This is for research clostridium difficile
The treatment of biological characteristics and the infection for seeking its initiation has great importance.
Embodiment
The present invention is further described below by way of specific embodiment, but the embodiment does not limit this hair in any way
The scope of bright patent protection.
The present invention of embodiment 1 is suitable for the culture medium of clostridium difficile
A kind of culture medium suitable for clostridium difficile, it is prepared by the component of following parts by weight：10 parts of peptone, takes off
Water small bovine brain 6 parts of powder of leaching, is dehydrated 10 parts of beef heart infusion, 1.0 parts of glucose, 0.1 part of magnesium sulfate, 1.5 parts of laminine, receives that he is mould
0.01 part of element, 0.5 part of sodium dihydrogen phosphate, 0.5 part of glycine .3 parts of vitaminB10,800 parts of purified water, pH is adjusted to 7.2.
The preparation method of culture medium is：The purified water of recipe quantity half amount is measured, adds peptone, dehydration thereto
Small bovine brain leaching powder, dehydration beef heart infusion and glucose, stir to dissolve, add surplus purified water, add sulfuric acid thereto
Magnesium and sodium dihydrogen phosphate, adjust its pH to 7.2,121 DEG C of sterilizing 20min, glycine, elder brother thereto after culture medium is cooled to room temperature
Cloth propylhomoserin and vitamin, up to the culture medium of clostridium difficile is suitable for.
The present invention of embodiment 2 is suitable for the culture medium of clostridium difficile
A kind of culture medium suitable for clostridium difficile, it is prepared by the component of following parts by weight：15 parts of peptone, takes off
Water small bovine brain 8 parts of powder of leaching, is dehydrated 15 parts of beef heart infusion, 1.5 parts of glucose, 0.5 part of magnesium sulfate, 2.5 parts of laminine, receives that he is mould
0.03 part of element, 0.9 part of sodium dihydrogen phosphate, 0.9 part of glycine .5 parts of vitaminB10,1000 parts of purified water, pH is adjusted to 7.4.
The preparation method of culture medium is：The purified water of recipe quantity half amount is measured, adds peptone, dehydration thereto
Small bovine brain leaching powder, dehydration beef heart infusion and glucose, stir to dissolve, add surplus purified water, add sulfuric acid thereto
Magnesium and sodium dihydrogen phosphate, adjust its pH to 7.4,121 DEG C of sterilizing 20min, glycine, elder brother thereto after culture medium is cooled to room temperature
Cloth propylhomoserin and vitamin, up to the culture medium of clostridium difficile is suitable for.
The present invention of embodiment 3 is suitable for the culture medium of clostridium difficile
A kind of culture medium suitable for clostridium difficile, it is prepared by the component of following parts by weight：12.5 parts of peptone,
Small 7 parts of powder of bovine brain leaching is dehydrated, is dehydrated 12.5 parts of beef heart infusion, 1.25 parts of glucose, 0.3 part of magnesium sulfate, 2 parts of laminine, receives
His 0.02 part of mycin, 0.7 part of sodium dihydrogen phosphate, 0.7 part of glycine .4 parts of vitaminB10,900 parts of purified water, pH is adjusted to
7.3。
The preparation method of culture medium is：The purified water of recipe quantity half amount is measured, adds peptone, dehydration thereto
Small bovine brain leaching powder, dehydration beef heart infusion and glucose, stir to dissolve, add surplus purified water, add sulfuric acid thereto
Magnesium and sodium dihydrogen phosphate, adjust its pH to 7.3,121 DEG C of sterilizing 20min, glycine, elder brother thereto after culture medium is cooled to room temperature
Cloth propylhomoserin and vitamin, up to the culture medium of clostridium difficile is suitable for.
The present invention of embodiment 4 is suitable for the culture medium of clostridium difficile
A kind of culture medium suitable for clostridium difficile, it is prepared by the component of following parts by weight：10 parts of peptone, takes off
Water small bovine brain 8 parts of powder of leaching, is dehydrated 10 parts of beef heart infusion, 1.5 parts of glucose, 0.1 part of magnesium sulfate, 2.5 parts of laminine, receives that he is mould
0.01 part of element, 0.9 part of sodium dihydrogen phosphate, 0.5 part of glycine .5 parts of vitaminB10,800 parts of purified water, pH is adjusted to 7.2.
The preparation method of culture medium is：The purified water of recipe quantity half amount is measured, adds peptone, dehydration thereto
Small bovine brain leaching powder, dehydration beef heart infusion and glucose, stir to dissolve, add surplus purified water, add sulfuric acid thereto
Magnesium and sodium dihydrogen phosphate, adjust its pH to 7.2,121 DEG C of sterilizing 20min, glycine, elder brother thereto after culture medium is cooled to room temperature
Cloth propylhomoserin and vitamin, up to the culture medium of clostridium difficile is suitable for.
The present invention of embodiment 5 is suitable for the culture medium of clostridium difficile
A kind of culture medium suitable for clostridium difficile, it is prepared by the component of following parts by weight：15 parts of peptone, takes off
Water small bovine brain 6 parts of powder of leaching, is dehydrated 15 parts of beef heart infusion, 1.0 parts of glucose, 0.5 part of magnesium sulfate, 1.5 parts of laminine, receives that he is mould
0.03 part of element, 0.5 part of sodium dihydrogen phosphate, 0.9 part of glycine .3 parts of vitaminB10,1000 parts of purified water, pH is adjusted to 7.4.
The preparation method of culture medium is：The purified water of recipe quantity half amount is measured, adds peptone, dehydration thereto
Small bovine brain leaching powder, dehydration beef heart infusion and glucose, stir to dissolve, add surplus purified water, add sulfuric acid thereto
Magnesium and sodium dihydrogen phosphate, adjust its pH to 7.4,121 DEG C of sterilizing 20min, glycine, elder brother thereto after culture medium is cooled to room temperature
Cloth propylhomoserin and vitamin, up to the culture medium of clostridium difficile is suitable for.
Comparative Examples 1
In addition to laminine is not included in prescription, other prescriptions and culture medium preparation method are the same as embodiment 3.
Comparative Examples 2
In addition to natamycin is not included in prescription, other prescriptions and culture medium preparation method are the same as embodiment 3.
Comparative Examples 3
In addition to glycine and vitamin B1 is not included in prescription, other prescriptions and culture medium preparation method are the same as embodiment 3.
The present invention of experimental example 6 is suitable for the culture effect observation of the culture medium of clostridium difficile
Stool collection：Selection Shanghai Medical Univ go to a doctor patient excrement as collection of specimens object, patient age
26 years old, occur diarrhea suddenly using antibiosis extract for treating after two weeks, defecate for watery stool.Take its excrement sample, by Saving specimen in
Sample is prepared into 20% glycerin bouillon tubule, takes 5mL samples to be observed for faecal microbiota, separately takes 2mL samples to be used to cultivate
Base culture, culture medium is respectively using the culture medium described in 1- of embodiment of the present invention embodiments 5, the training described in Comparative Examples 1-3
Support base.Control medium is concurrently set, control medium uses CDMN Selective agar mediums, speeds the limited public affairs of science and technology purchased from Beijing friend's profit
Department.
Through examining, flora and ratio are clostridium difficile in patient's excrement：Escherichia coli：Streptococcus fecalis：Salmonella
Bacterium=1:50:10:8, total colony count is 5.2 × 108Cfu/mL, by different Pseudomonas ratios and bacterium after medium culture
It is as shown in table 1 to fall number.
Different Pseudomonas ratios and colony count after 1 different culture media culture fecal sample of table
As can be seen from Table 1, after culture, clostridium difficile is changed into excellent the culture medium in the present invention by weak tendency bacterial strain
Gesture bacterial strain, and colony count tool compared with before has greatly improved after culture.This shows culture medium phase of the present invention
Screened for dominant strain of the control medium more suitable for clostridium difficile, wherein the culture with 3 corresponding culture medium of embodiment
Effect is best.For Comparative Examples 1- Comparative Examples 3 in addition to constituent part is different from embodiment 3, remaining is same as Example 3,
But after the medium culture using Comparative Examples 1- Comparative Examples 3, not only overall culture clump count declines, but also difficult
The superiority of clostridium is remarkably decreased, this shows laminine, natamycin in instant component, glycine and vitamin
B1 has key effect in culture clostridium difficile and its established superiority, and embodies significant synergistic effect wherein.
Secondary culture is carried out using culture medium of the present invention and control medium, secondary culture is solid culture
Base, including 1.8% agar powder.Different algebraically measure are passed on into the time change of logarithmic phase, the change of sporulation time
Change and the change of toxin yield.
Clostridium difficile vigor changes after the different number passages of table 2
As can be seen from Table 2, as the increase of passage number, the vigor of clostridium difficile are gradually reduced, be mainly manifested in into
Entering the time lengthening of exponential phase, sporulation time lengthening, and toxin yield reduces.But culture of the present invention
The vigor rate of descent of base is significantly reduced compared with control medium, that is to say, that using culture medium of the present invention relative to existing culture
The time of rest period can be greatly lowered in base, and can maintain the Production of Toxin amount of clostridium difficile and prevent the sporulation time
Extension, wherein 3 corresponding culture medium of embodiment best results.Comparative Examples 1- Comparative Examples 3 are only in 3 base of embodiment
Constituent part is changed on plinth, but its dormancy time, toxin yield and sporulation time greatly prolong, and this shows this
Laminine, natamycin in invention component, glycine and vitamin B1 maintain its vigor in clostridium difficile preserving process
In there is key effect, and embody significant synergistic effect wherein.
Claims (5)
1. a kind of culture medium suitable for clostridium difficile, it is prepared by the component of following parts by weight：10-15 parts of peptone,
Small 6-8 parts of powder of bovine brain leaching is dehydrated, is dehydrated 10-15 parts of beef heart infusion, 1.0-1.5 parts of glucose, 0.1-0.5 parts of magnesium sulfate, elder brother
1.5-2.5 parts of cloth propylhomoserin, 0.01-0.03 parts of natamycin, 0.5-0.9 parts of sodium dihydrogen phosphate, 0.5-0.9 parts of glycine, dimension
Raw 0.3-0.5 parts of element B1,800-1000 parts of purified water, pH is adjusted to 7.2-7.4.
2. the culture medium according to claim 1 suitable for clostridium difficile, it is characterised in that the following weight of culture medium
The component of part is prepared：12.5 parts of peptone, is dehydrated small 7 parts of powder of bovine brain leaching, is dehydrated 12.5 parts of beef heart infusion, glucose
1.25 parts, 0.3 part of magnesium sulfate, 2 parts of laminine, 0.02 part of natamycin, 0.7 part of sodium dihydrogen phosphate, 0.7 part of glycine,
0.4 part of vitamin B1,900 parts of purified water, pH is adjusted to 7.3.
3. the preparation method of a kind of claim 1 or 2 culture medium suitable for clostridium difficile, it is characterised in that described
Preparation method comprises the following steps：The purified water of recipe quantity half amount is measured, adds peptone, the small bovine brain of dehydration thereto
Powder, dehydration beef heart infusion and glucose are soaked, is stirred to dissolve, adds surplus purified water, adds magnesium sulfate and phosphorus thereto
Acid dihydride sodium, adjusts its pH to 7.2-7.4,121 DEG C of sterilizing 20min, and culture medium adds thereto after being cooled to room temperature receives that he is mould
Element, glycine, laminine and vitamin B1, up to the culture medium of clostridium difficile is suitable for.
4. the culture medium suitable for clostridium difficile described in claim 1 or 2 is in the preservation of its bacterial strain, activation and separation
Purposes.
5. according to the purposes described in claim 4, it is characterised in that during for clostridium difficile preservation, further included in the culture medium
The agar of 1.5%-2%, the addition time of agar is before sterilizing.
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| CN114540238B (en) * | 2022-03-10 | 2023-08-08 | 江南大学 | Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof |
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| WO2010036826A1 (en) * | 2008-09-24 | 2010-04-01 | Sanofi Pasteur Biologics Co. | Methods and compositions for increasing toxin production |
| CN104946723A (en) * | 2015-06-05 | 2015-09-30 | 中国检验检疫科学研究院 | General medium for determination of lactobacillus resistance to drugs and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010036826A1 (en) * | 2008-09-24 | 2010-04-01 | Sanofi Pasteur Biologics Co. | Methods and compositions for increasing toxin production |
| CN104946723A (en) * | 2015-06-05 | 2015-09-30 | 中国检验检疫科学研究院 | General medium for determination of lactobacillus resistance to drugs and use thereof |
Non-Patent Citations (1)
| Title |
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| "艰难梭菌分离培养与病原学分子特征研究";金慧等;《浙江预防医学》;20140630;第26卷(第6期);第602-609页 * |
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Address after: 530031 3rd floor, B-4 office building, phase I project of Beibu Gulf science and Technology Park headquarters base, east side of Zhuangjin Avenue and south side of Guokai Avenue, Nanning City, Guangxi Zhuang Autonomous Region Patentee after: GUANGXI HUAYIN MEDICAL LABORATORY Co.,Ltd. Patentee after: Guangzhou Huayin health care group Co.,Ltd. Address before: 530031 3rd floor, B-4 office building, phase I project of Beibu Gulf science and Technology Park headquarters base, east side of Zhuangjin Avenue and south side of Guokai Avenue, Nanning City, Guangxi Zhuang Autonomous Region Patentee before: GUANGXI HUAYIN MEDICAL LABORATORY Co.,Ltd. Patentee before: GUANGZHOU HUAYIN HEALTH TECHNOLOGY Co.,Ltd. |
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Address after: 530031 3rd floor, B-4 office building, phase I project of Beibu Gulf science and Technology Park headquarters base, east side of Zhuangjin Avenue and south side of Guokai Avenue, Nanning City, Guangxi Zhuang Autonomous Region Patentee after: GUANGXI HUAYIN MEDICAL LABORATORY CO.,LTD. Patentee after: Guangzhou huayinkang Medical Group Co., Ltd Address before: 530031 3rd floor, B-4 office building, phase I project of Beibu Gulf science and Technology Park headquarters base, east side of Zhuangjin Avenue and south side of Guokai Avenue, Nanning City, Guangxi Zhuang Autonomous Region Patentee before: GUANGXI HUAYIN MEDICAL LABORATORY CO.,LTD. Patentee before: Guangzhou Huayin health care group Co., Ltd |