CN108277257A - A kind of culture medium - Google Patents
A kind of culture medium Download PDFInfo
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- CN108277257A CN108277257A CN201710600643.3A CN201710600643A CN108277257A CN 108277257 A CN108277257 A CN 108277257A CN 201710600643 A CN201710600643 A CN 201710600643A CN 108277257 A CN108277257 A CN 108277257A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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Abstract
The present invention relates to bioengineering field, more particularly to a kind of culture medium.The culture medium is by cattle heart extract, bovine brain extract, cysteine, Na2HPO4, peptone, NaCl and agar powder composition.So that the culture susceptibility of HP can reach 98.88%, specificity 100%, improves the application of HP cultural methods at the shortcomings that greatly improved the prior art.
Description
Technical field
The present invention relates to bioengineering field, more particularly to a kind of culture medium.
Background technology
Gastritis (gastritis) is stomach lining inflammation caused by a variety of causes, is one of most common disease of digestive system.
By the emergency of clinical onset, acute and chronic gastritis two major classes type generally can be divided into；It can be divided into helicobacter pylori by cause of disease difference
Gastritis, irritability gastritis, autoimmune gastritis etc..Its pathology of gastritis caused by different pathogeny and Histological change are also
Difference generally includes three processes i.e. epithelial damage, mucosal inflammation reaction and epithelium regeneration.Acute gastritis is according to its pathological change
Pure, erosive hemorrhagic, corrosivity, purulent gastritis etc. can be divided into again, chronic gastritis can be divided into non-according to its pathological change
Atrophic, atrophic and specific type gastritis three categories.The diagnosis and differential diagnosis Main Basiss gastrocopy of various gastritis.Its
In, the main pathogenic bacteria of chronic gastritis is helicobacter pylori, and 90% or more Patients with Chronic Gastritis has helicobacter pylori infections.
Helicobacter pylori is a kind of pathogen being colonized in gastric mucosa, and world wide is interior, and there are about the up to senses of 50%-70%
Dye rate.The main reason for HP infection is chronic gastritis.Long-term HP infection simultaneously can also cause serious atrophic gastritis, adjoint
Property intestinal metaplasia, it is serious to lead to gastric cancer.Include at present a variety of methods for the detection of HP infection：Histopathology and
Immunohistochemistry, rapid urease test, tissue bacterial culture, urea breath test, the detection of excrement HP antigens and serology are anti-
Physical examination survey etc., but still without a kind of absolute goldstandard detection method.
The culture of tissue bacterial is still a kind of effective ways that can directly judge HP infection, and due to its high specific
And its important function in the directing study of antibiotic resistance, make it in research HP infection in occupation of important position
It sets.The culture of helicobacter pylori (H.Pylori, HP) is to diagnose a highly important technology for whether infecting helicobacter pylori
Means, but at present by technology restriction, this kind of method is relatively low to the culture positive rate of HP, and sensibility is inadequate, however at present to HP's
The existing maximum defect of culture is exactly that sensibility is relatively low, and the positive rate generally cultivated only has 50-70%, is even extremely having
Under the laboratory of experience and the sampling and culture of operating personnel, positive rate can be increased between 70-90% (with immune group reluctantly
It is turned to the standard of reference).
Invention content
In view of this, the present invention provides a kind of culture medium.Present invention is generally directed to the low defects of the positive rate of culture, prepare
A kind of advantage culture medium so that the culture positive rate of HP has reached 98.88%, and specificity 100% greatly improved existing
The shortcomings that technology, improves the application of HP cultural methods.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of basal mediums, by cattle heart extract, bovine brain extract, cysteine, Na2HPO4, egg
White peptone, NaCl and agar powder composition.
In some specific embodiments, in basal medium, in terms of g/g/g/g/g/mL/mL, cysteine,
Na2HPO4, peptone, NaCl, agar and cattle heart extract, bovine brain extract mass volume ratio be (1~5):(10~12):
(4~8):(0.5~2):(25~30):250:200.
In some specific embodiments, contain per 1000mL agar plates：
In some specific embodiments, contain per 1000mL agar plates：
The present invention also provides application of the basal medium in cultivating helicobacter pylori.
The present invention also provides a kind of complete mediums, including the basal medium, antibiotic and animal blood.
In some specific embodiments of the present invention, the antibiotic includes vancomycin, methoxybenzyl aminopyrimidine, both sexes
Mixture more than one or both of mycin or polymyxins.
In some specific embodiments of the present invention, cysteine and vancomycin, methoxybenzyl aminopyrimidine, anphotericin
Mass ratio with polymyxins is (1000~5000):(9~12):(3~6):(3~6):(3~6).
The present invention also provides application of the complete medium in cultivating helicobacter pylori.
The present invention also provides the preparation methods of the complete medium, include the following steps：
Step 1：Take cattle heart extract, bovine brain extract, cysteine, peptone, sodium chloride, the phosphoric acid hydrogen two of formula ratio
Sodium is mixed with water, is dissolved by heating, filtering and collecting filter liquid, and adjustment pH value is 7.1~7.3, is dispensed, and sterilizing seals after cooling,
Basal medium is made；
Step 2：Basal medium made from step 1 is taken, the agar powder of formula ratio is added, is sterilized；
Step 3：45 DEG C~50 DEG C are cooled to, the antibiotic of formula ratio is added；
Step 4：The animal blood (preferably fresh sheep blood) of 10% (V/V) is added, is down flat plate after mixing, after cooled and solidified
Sealing.
Specifically, the preparation method of the complete medium, includes the following steps：
It after mixing, dissolves by heating, is then filtered with filter paper, adjustment pH value to 7.2 ± 0.1 dispenses 800mL/ bottles, Yu Gao
High pressure sterilization in steam sterilization pan, 121 DEG C of sterilizing 20min is pressed to be sealed after being cooled to room temperature, 4 DEG C save backup.
When preparing plate：
1) basal medium for taking a certain amount of above 4 DEG C of preservations, agar is added by the amount that 25~30g agar powders are added per 1L
Powder, then 121 DEG C, high pressure sterilization 20min；
2) wait for that culture medium temperature is down to 45 DEG C -50 DEG C or so, according to every 1L culture mediums be added 9~12mg vancomycins, 3~
The stoste of various antibiotic is added in the concentration of 6mg anphotericins, 3~6mg polymyxins and 3~6mg methoxy ammonia benzyl pyrimidines；
3) add the amount of the fresh sheep blood of 100mL that fresh sheep blood is added by every 1L culture mediums；
4) plate is down flat after mixing well；(tablet of each diameter 90mm needs 20mL or so culture mediums, pays attention to avoiding producing
Anger bubble, flatbed horizontal is placed, be swift in motion after getting well, in case fluid nutrient medium solidification can not be down flat plate.)
5) after waiting for tablet cooled and solidified, sterile sealing bag, vacuum sealing, 4 DEG C of storages are packed into.
Detection method：
1. preparing before experiment：Culture medium needed for taking-up restores to room temperature (18 DEG C -25 DEG C).
2. direct view under endoscope, pincers take different parts mucosa tissue.
3. on superclean bench, the mucosa tissue face removed coating is directly seeded to HP selective separation cultures
Base.
4. culture medium is placed in anaerobic jar, it is slowly injected into and mixes gas (5%O2, 10%CO2, 85%N2), 37 DEG C are continuous
Culture 5~7 days；
5. observing colonial morphology.
Preferably, the preparation method of the complete medium, includes the following steps：
It after mixing, dissolves by heating, is then filtered with filter paper, adjustment pH value to 7.2 ± 0.1 dispenses 800mL/ bottles, Yu Gao
High pressure sterilization in steam sterilization pan, 121 DEG C of sterilizing 20min is pressed to be sealed after being cooled to room temperature, 4 DEG C save backup.
When preparing plate：
6) basal medium for taking a certain amount of above 4 DEG C of preservations, agar powder is added by the amount that 25g agar powders are added per 1L,
Then 121 DEG C, high pressure sterilization 20min；
7) it waits for that culture medium temperature is down to 45 DEG C -50 DEG C or so, 10mg vancomycins, 5mg two is added according to every 1L culture mediums
The stoste of various antibiotic is added in the concentration of property mycin, 5mg polymyxins and 5mg methoxy ammonia benzyl pyrimidines；
8) add the amount of the fresh sheep blood of 100mL that fresh sheep blood is added by every 1L culture mediums；
9) plate is down flat after mixing well；(tablet of each diameter 90mm needs 20mL or so culture mediums, pays attention to avoiding producing
Anger bubble, flatbed horizontal is placed, be swift in motion after getting well, in case fluid nutrient medium solidification can not be down flat plate.)
10) after waiting for tablet cooled and solidified, sterile sealing bag, vacuum sealing, 4 DEG C of storages are packed into.
Detection method：
1. preparing before experiment：Culture medium needed for taking-up restores to room temperature (18 DEG C -25 DEG C).
2. direct view under endoscope, pincers take different parts mucosa tissue.
3. on superclean bench, the mucosa tissue face removed coating is directly seeded to HP selective separation cultures
Base.
4. culture medium is placed in anaerobic jar, it is slowly injected into and mixes gas (5%O2, 10%CO2, 85%N2), 37 DEG C are continuous
Culture 5~7 days；
5. observing colonial morphology.
The present invention is prepared for a kind of culture medium for the low defect of positive rate of heiicobacter pylori cultivation, the culture medium by
Cattle heart extract, bovine brain extract, cysteine, Na2HPO4, peptone, NaCl and agar powder composition, while being added and matching in right amount
The antibiotic and fresh sheep blood of side.So that the culture susceptibility of HP can reach 98.88%, specificity 100% greatly improved
The shortcomings that prior art, improves the application of HP cultural methods.
Specific implementation mode
The invention discloses a kind of culture medium, those skilled in the art can use for reference present disclosure, be suitably modified technique ginseng
Number is realized.In particular, it should be pointed out that all similar substitutions and modifications are apparent to those skilled in the art,
They are considered as being included in the present invention.The method of the present invention and application are described by preferred embodiment, related
Personnel can obviously not depart from the content of present invention, be modified to method described herein and application in spirit and scope or suitably
It changes and combines, to realize and apply the technology of the present invention.
Raw materials used and reagent is available on the market in a kind of culture medium provided by the invention.
With reference to embodiment, the present invention is further explained：
Embodiment 1
Cultivate the culture medium prescription of helicobacter pylori：
Contain per 1000mL agar plates：
Specific preparation steps：
By formula：
It after mixing, dissolves by heating, is then filtered with filter paper, adjustment pH value to 7.2 ± 0.1 dispenses 800mL/ bottles, Yu Gao
High pressure sterilization in steam sterilization pan, 121 DEG C of sterilizing 20min is pressed to be sealed after being cooled to room temperature, 4 DEG C save backup.
When preparing plate：
1) basal medium for taking a certain amount of above 4 DEG C of preservations, agar powder is added by the amount that 25g agar powders are added per 1L,
Then 121 DEG C, high pressure sterilization 20min；
2) it waits for that culture medium temperature is down to 45 DEG C -50 DEG C or so, 12mg vancomycins, 6mg two is added according to every 1L culture mediums
The stoste of various antibiotic is added in the concentration of property mycin, 3mg polymyxins and 3mg methoxy ammonia benzyl pyrimidines；
3) add the amount of the fresh sheep blood of 100mL that fresh sheep blood is added by every 1L culture mediums；
4) plate is down flat after mixing well；(tablet of each diameter 90mm needs 20mL or so culture mediums, pays attention to avoiding producing
Anger bubble, flatbed horizontal is placed, be swift in motion after getting well, in case fluid nutrient medium solidification can not be down flat plate.)
5) after waiting for tablet cooled and solidified, sterile sealing bag, vacuum sealing, 4 DEG C of storages are packed into.
Embodiment 2
Cultivate the culture medium prescription of helicobacter pylori：
Contain per 1000mL agar plates：
Specific preparation steps：
By formula：
It after mixing, dissolves by heating, is then filtered with filter paper, adjustment pH value to 7.2 ± 0.1 dispenses 800mL/ bottles, Yu Gao
High pressure sterilization in steam sterilization pan, 121 DEG C of sterilizing 20min is pressed to be sealed after being cooled to room temperature, 4 DEG C save backup.
When preparing plate：
1) basal medium for taking a certain amount of above 4 DEG C of preservations, agar powder is added by the amount that 25g agar powders are added per 1L,
Then 121 DEG C, high pressure sterilization 20min；
2) it waits for that culture medium temperature is down to 45 DEG C -50 DEG C or so, 10mg vancomycins, 5mg two is added according to every 1L culture mediums
The stoste of various antibiotic is added in the concentration of property mycin, 5mg polymyxins and 5mg methoxy ammonia benzyl pyrimidines；
3) add the amount of the fresh sheep blood of 100mL that fresh sheep blood is added by every 1L culture mediums；
4) plate is down flat after mixing well；(tablet of each diameter 90mm needs 20mL or so culture mediums, pays attention to avoiding producing
Anger bubble, flatbed horizontal is placed, be swift in motion after getting well, in case fluid nutrient medium solidification can not be down flat plate.)
5) after waiting for tablet cooled and solidified, sterile sealing bag, sealing, 4 DEG C of storages are packed into.
Embodiment 3
Cultivate the culture medium prescription of helicobacter pylori：
Contain per 1000mL agar plates：
Specific preparation steps：
By formula：
It after mixing, dissolves by heating, is then filtered with filter paper, adjustment pH value to 7.2 ± 0.1 dispenses 800mL/ bottles, Yu Gao
High pressure sterilization in steam sterilization pan, 121 DEG C of sterilizing 20min is pressed to be sealed after being cooled to room temperature, 4 DEG C save backup.
When preparing plate：
1) basal medium for taking a certain amount of above 4 DEG C of preservations, agar powder is added by the amount that 30g agar powders are added per 1L,
Then 121 DEG C, high pressure sterilization 20min；
2) it waits for that culture medium temperature is down to 45 DEG C -50 DEG C or so, 9mg vancomycins, 3mg both sexes is added according to every 1L culture mediums
The stoste of various antibiotic is added in the concentration of mycin, 6mg polymyxins and 6mg methoxy ammonia benzyl pyrimidines；
3) add the amount of the fresh sheep blood of 100mL that fresh sheep blood is added by every 1L culture mediums；
4) plate is down flat after mixing well；(tablet of each diameter 90mm needs 20mL or so culture mediums, pays attention to avoiding producing
Anger bubble, flatbed horizontal is placed, be swift in motion after getting well, in case fluid nutrient medium solidification can not be down flat plate.)
5) after waiting for tablet cooled and solidified, sterile sealing bag, vacuum sealing, 4 DEG C of storages are packed into.
Embodiment 4
Clinical sample：The positive and the negative patient of helicobacter pylori infections are confirmed as in gastrocopy, and in direct view under endoscope,
Clamp the stomach mucosa tissue samples taken.
Experimental method：
Prepare before experiment：The culture medium that the embodiment of the present invention 1~3 provides is taken out, is restored to room temperature (18 DEG C -25 DEG C).
Direct view under endoscope, pincers take different parts mucosa tissue.
On superclean bench, the mucosa tissue face removed coating is directly seeded to HP Pseudonocardias.
Be placed in anaerobic jar, be slowly injected into and mix gas (5%O2, 10%CO2, 85%N2), 37 DEG C are continuously cultivated 5-7 days；
Observe cultivation results：It can determine whether as helicobacter pylori if growing tip-like, canescence, translucent tiny bacterium colony
Infection is positive, negative for helicobacter pylori infections if continuous culture does not have germy growth for 7 days.
Data analysis：
The knot of the 89 positive clinical samples of medium culture provided with the embodiment of the present invention 2 and 86 negative clinical samples
Fruit is shown in Table 1.
Table 1
It is detected as goldstandard with gastroscope, the result positive rate of medium culture of the invention is 98.88%, i.e. sensibility can
Up to 98.88%, the cultivation results of negative sample are consistent with gastroscope testing result, i.e., specificity is up to 100%.
The culture medium prepared with embodiment 1, embodiment 3 carries out above-mentioned experiment, the culture that experimental result is prepared with embodiment 2
The testing result of base is without significant difference (P ＞ 0.05).
In summary experiment conclusion, culture medium provided by the invention are up to the culture susceptibility of the HP of clinical sample
98.88%；Its specificity reaches 100%.
Embodiment 5
Control group：SKIRROW culture mediums, Brain Heart Infusion (7% calf serums of BHI w ith), brother's human relations
Than sub- blood agar culture-medium etc.；
Experimental group：Culture medium prepared by 1~embodiment of the embodiment of the present invention 3.
It is control with Columbia Blood Agar culture medium, compared the HP for the culture medium that it is prepared with the embodiment of the present invention 2
Blood agar culture-medium is shown in Table 2 to the cultivation results of above-mentioned 175 clinical samples：
Table 2
It can be seen that from contrast and experiment, the culture susceptibility of HP blood agar culture-mediums of the invention to the HP of clinical sample
Up to 98.88%, greatly it is higher than the susceptibility (70.79%) of control medium；Its specificity 100% is also cultivated than control
The specificity (98.84%) of base is more preferable.
SKIRROW culture mediums, Brain Heart Infusion (7% calf serums of BHI with) is taken to carry out above-mentioned reality
It tests, experimental result is with Columbia Blood Agar culture medium without significant difference (P ＞ 0.05).
The culture medium prepared with embodiment 1, embodiment 3 carries out above-mentioned experiment, the culture that experimental result is prepared with embodiment 2
The testing result of base is without significant difference (P ＞ 0.05).
In summary the experimental results showed that, culture medium provided by the invention and SKIRROW culture mediums, Brain Heart
Infusion (7% calf serums of BHI with), Columbia Blood Agar culture medium are compared, and can significantly (P ＜ 0.05) be improved
Susceptibility is up to 98.88% to the susceptibility that is separately cultured of clinical sample HP, is significantly higher than control medium；Its specificity
100%, also significantly improved (P ＜ 0.05) than the specificity of control medium (98.84%).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of basal medium, which is characterized in that by cattle heart extract, bovine brain extract, cysteine, Na2HPO4, albumen
Peptone, NaCl and agar powder composition.
2. basal medium according to claim 1, which is characterized in that in terms of g/g/g/g/g/mL/mL, cysteine,
Na2HPO4, peptone, NaCl, agar and cattle heart extract, bovine brain extract mass volume ratio be (1~5):(10~12):
(4~8):(0.5~2):(25~30):250:200.
3. basal medium according to claim 1 or 2, which is characterized in that contain per 1000mL culture mediums：
4. basal medium according to any one of claims 1 to 3, which is characterized in that contain per 1000mL culture mediums：
5. application of the basal medium according to any one of claims 1 to 4 in cultivating helicobacter pylori.
6. a kind of complete medium, which is characterized in that including basal medium as described in any one of claims 1 to 3, antibiosis
Element and animal blood.
7. complete medium according to claim 6, which is characterized in that the antibiotic includes vancomycin, methoxy benzyl
Mixture more than one or both of Aminometradine, anphotericin or polymyxins.
8. the complete medium described according to claim 6 or 7, which is characterized in that cysteine and vancomycin, methoxy benzyl ammonia
Pyrimidine, anphotericin and the mass ratio of polymyxins are (1000~5000):(9~12):(3~6):(3~6):(3~6).
9. according to application of claim 6 to 8 any one of them complete medium in cultivating helicobacter pylori.
10. according to the preparation method of claim 6 to 8 any one of them complete medium, which is characterized in that including walking as follows
Suddenly：
Step 1：Cattle heart extract, bovine brain extract, cysteine, peptone, sodium chloride, the disodium hydrogen phosphate of formula ratio are taken,
It mixes, dissolves by heating, filtering and collecting filter liquid with water, adjustment pH value is 7.1~7.3, is dispensed, and sterilizing seals after cooling, is made
Basal medium；
Step 2：Basal medium made from step 1 is taken, the agar powder of formula ratio is added, is sterilized；
Step 3：45 DEG C~50 DEG C are cooled to, the antibiotic of formula ratio is added；
Step 4：The animal blood of 10% (V/V) is added, plate is down flat after mixing, is sealed after cooled and solidified.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710600643.3A CN108277257B (en) | 2017-07-21 | 2017-07-21 | Culture medium |
| PCT/CN2018/081701 WO2019015357A1 (en) | 2017-07-21 | 2018-04-03 | Medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710600643.3A CN108277257B (en) | 2017-07-21 | 2017-07-21 | Culture medium |
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| CN108277257A true CN108277257A (en) | 2018-07-13 |
| CN108277257B CN108277257B (en) | 2021-06-22 |
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| CN201710600643.3A Active CN108277257B (en) | 2017-07-21 | 2017-07-21 | Culture medium |
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| CN (1) | CN108277257B (en) |
| WO (1) | WO2019015357A1 (en) |
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| CN101659979A (en) * | 2008-08-25 | 2010-03-03 | 薄涛 | Brain-heart-infusion neutral nutrient medium |
| CN102888442A (en) * | 2011-07-20 | 2013-01-23 | 农高惠 | Kit for rapid culture, identification and drug sensitivity of gastric helicobacter pylori and inspection method thereof |
| CN104195086A (en) * | 2014-08-27 | 2014-12-10 | 博奥生物集团有限公司 | Composite enrichment medium for five bacteria and preparation method for composite enrichment medium |
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|---|---|---|---|---|
| CN1077750A (en) * | 1993-05-25 | 1993-10-27 | 海拉尔市卫生防疫站 | Food micro-organism detecting plate and preparation thereof and application |
| JP4811760B2 (en) * | 2004-12-27 | 2011-11-09 | 独立行政法人農業・食品産業技術総合研究機構 | Enterobacteria and its utilization to improve equol production by utilization of daidzein |
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2017
- 2017-07-21 CN CN201710600643.3A patent/CN108277257B/en active Active
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2018
- 2018-04-03 WO PCT/CN2018/081701 patent/WO2019015357A1/en active Application Filing
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| CN101659979A (en) * | 2008-08-25 | 2010-03-03 | 薄涛 | Brain-heart-infusion neutral nutrient medium |
| CN102888442A (en) * | 2011-07-20 | 2013-01-23 | 农高惠 | Kit for rapid culture, identification and drug sensitivity of gastric helicobacter pylori and inspection method thereof |
| CN104195086A (en) * | 2014-08-27 | 2014-12-10 | 博奥生物集团有限公司 | Composite enrichment medium for five bacteria and preparation method for composite enrichment medium |
| CN106635862A (en) * | 2015-10-30 | 2017-05-10 | 杭州致远医学检验所有限公司 | Isolation medium for helicobacter pylori |
| CN105820975A (en) * | 2016-03-25 | 2016-08-03 | 美利泰格诊断试剂(嘉兴)有限公司 | Oral cavity helicobacter pylori culture method |
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| Publication number | Publication date |
|---|---|
| WO2019015357A1 (en) | 2019-01-24 |
| CN108277257B (en) | 2021-06-22 |
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