CN102409076A - Solid culture medium for quick detection of mycoplasma, and preparation method thereof - Google Patents
Solid culture medium for quick detection of mycoplasma, and preparation method thereof Download PDFInfo
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- CN102409076A CN102409076A CN2011104290739A CN201110429073A CN102409076A CN 102409076 A CN102409076 A CN 102409076A CN 2011104290739 A CN2011104290739 A CN 2011104290739A CN 201110429073 A CN201110429073 A CN 201110429073A CN 102409076 A CN102409076 A CN 102409076A
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Abstract
The invention discloses a solid culture medium for quick detection of mycoplasma, and a preparation method thereof. By using the solid culture medium disclosed by the invention, the existence of ureaplasma urealyticum and mycoplasma hominis can be accurately detected, and the content of mycoplasmas can be quantitatively concluded by colony counting, thus greatly improving the accuracy and reliability. Simultaneously, by using the culture medium disclosed by the invention for detecting mycoplasmas, the result can be read after only 18 hours, thereby realizing quick detection of mycoplasmas, and finally confirming diagnosis of urogenital tract infection caused by ureaplasma urealyticum and mycoplasma hominis.
Description
Technical field
The present invention relates to a kind of mycoplasma culture medium and preparation method thereof, solid medium of particularly a kind of rapid detection mycoplasma and preparation method thereof.
Background technology
The urogenital infections that ureaplasma urealyticum (UU) and mycoplasma hominis (MH) cause accounts for more than 1/3 of urogenital infections, and the detection of ureaplasma urealyticum and mycoplasma hominis has great importance clinically.
The method that detects ureaplasma urealyticum and mycoplasma hominis has specific antibody detection method, PCR method, Southern Blot method and isolated culture etc.Cross reaction appears in immunological detection method easily, and false negative and false positive appear in PCR method easily, and Southern Blot method is had relatively high expectations to experimental technique, is not suitable in hospital, using.Detection method commonly used clinically is an isolated culture.Domestic practical application and research of cultivating about the urogenital tract mycoplasma shows that liquid culture method is mainly adopted in the evaluation of mycoplasma.Though liquid culture method judgement easy to use, quick, easy, this method can only infer whether mycoplasma exists from the change of liquid color, and false positive rate is high, and accuracy is not high.And the solid culture rule can arrive the bacterium colony of mycoplasma hominis and ureaplasma urealyticum through microscopic examination, thereby the existence of conclusive evidence mycoplasma solves the high problem of clinical samples false positive rate preferably.But the time of plate method sentence read result is longer, thereby needs the time could make a definite diagnosis the urinary tract RTI that UU and MH cause than length, is unfavorable for the diagnosis and the treatment of disease.
Summary of the invention
The object of the present invention is to provide solid medium of a kind of rapid detection mycoplasma and preparation method thereof.
The technical scheme that the present invention adopted is:
A kind of solid medium of rapid detection mycoplasma, it is filled a prescription as follows:
Preferably, the peptone in the above-mentioned substratum is soy peptone and casein peptone.
Preferably, the microbiotic in the above-mentioned substratum is the mixing microbiotic of amphotericin B, PXB and Veticillin.
Preferably, above-mentioned substratum also is added with potassium primary phosphate.
Preferably, the addition of potassium primary phosphate is 2g～6g/L zero(ppm) water in the above-mentioned substratum.
A kind of method for preparing the solid medium of above-mentioned rapid detection mycoplasma may further comprise the steps:
(1) agar powder is dissolved in the zero(ppm) water, is placed in the warm water through autoclaving, subsequent use;
(2) special peptone and glucose are added in the entry, after the heating for dissolving, be cooled to room temperature, add HEPES buffer reagent, L-halfcystine, l-arginine, urea and RPMI1640 cell culture medium, mixing, after waiting to dissolve, impurity screening;
(3) pH value with above-mentioned solution transfers to 6.0 ± 0.2, and filtration sterilization is subsequent use;
(4) horse serum is added in the solution of above-mentioned degerming, mixing is put into the warm water rewarming;
(5) above-mentioned solution is mixed with sterilized agar, add manganous sulfate and microbiotic, shake up, be poured in the aseptic plate, treat that it solidifies.
The invention has the beneficial effects as follows:
Use solid medium of the present invention optionally to cultivate ureaplasma urealyticum and mycoplasma hominis; Prove conclusively the existence of mycoplasma through the formation of bacterium colony; And can judge the type of mycoplasma from colonial morphology; Therefore, with respect to the false-positive situation of the normal appearance of liquid nutrient medium, solid medium of the present invention improves in the accuracy aspect the diagnosis urogenital infections greatly.Use solid medium of the present invention can also directly infer the real content of mycoplasma in the sample through enumeration; Accurately judging sample from quantitative angle is in carrier state or has reached pathogenic concentration; And use liquid nutrient medium can only judge the existence of mycoplasma qualitatively, thereby the accuracy of detected result of the present invention is further enhanced.In addition; On mycoplasma bacterium colony formation time, the time of interpretation as a result of solid medium is 24 hours at present, and uses mycoplasma culture medium sentence read result of the present invention only to need 18 hours; Shortened detection time greatly; Realized the rapid detection of ureaplasma urealyticum and mycoplasma hominis, and formed bacterium colony is more much bigger than the bacterium colony that on commercially available substratum, forms, the rapid and reliable assay is provided for the doctor works out the proper treatment scheme.
Description of drawings
Fig. 1 is the bacterium colony (amplifying 400 times) that ureaplasma urealyticum forms on solid medium of the present invention is cultivated;
Fig. 2 is the bacterium colony (amplifying 400 times) that mycoplasma hominis forms on solid medium of the present invention;
Fig. 3 is the bacterium colony (amplifying 400 times) that ureaplasma urealyticum and mycoplasma hominis form on solid medium of the present invention;
Fig. 4 is the bacterium colony that ureaplasma urealyticum forms on commercially available substratum among the embodiment 5 (amplifying 400 times).
Embodiment
The invention provides a kind of mycoplasma culture medium and preparation method thereof, this substratum can detect ureaplasma urealyticum and mycoplasma hominis quickly and accurately.
A kind of solid medium of rapid detection mycoplasma, it is filled a prescription as follows:
Preferably, the peptone in this substratum is soy peptone and casein peptone.
Preferably, the microbiotic in this substratum is the mixing microbiotic of amphotericin B, PXB and Veticillin.
Preferably, this substratum also is added with potassium primary phosphate.
Preferably, the addition of potassium primary phosphate is 2g～6g/L zero(ppm) water in this substratum.
The method of domestic detection mycoplasma commonly used is a liquid culture, can only infer that whether mycoplasma exists from the change of liquid color, and false positive rate is high, and accuracy is not high.Mycoplasma solid medium of the present invention can optionally be cultivated ureaplasma urealyticum and mycoplasma hominis, is used to diagnose urogenital infections, and accuracy is high.Can observe the brownish black sea urchin shape bacterium colony (as shown in Figure 1) of ureaplasma urealyticum on the solid medium and the fried egg shape bacterium colony (as shown in Figure 2) of mycoplasma hominis through microscope, thus the existence of conclusive evidence mycoplasma.In addition, this substratum contains rich nutrient substances, mixes microbiotic and developer.Ureaplasma urealyticum can make the manganous sulfate oxidation in the substratum, thereby makes its colony colour become coffee to black; Mycoplasma hominis then can not make the manganous sulfate oxidation, thereby its bacterium colony does not develop the color.Through different (as shown in Figure 3) of colonial morphology and color, can accurately clearly differentiate the mycoplasma type.Simultaneously, can infer the real content (CFU) of mycoplasma in the sample, judge that accurately sample is carrier state or has reached pathogenic concentration through enumeration.This substratum has solved the high problem of clinical samples false positive rate, also from before qualitative detection upgrading become detection by quantitative.Simultaneously, also shorten greatly detection time, and in general, using the interpretation time of solid medium is 24 hours, and uses mycoplasma culture medium sentence read result of the present invention only to need 18 hours.Therefore, solid medium of the present invention has rapid detection and result characteristics accurately and reliably.
A kind of method for preparing the solid medium of above-mentioned rapid detection mycoplasma may further comprise the steps:
(1) agar powder is dissolved in the zero(ppm) water, is placed in the warm water through autoclaving, subsequent use;
(2) peptone and glucose are added in the nutritive medium, after the heating for dissolving, be cooled to room temperature, add HEPES buffer reagent, L-halfcystine, l-arginine, urea and RPMI1640 cell culture medium, mixing, after waiting to dissolve, impurity screening;
(3) pH value with above-mentioned solution transfers to 6.0 ± 0.2, and filtration sterilization is subsequent use;
(4) horse serum is added in the solution of above-mentioned degerming, mixing is put into the warm water rewarming;
(5) above-mentioned solution is mixed with sterilized agar, add manganous sulfate and microbiotic, shake up, be poured in the aseptic plate, treat that it solidifies.
Further specify the present invention below in conjunction with embodiment:
Embodiment 1
A kind of solid medium of rapid detection mycoplasma, its component is following:
Embodiment 2
A kind of solid medium of rapid detection mycoplasma, its component is following:
Embodiment 3
A kind of solid medium of rapid detection mycoplasma, its component is following:
Embodiment 4
A kind of method for preparing the solid medium of embodiment 1 may further comprise the steps:
(1) agar powder is dissolved in the zero(ppm) water,, remains on after the cooling in 50 ℃ the warm water through 121 ℃ of HP steams sterilization 15min, subsequent use;
(2) peptone, glucose and potassium primary phosphate are added in the entry, after the heating for dissolving, be cooled to room temperature, add HEPES buffer reagent, L-halfcystine, l-arginine, urea and RPMI1640 cell culture medium, mixing after waiting to dissolve, is used filter paper filtering impurity;
(3) pH value with above-mentioned solution transfers to 6.0, and is through 0.22 μ m filtration sterilization, subsequent use;
(4) under aseptic technique, serum is added in the solution of above-mentioned degerming, mixing is put into 50 ℃ warm water rewarming;
(5) treat solution rewarming to 50 ℃ after, said solution and sterilized agar are mixed under aseptic condition, add manganous sulfate and mix microbiotic, shake up, be poured in the aseptic plate, treat that it solidifies;
(6) solidifying the back, to place 4 ℃ of stored refrigerated subsequent use.
Embodiment 5
The positive sample of same concentrations is seeded on the solid medium that makes among the embodiment 4 and the commercially available substratum cultivates and observe, the result is as shown in the table:
Visible from table, use the substratum that makes among the embodiment 4 can make the time of interpretation as a result shorten 6h, rapid detection ureaplasma urealyticum and mycoplasma hominis.In addition, owing to increased carbon source in the substratum, ureaplasma urealyticum decomposes carbon source when growth, make the bacterium colony size of ureaplasma urealyticum increase greatly, more helps the observation of ureaplasma urealyticum.
Claims (6)
1. the solid medium of a rapid detection mycoplasma, it is characterized in that: it is filled a prescription as follows:
2. the solid medium of a kind of rapid detection mycoplasma according to claim 1 is characterized in that: said peptone is soy peptone and casein peptone.
3. the solid medium of a kind of rapid detection mycoplasma according to claim 1 is characterized in that: said microbiotic is the mixing microbiotic of amphotericin B, PXB and Veticillin.
4. the solid medium of a kind of rapid detection mycoplasma according to claim 1 is characterized in that: said substratum also is added with potassium primary phosphate.
5. the solid medium of a kind of rapid detection mycoplasma according to claim 2 is characterized in that: the addition of said potassium primary phosphate is 2g～6g/L zero(ppm) water.
6. method for preparing the solid medium of claim 1 rapid detection mycoplasma may further comprise the steps:
(1) agar powder is dissolved in the zero(ppm) water, is placed in the warm water through autoclaving, subsequent use;
(2) peptone and glucose are added in the entry, after the heating for dissolving, be cooled to room temperature, add HEPES buffer reagent, L-halfcystine, l-arginine, urea and RPMI1640 cell culture medium, mixing, after waiting to dissolve, impurity screening;
(3) pH value with above-mentioned solution transfers to 6.0 ± 0.2, and filtration sterilization is subsequent use;
(4) horse serum is added in the solution of above-mentioned degerming, mixing is put into the warm water rewarming;
(5) above-mentioned solution is mixed with sterilized agar, add manganous sulfate and microbiotic, shake up, be poured in the aseptic plate, treat that it solidifies.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694425A (en) * | 2015-02-12 | 2015-06-10 | 众爱生河北生物科技有限公司 | Isolation medium of genitourinary tract mycoplasma Un and Mh and application of Un and Mh isolation medium |
| CN105331671A (en) * | 2015-11-20 | 2016-02-17 | 刘慧� | Rapid culture method and culture medium of ureaplasma urealyticum |
| CN106282300A (en) * | 2015-05-26 | 2017-01-04 | 临沂大学 | A kind of chicken virus mycoplasma detection method |
| CN109385381A (en) * | 2018-11-16 | 2019-02-26 | 山东恒辰生物科技有限公司 | A kind of Urogenital Mycoplasma biphasic culture |
| CN111304279A (en) * | 2020-03-09 | 2020-06-19 | 珠海市银科医学工程股份有限公司 | Mycoplasma identification agar culture medium and preparation method thereof |
-
2011
- 2011-12-16 CN CN 201110429073 patent/CN102409076B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 《湖南师范大学学报(医学版)》 20071231 易宙进等 "固体培养基分离解脲支原体和人型支原体探讨" 47-49 1-6 第4卷, 第3期 * |
| 易宙进等: ""固体培养基分离解脲支原体和人型支原体探讨"", 《湖南师范大学学报(医学版)》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694425A (en) * | 2015-02-12 | 2015-06-10 | 众爱生河北生物科技有限公司 | Isolation medium of genitourinary tract mycoplasma Un and Mh and application of Un and Mh isolation medium |
| CN106282300A (en) * | 2015-05-26 | 2017-01-04 | 临沂大学 | A kind of chicken virus mycoplasma detection method |
| CN106282300B (en) * | 2015-05-26 | 2019-12-24 | 临沂大学 | Mycoplasma gallisepticum detection method |
| CN105331671A (en) * | 2015-11-20 | 2016-02-17 | 刘慧� | Rapid culture method and culture medium of ureaplasma urealyticum |
| CN109385381A (en) * | 2018-11-16 | 2019-02-26 | 山东恒辰生物科技有限公司 | A kind of Urogenital Mycoplasma biphasic culture |
| CN109385381B (en) * | 2018-11-16 | 2019-10-08 | 山东恒辰生物科技有限公司 | A kind of Urogenital Mycoplasma biphasic culture |
| CN111304279A (en) * | 2020-03-09 | 2020-06-19 | 珠海市银科医学工程股份有限公司 | Mycoplasma identification agar culture medium and preparation method thereof |
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