CN101643713B - Legionnella selective separation medium - Google Patents
Legionnella selective separation medium Download PDFInfo
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- CN101643713B CN101643713B CN2009100402827A CN200910040282A CN101643713B CN 101643713 B CN101643713 B CN 101643713B CN 2009100402827 A CN2009100402827 A CN 2009100402827A CN 200910040282 A CN200910040282 A CN 200910040282A CN 101643713 B CN101643713 B CN 101643713B
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Abstract
The invention discloses a legionnella selective separation medium. Dyes such as bromothymol blue and bromocresol purple are added to the basic formulation of the basic medium of legionnella BCYE alpha and legionnella selective antibiotics of glycin, vancocin, polymyxin B and actinone are also added. The added dyes produce pigment in the growth process of legionnella; the shape and the color of a colony are beneficial to identifying and distinguishing a target bacterium of the legionnella; the concentrations of the selective antibiotics are added and regulated, the inhibiting capability to non-legionnella and microbial class is enhanced and the inhibiting performance for the legionnella is reduced, thereby enhancing the positive rate for the separation culture of the legionnella; the detection cost is reduced and the legionnella selective separation medium is economic and practical and is convenient for popularization and application for clinical and health and epidemic prevention departments.
Description
Technical field
The present invention relates to a kind of substratum, specifically is a kind of separation and Culture that is fit to legionella in environment and the clinical samples, can reduce the legionnella selective separation medium of living contaminants, raising legionella separation and Culture positive rate.
Background technology
At present, the legionella isolation medium of generally acknowledging both at home and abroad is the BCYE α-CCVC, the BCYE α-GVPC that produce such as French biological Mei Liai (bioM é rieux), Britain OXOID, U.S. BD, DIFCO company and BCYE α-GPVA etc.
[1], these substratum cost an arm and a leg by (18～25 yuan/1 plate) mostly, when hospital clinical and health and epidemic prevention department's extensive application, detect the cost height, and legionella separation and Culture positive rate is generally 35.1%～65.0%
[2-3], positive rate is lower.So existing legionella isolation medium is unfavorable for clinical and health and epidemic prevention department applies.
Summary of the invention
The objective of the invention is deficiency, the legionnella selective separation medium that a kind of cost is low, the separation and Culture positive rate is higher is provided at the above legionella isolation medium existence.
The present invention is achieved in that legionnella selective separation medium, and the component parts by weight that it comprises are as follows:
Basal liquid:
Protein liquid:
Glycine 2.5～3.5
L-halfcystine 0.3～0.8
Albumin 0.05～0.15
Dye solution:
Purpurum bromocresolis 0.005～0.02
Tetrabromophenol sulfonphthalein 0.005～0.02
Microbiotic liquid:
Vancomycin 0.0005～0.002
PXB 0.006～0.010
Cycloheximide 0.006～0.010
Preparation process is as follows:
(1) yeast powder in the basal liquid, agar, carbon dust, ACES and a-ketoglutaric acid are placed in the container by above-mentioned parts by weight weighing, add 900～1000 parts in water, with KOH the pH value are transferred to 6.85; Boil 1 minute, and then 121 ℃ of high-temperature sterilizations 15 minutes; Ferric pyrophosphate is dissolved in 8～12 parts of water filtration sterilization; Yeast powder, agar, carbon dust, ACES and a-ketoglutaric acid behind 121 ℃ of high-temperature sterilizations are cooled to the ferric pyrophosphate solution mixing that adds filtration sterilization after 55 ℃;
(2) glycine, L-halfcystine and albumin are dissolved by adding 27～31 parts of water in the above-mentioned parts by weight weighing placement container;
(3) purpurum bromocresolis and tetrabromophenol sulfonphthalein are dissolved by adding in 8～12 parts of ethanol in the above-mentioned parts by weight weighing placement container;
(4) vancomycin, PXB and cycloheximide are dissolved by adding in 0.8～1.2 part of water in the above-mentioned parts by weight weighing placement container;
(5) solution of (1) step is added in the solution that (2) step obtains, resulting mixed solution adds in the solution that (3) step obtains again, at last resulting mixed solution is added mixing in the resulting solution of (4) step.
The present invention is on the basic components that comprises the necessary nutritive ingredient of growing with legionella such as yeast powder, gac, agar, ACES (N-2-acetylaminohydroxyphenylarsonic acid 2-aminoethane sulphonic acid) buffer reagent, α-Tong Wuersuan, soluble ferric pyrophosphate and L-halfcystines, add dyestuff bromothymol blue and purpurum bromocresolis, legionella is carried out selective separation can be when cultivating so that the bacterium colony colour developing, according to colonial morphology and color identification and distinguishing legionnella object bacteria; Add the selectivity microbiotic and can suppress non-legionella bacterium such as gram positive coccus, gram negative bacilli and mould fungi growth, thereby improve legionella separation and Culture positive rate.
Compared with prior art, legionnella selective separation medium beneficial effect of the present invention is: the dyestuff that add (1) makes chromogenesis in the legionella process of growth, helps identifying and the distinguishing legionnella object bacteria according to colonial morphology and color; (2) add and adjust the selectivity antibiotic concentration, improve inhibition ability, reduce inhibition, thereby improved legionella separation and Culture positive rate to species of legionella to non-legionella and assorted mushroom; (3) reduction detection cost, economical and practical is convenient to clinical and health and epidemic prevention department applies.
Description of drawings
Fig. 1 is the figure of blank substratum.
Fig. 2 is four a zoning collimation methods inoculation legionella ATCC33153 reference culture, cultivates the picture after 72 hours.
Embodiment
Below in conjunction with embodiment specific embodiments of the invention are elaborated.
Embodiment 1
Legionnella selective separation medium, the component parts by weight that it comprises are as follows:
Basal liquid:
Protein liquid:
Glycine 2.8～3.2
L-halfcystine 0.4～0.6
Albumin 0.08～0.12
Dye solution:
Purpurum bromocresolis 0.008～0.012
Tetrabromophenol sulfonphthalein 0.008～0.012
Microbiotic liquid:
Vancomycin 0.0008～0.0012
PXB 0.0070.009
Cycloheximide 0.007～0.009
Preparation process is as follows:
(1) yeast powder in the basal liquid, agar, carbon dust, ACES and a-ketoglutaric acid are placed in the container by above-mentioned parts by weight weighing, add 930～980 parts in water, with KOH the pH value are transferred to 6.85; Boil 1 minute, and then 121 ℃ of high-temperature sterilizations 15 minutes; Ferric pyrophosphate is dissolved in 9～11 parts of water filtration sterilization; Yeast powder, agar, carbon dust, ACES and a-ketoglutaric acid behind 121 ℃ of high-temperature sterilizations are cooled to the ferric pyrophosphate solution mixing that adds filtration sterilization after 55 ℃;
(2) glycine, L-halfcystine and albumin are dissolved by adding 28～30 parts of water in the above-mentioned parts by weight weighing placement container;
(3) purpurum bromocresolis and tetrabromophenol sulfonphthalein are dissolved by adding in 9～11 parts of ethanol in the above-mentioned parts by weight weighing placement container;
(4) vancomycin, PXB and cycloheximide are dissolved by adding in 0.9～1.1 part of water in the above-mentioned parts by weight weighing placement container;
(5) solution of (1) step is added in the solution that (2) step obtains, resulting mixed solution adds in the solution that (3) step obtains again, at last resulting mixed solution is added mixing in the resulting solution of (4) step.
Embodiment 2
Legionnella selective separation medium (BCYE α+DGVPC), the component parts by weight that comprise are as follows:
Basal liquid:
Protein liquid:
Glycine 3.0
L-halfcystine 0.5
Albumin 0.1
Dye solution:
Purpurum bromocresolis 0.01
Tetrabromophenol sulfonphthalein 0.01
Microbiotic liquid:
Vancomycin 0.001
PXB 0.008
Cycloheximide 0.008
Preparation process is as follows:
(1) yeast powder in the basal liquid, agar, carbon dust, ACES and a-ketoglutaric acid are placed in the container by above-mentioned parts by weight weighing, add the water of 950 parts in water, with KOH the pH value are transferred to 6.85; Boil 1 minute, and then 121 ℃ of autoclavings (15 pounds) 15 minutes; Ferric pyrophosphate is dissolved in 10 parts of water filtration sterilization; With the yeast powder behind the autoclaving, agar, carbon dust, ACES and a-ketoglutaric acid, be cooled to the ferric pyrophosphate solution mixing that adds filtration sterilization after 55 ℃;
(2) glycine, L-halfcystine, albumin being placed the water that adds 29 parts in the container by above-mentioned parts by weight weighing dissolves;
(3) will dissolve in purpurum bromocresolis, the ethanol of tetrabromophenol sulfonphthalein by 10 parts of addings in the above-mentioned parts by weight weighing placement container;
(4) vancomycin, PXB, cycloheximide are dissolved by adding in 1 part of water in the above-mentioned parts by weight weighing placement container;
(5) solution of (1) step is added in the solution that (2) step obtains, resulting mixed solution adds in the solution that (3) step obtains again, at last resulting mixed solution is added mixing in the resulting solution of (4) step.
Embodiment 3
Legionnella selective separation medium, the component parts by weight that it comprises are as follows:
Basal liquid:
Protein liquid:
Glycine 2.5
L-halfcystine 0.3
Albumin 0.05
Dye solution:
Purpurum bromocresolis 0.005
Tetrabromophenol sulfonphthalein 0.005
Microbiotic liquid:
Vancomycin 0.0005
PXB 0.006
Cycloheximide 0.006
Preparation process is as follows:
(1) yeast powder in the basal liquid, agar, carbon dust, ACES and a-ketoglutaric acid are placed in the container by above-mentioned parts by weight weighing, add 900 parts in water, with KOH the pH value are transferred to 6.85; Boil 1 minute, and then 121 ℃ of high-temperature sterilizations 15 minutes; Ferric pyrophosphate is dissolved in 8 parts of water filtration sterilization; Yeast powder, agar, carbon dust, ACES and a-ketoglutaric acid behind 121 ℃ of high-temperature sterilizations are cooled to the ferric pyrophosphate solution mixing that adds filtration sterilization after 55 ℃;
(2) glycine, L-halfcystine and albumin are dissolved by adding 27 parts of water in the above-mentioned parts by weight weighing placement container;
(3) purpurum bromocresolis and tetrabromophenol sulfonphthalein are dissolved by adding in 8 parts of ethanol in the above-mentioned parts by weight weighing placement container;
(4) vancomycin, PXB and cycloheximide are dissolved by adding in 0.8 part of water in the above-mentioned parts by weight weighing placement container;
(5) solution of (1) step is added in the solution that (2) step obtains, resulting mixed solution adds in the solution that (3) step obtains again, at last resulting mixed solution is added mixing in the resulting solution of (4) step.
Embodiment 4
Legionnella selective separation medium, the component parts by weight that it comprises are as follows:
Basal liquid:
Protein liquid:
Glycine 3.5
L-halfcystine 0.8
Albumin 0.15
Dye solution:
Purpurum bromocresolis 0.02
Tetrabromophenol sulfonphthalein 0.02
Microbiotic liquid:
Vancomycin 0.002
PXB 0.010
Cycloheximide 0.010
Preparation process is as follows:
(1) yeast powder in the basal liquid, agar, carbon dust, ACES and a-ketoglutaric acid are placed in the container by above-mentioned parts by weight weighing, add 1000 parts in water, with KOH the pH value are transferred to 6.85; Boil 1 minute, and then 121 ℃ of high-temperature sterilizations 15 minutes; Ferric pyrophosphate is dissolved in 12 parts of water filtration sterilization; Yeast powder, agar, carbon dust, ACES and a-ketoglutaric acid behind 121 ℃ of high-temperature sterilizations are cooled to the ferric pyrophosphate solution mixing that adds filtration sterilization after 55 ℃;
(2) glycine, L-halfcystine and albumin are dissolved by adding 31 parts of water in the above-mentioned parts by weight weighing placement container;
(3) purpurum bromocresolis and tetrabromophenol sulfonphthalein are dissolved by adding in 12 parts of ethanol in the above-mentioned parts by weight weighing placement container;
(4) vancomycin, PXB and cycloheximide are dissolved by adding in 1.2 parts of water in the above-mentioned parts by weight weighing placement container;
(5) solution of (1) step is added in the solution that (2) step obtains, resulting mixed solution adds in the solution that (3) step obtains again, at last resulting mixed solution is added mixing in the resulting solution of (4) step.
The present invention adds bromothymol blue and purpurum bromocresolis fuel fluid and selectivity microbiotic in the formula system of legionella selectivity basic medium.Bromothymol blue and purpurum bromocresolis legionella is carried out selective separation can be when cultivating so that the bacterium colony colour developing, according to colonial morphology and color identification and distinguishing legionnella object bacteria.The selectivity microbiotic can suppress non-legionella bacterium such as gram positive coccus, gram negative bacilli and mould fungi growth, thereby improves legionella separation and Culture positive rate.Wherein glycine (glycine) is respectively 1280 μ g/ml and 160 μ g/ml to the minimum inhibitory concentration of streptococcus aureus; Minimum inhibitory concentration to dysentery bacterium is 640 μ g/ml; Minimum inhibitory concentration to Corynebacterium diphtheriae is 1280 μ g/ml and 640 μ g/ml; To colibacillary minimum inhibitory concentration is 1280 μ g/ml; Vancomycin (vancomycin) has stronger restraining effect to streptococcus aureus; PXB (polymixin B) has obvious restraining effect to Pseudomonas aeruginosa and other gram negative bacillis; Cycloheximide (Cycloheximide) has obvious restraining effect to mould fungi.
The clinical detection test
To detect the legionella sample after treatment, be seeded in the substratum of the present invention, in 36 ℃, 5%CO
2In the incubator, cultivated 2～3 days, the growth of legionella is obvious generally speaking, and as depicted in figs. 1 and 2, Fig. 2 can obviously see the growing state of legionella; Also may be cultured to and 1 week can observe legionella.The characteristics of this selective medium are when carrying out the selective separation cultivation, can make the bacterium colony colour developing, according to colonial morphology and color identification and distinguishing legionnella object bacteria; In addition, adjusted antibiotic concentration, strengthened inhibition ability, reduced inhibition, thereby improved positive rate legionella bacterium separation and Culture to species of legionella to other non-legionella.
206 parts of environmental water samples this area being gathered by the present invention carry out that legionella is separated and and some substratum of using always compare test, as shown in the table, legionella separation and Culture positive rate is by 35.1%～65.0%
[2,3]Bring up to 50%～80%, and the 3-5 doubly (3～5 yuan/1 plate) that reduces cost.
Substratum of the present invention both can be used as common BCYE α-basic medium and use on the one hand, prevented in the culturing process pollution of other bacteriums; On the other hand, be beneficial to, can suppress varied bacteria growing, improve the legionella separation and Culture greatly and detect positive rate by colonial morphology and color identification legionella.
Claims (1)
1. legionnella selective separation medium is characterized in that the component parts by weight that comprise are as follows:
Basal liquid:
Protein liquid:
Glycine 3.0
L-halfcystine 0.5
Albumin 0.1
Dye solution:
Purpurum bromocresolis 0.01
Tetrabromophenol sulfonphthalein 0.01
Microbiotic liquid:
Vancomycin 0.001
PXB 0.008
Cycloheximide 0.008
Preparation process is as follows:
(1) yeast powder in the basal liquid, agar, carbon dust, ACES and a-ketoglutaric acid are placed in the container by above-mentioned parts by weight weighing, add 950 parts water, with KOH the pH value are transferred to 6.85; Boil 1 minute, and then 121 ℃ of high-temperature sterilizations 15 minutes; Ferric pyrophosphate is dissolved in 10 parts of water filtration sterilization; With yeast powder, agar, carbon dust, ACES and the a-ketoglutaric acid behind 121 ℃ of high-temperature sterilizations, be cooled to the ferric pyrophosphate solution mixing that adds filtration sterilization after 55 ℃;
(2) glycine, L-halfcystine and albumin being placed the water that adds 29 parts in the container by above-mentioned parts by weight weighing dissolves;
(3) purpurum bromocresolis and tetrabromophenol sulfonphthalein are dissolved in the ethanol by 10 parts of addings in the above-mentioned parts by weight weighing placement container;
(4) vancomycin, PXB and cycloheximide are dissolved by adding in 1 part of water in the above-mentioned parts by weight weighing placement container;
(5) solution of (1) step is added in the solution that (2) step obtains, resulting mixed solution adds in the solution that (3) step obtains again, at last resulting mixed solution is joined mixing in the resulting solution of (4) step.
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| CN2009100402827A CN101643713B (en) | 2009-06-16 | 2009-06-16 | Legionnella selective separation medium |
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| CN2009100402827A CN101643713B (en) | 2009-06-16 | 2009-06-16 | Legionnella selective separation medium |
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| CN101921719B (en) * | 2010-05-31 | 2013-08-07 | 广州金域医学检验中心有限公司 | Legionella transport and enrichment medium |
| CN102971433A (en) * | 2010-06-03 | 2013-03-13 | 巴斯夫欧洲公司 | Detection and enumeration of microorganisms |
| CN109355226A (en) * | 2018-11-19 | 2019-02-19 | 上海申启生物科技有限公司 | Zengjing Granule for Legionella and the biphasic culture and preparation method thereof that is separately cultured |
| CN113308422A (en) * | 2021-07-30 | 2021-08-27 | 山东格研生物技术有限公司 | Separation culture medium for boltzmann legionella and preparation method thereof |
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Non-Patent Citations (11)
| Title |
|---|
| 刘本荣 |
| 朱庆义 |
| 杨瑞馥 |
| 杨瑞馥;王菊英;黄留玉;高树德.从外环境分离军团杆菌的研究.《中国公共卫生》.1989,全文. * |
| 梁耀铭.军团菌选择性分离培养基及其实用性比较研究.《国际检验医学杂志》.2006,第27卷(第3期),全文. |
| 潘文刚 |
| 王菊英 |
| 胡朝晖 |
| 胡朝晖;朱庆义;刘本荣;潘文刚;梁耀铭.军团菌选择性分离培养基及其实用性比较研究.《国际检验医学杂志》.2006,第27卷(第3期),全文. * |
| 高树德.从外环境分离军团杆菌的研究.《中国公共卫生》.1989,全文. |
| 黄留玉 |
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