CN101280337B - Improved rapid Lo's wenstein-Jensen culture medium and preparation theroef - Google Patents
Improved rapid Lo's wenstein-Jensen culture medium and preparation theroef Download PDFInfo
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- CN101280337B CN101280337B CN 200810099812 CN200810099812A CN101280337B CN 101280337 B CN101280337 B CN 101280337B CN 200810099812 CN200810099812 CN 200810099812 CN 200810099812 A CN200810099812 A CN 200810099812A CN 101280337 B CN101280337 B CN 101280337B
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Abstract
The invention discloses a modified rapid L (o) wenstein-Jensen culture medium and a preparation method thereof. The culture medium is made of the following material: basic liquid (potassium dihydrogen phosphate, asparagines, magnesium citrateusp, magnesium sulphate, pure glycerin, 2.4-dichlorophenoxyacetic acid, 6-furfurylaminopurine and distilled water), potato powder, fresh eggs and malachite green solution; the culture medium preparation method comprises the following steps: firstly, all the components of the <basic liquid> are mixed and the liquid is heated into boiled water to solubilize; secondly, the potato powder is added and stirring is performed while adding, and attention is paid to ensure the powder not to be caked and to the heating for the liquid in the boiled water is continued for half an hour and occasionally stirring is performed; thirdly, the whole egg liquid and the malachite green solution are not added until the liquid is cooled at 65 DEG C, then the liquid is evenly and fully stirred; fourthly, the liquid is filled in aseptic tubes and the tubes are plugged; fifthly, the tubes are positioned into serum coagulators to form a bevel, sterilizing is performed at90 DEG C for one hour or twenty minutes under high pressure, and the tubes are prepared for using after aseptic experiments.
Description
Technical field
The present invention relates to a kind of improvement Luo Shi fast culture media, also relate to a kind of preparation method of substratum.
Background technology
At present China generally uses the solid tubercule bacillus substratum of a kind of being referred to as " Russell medium " to come tubercule bacillus is carried out the vitro human work point from cultivation, and these are documented on " clinical medical inspection " that army hospital general, Foochow volume, Zhu Zhongyong, Chen Zhihang chief editor, Shanghai science tech publishing house publish in April, 1978 the 454th page.Because existing solid tubercule bacillus substratum all is can purchase without off-the-shelf by each hospital's deallocation system, this just needs school to acquire separately the corresponding production unit of a cover for this reason, has both strengthened the equipment expense of hospital, again the corresponding preparation workload that increased.Through the retrieval Chinese patent, number of patent application 98 1 10068 its technical schemes are: formed by basic substance, energy matter, plant growth stimulating material mixed preparing, potassium primary phosphate, sal epsom (MgSO are arranged in the basic substance.7HZo), structure list acid magnesium, ironic citrate is pressed, Radix Asparagi phthalein amine, neutral glycerine, potato powder, Fresh Egg liquid, 2% malachite green solution, water, animal serum, coenzyme A is arranged in the energy matter, spartofam, Nitrosylferricytochrome C, Q one Cai's acetic acid is arranged in the plant growth stimulating material, and the content of each component is in per 1000 gram fast culture media for mycobaterium tuberculosis: potassium primary phosphate 1.36～1.4 grams, Fresh Egg liquid 560～600 grams, sal epsom 0.136～0.14 gram 2% malachite green solution 11～13 gram structure list acid magnesium 0.3～0.4 gram water 300～340 gram ironic citrate money 0.028～0.03 gram animal serum 56～60 gram Radix Asparagi phthalein amine 2.06～2.1 gram coenzyme As 0.4～0.8 gram neutral glycerine 6～8 gram spartofams 0.04～0.08 gram potato powder 17～17.5 gram Nitrosylferricytochrome Cs 0.045～0.075 gram.0.05～0.1 milligram of one Cai's acetic acid.The steps include: A, paste producing: after the potato powders of 17～17.5 grams are added 150～170 gram water, place boiling water bath to heat, the time add stirring, make into even pasty state, naturally be down to after the room temperature for subsequent use; B, mixing preparation: with the potassium primary phosphate of 1.36～1.4 grams, 0.136 the sal epsom of～0.14 gram, a 0.3 structure sour magnesium of～0.4 gram, 0.028 the ironic citrate money of～0.03 gram, 2.06 the Radix Asparagi phthalein amine of～2.1 grams, after the neutral glycerine of 6～8 grams adds 150～170 gram water, place the boiling water bath heating for dissolving, when waiting to be chilled to below 50 ℃, the Fresh Egg liquid that adds 560～600 grams, the animal serum of 56～60 grams, 0.4 the coenzyme A of～0.8 gram, 0.04 the spartofam of～0.08 gram, 0.045～0.075 gram Nitrosylferricytochrome C, 0.05 Q one Cai's acetic acid of～0.1 milligram, after stirring, add again the potato powder of 167～187.5 grams and 2% malachite green solution of 11-13 gram, and after fully stirring evenly, get 1000 gram mixtures; C, packing and sealing; Mixture is sub-packed in the Boiling tube, every pipe packing 5 grams, be set to long inclined-plane after, sealing by fusing; D, sterilization; Adopt serum coagulator that Boiling tube is sterilized, sterilising conditions is: head day, 85 ℃, 30 minutes; Next day, 80 ℃, 30 minutes; The 3rd is, 80 ℃, and 30 minutes.By the above as can be known, the existing process for preparation of solid tubercule bacillus substratum is complexity and trouble comparatively, therefore hospital can only give according to the roughly consumption of solid tubercule bacillus substratum at no distant date and prepare first that some are for subsequent use, but, several thousand the solid tubercule bacillus substratum that hospital disperses to prepare separately in the whole nation, exist all that quality standard is inconsistent, yield rate is not high, be difficult for long storage and storage period slightly long because moisture content scatters and disappears, medicine decomposes the defective that makes detected result inaccurate.Clinical detection is to find that there is following defective in existing tubercule bacillus substratum: 1, because existing solid tuberculosis substratum is deposited in the Boiling tube, mouth of pipe soft rubber ball jam-pack, therefore the sealed degree of this substratum is relatively poor, moisture content in it easily scatters and disappears, so that the storage period very short (only 3 months) of this substratum, so just, can not be by in its substratum of suitability for industrialized production hand the multiple nutrients material being arranged, and as mentioned above as can be known, the kind of nutritive substance is few in the existing solid tubercule bacillus substratum, there is not in addition to accelerate the tubercule bacillus metabolic activity in it to improve energy matter and the plant growth stimulating material of tubercule bacillus growth and breeding speed yet, like this so that tubercule bacillus can not carry out well growth and breeding in existing solid tubercule bacillus substratum, mitotic cycle reaches 12～16 hours, for 4～6 weeks can see bacterium colony.3, some liquid culture based formulas is complicated, and method is loaded down with trivial details, and Russell medium needs 35 days.Therefore, come tubercule bacillus is carried out the vitro human work point when cultivating with existing tubercule bacillus substratum, its detection speed is quite slow, and positive rate is low, is unfavorable for early diagnosis and therapy lungy.
Summary of the invention
The object of the present invention is to provide a kind of simply, realize fast culture tubercule bacillus substratum, solved clinical early diagnosis and the problem for the treatment of effectively.
Another object of the present invention has provided a kind of easy to implement the method, easy to operate a kind of preparation method who improves the Luo Shi fast culture media.
In order to achieve the above object, the present invention adopts following technical measures:
A kind of improvement Luo Shi fast culture media, the substratum that it is made by the raw material of following weight proportioning: potassium primary phosphate (anhydrous) 0.96g, asparagine (asparagine) 0.27g, Citric Acid 0.21g, sal epsom (MgSO
4, 7H
2O) 0.084g, pure glycerin (neutrality) 4.2ml, 2.4-Dichlorophenoxyacetic acid sodium 0.1g, 6-Furfurylaminopurine 0.1g, distilled water 120ml, potato powder 0.6g, Fresh Egg 3-4, the malachite green solution 8ml of 10g/L.
A kind of preparation method who improves the Luo Shi fast culture media, the method comprises the following steps:
A, with " potassium primary phosphate (anhydrous) 0.96g, asparagine (asparagine) 0.27g, citrate of magnesia 0.21g, sal epsom (MgSO
4, 7H
2O) 0.084g, pure glycerin (neutral 4.2ml, 2.4-Dichlorophenoxyacetic acid sodium 0.1g, 6-Furfurylaminopurine 0.1g and distilled water 120ml, " each composition mix, put heating for dissolving in the boiling water;
B, add potato powder, the limit edged stirs, and notes not forming piece, and continues to heat half an hour in boiling water bath, the time add stirring;
C, add liquid whole egg 200ml and 10g/L malachite green solution 8ml when waiting to be chilled to 65 ℃, fully mixing.
D, packing sterile test tube, every pipe is 5～6ml approximately, beyond the Great Wall plug;
E, put bevel in the serum coagulator, through 90 ℃ of 1 hour or high pressure (103.43kpa), sterilize after 20 minutes, remake after the sterility test for subsequent use.
A kind of improvement Luo Shi fast culture media, described basal culture medium PH is about 6.0; A kind of using method that improves the Luo Shi fast culture media, the method comprise the following steps: that 2～3 of the good sample direct inoculation of pre-treatment the full inclined-plane of lid through 37 ℃ of cultivations, is seen and looked into growing state every day.Approximately just can see the tiny bacterium colony of needle point sample in two～three days, just can see the shaping bacterium colony in six～seven days, seven～fortnight just can be seen plentiful bacterium colony.Can do the test of bacterial type identification and drug sensitivity.
The present invention compared with prior art has the following advantages and effect, and prescription rationally, and is easy to implement the method, easy to operate, detects fast, accurately and reliably, and is cheap, by clinical trial certificate, is particularly useful for basic hospital, has good society and economic benefit.
Embodiment
A kind of improvement Luo Shi fast culture media, the substratum that it is made by the raw material of following weight proportioning: potassium primary phosphate (anhydrous) 0.96g, asparagine (asparagine) 0.27g, citrate of magnesia 0.21g, sal epsom (MgSO
4, 7H
2O) 0.084g, pure glycerin (neutrality) 4.2ml, 2.4-Dichlorophenoxyacetic acid sodium 0.1g, 6-Furfurylaminopurine 0.1g, distilled water 120ml, potato powder 0.6g, new freshly-slaughtered poultry 3-4, the malachite green solution 8ml of 10g/L;
A kind of preparation method who improves the Luo Shi fast culture media, the method comprises the following steps:
A, with " potassium primary phosphate (anhydrous) 0.96g, asparagine (asparagine) 0.27g, citrate of magnesia 0.21g, sal epsom (MgSO
4, 7H
2O) 0.084g, pure glycerin (neutrality) 4.2ml, 2.4-Dichlorophenoxyacetic acid sodium 0.1g, 6-Furfurylaminopurine 0.1g and distilled water 120ml " each composition mix, put heating for dissolving in the boiling water;
B, add potato powder, the limit edged stirs, and notes not forming piece, and continues to heat half an hour in boiling water bath, the time add stirring;
C, add liquid whole egg 200ml and 10g/L malachite green solution 8ml when waiting to be chilled to 65 ℃, fully mixing.
D, packing sterile test tube, every pipe is 5～6ml approximately, beyond the Great Wall plug;
E, put bevel in the serum coagulator, through 90 ℃ of 1 hour or high pressure (103.43kpa), sterilize after 20 minutes, remake after the sterility test for subsequent use.
A kind of improvement Luo Shi fast culture media, described basal culture medium PH is about 6.0; A kind of using method that improves the Luo Shi fast culture media, the method comprise the following steps: that 2～3 of the good sample direct inoculation of pre-treatment the full inclined-plane of lid through 37 ℃ of cultivations, is seen and looked into growing state every day.Approximately just can see the tiny bacterium colony of needle point sample in two～three days, just can see the shaping bacterium colony in six～seven days, seven～fortnight just can be seen plentiful bacterium colony.Can do the test of bacterial type identification and drug sensitivity.
The present invention compared with prior art has the following advantages and effect, and prescription rationally, and is easy to implement the method, easy to operate, detects fast, accurately and reliably, and is cheap, by clinical trial certificate, is particularly useful for basic hospital, has good society and economic benefit.
Claims (3)
1. an improvement Luo Shi fast culture media is characterized in that: the substratum that it is made by the raw material of following weight proportioning: anhydrous potassium dihydrogenphosphate 0.96g, asparagine 0.27g, citrate of magnesia 0.21g, MgSO
47H
2O 0.084g, neutral pure glycerin 4.2ml, 2.4-Dichlorophenoxyacetic acid sodium 0.1g, 6-chaff aminopurine 0.1g, distilled water 120ml, potato powder 0.6g, 3-4 of Fresh Eggs, the malachite green solution 8ml of 10g/L.
2. realize a kind of preparation method who improves the Luo Shi fast culture media claimed in claim 1 for one kind, it is characterized in that: the method comprises the following steps:
A, with " anhydrous potassium dihydrogenphosphate 0.96g, asparagine 0.27g, citrate of magnesia 0.21g, MgSO
47H
2O 0.084g, neutral pure glycerin 4.2ml, 2.4-Dichlorophenoxyacetic acid sodium 0.1g, 6-chaff aminopurine 0.1g and distilled water 120ml " each composition mix, put heating for dissolving in the boiling water;
B, add potato powder, the limit edged stirs, and notes not forming piece, and continues to heat half an hour in boiling water bath, the time add stirring;
C, add liquid whole egg 200ml and 10g/L malachite green solution 8ml when waiting to be chilled to 65 ℃, fully mixing;
D, packing sterile test tube, every pipe is 5～6ml approximately, beyond the Great Wall plug;
E, put bevel in the serum coagulator, through 90 ℃ of 1 hour or 103.43kpa, sterilize after 20 minutes, remake after the sterility test for subsequent use.
3. a kind of improvement Luo Shi fast culture media according to claim 1, it is characterized in that: described Medium's PH Value is 6.0.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1226602A (en) * | 1998-02-20 | 1999-08-25 | 刘照云 | Fast culture media for mycobaterium tuberculosis and preparation thereof |
| CN1699592A (en) * | 2005-04-29 | 2005-11-23 | 胡忠义 | Two phase Roe's culture medium and preparation method thereof |
| CN101168766A (en) * | 2007-11-13 | 2008-04-30 | 上海市肺科医院 | Diphase Roe's authentication or drug sensitive culture medium |
-
2008
- 2008-05-25 CN CN 200810099812 patent/CN101280337B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1226602A (en) * | 1998-02-20 | 1999-08-25 | 刘照云 | Fast culture media for mycobaterium tuberculosis and preparation thereof |
| CN1699592A (en) * | 2005-04-29 | 2005-11-23 | 胡忠义 | Two phase Roe's culture medium and preparation method thereof |
| CN101168766A (en) * | 2007-11-13 | 2008-04-30 | 上海市肺科医院 | Diphase Roe's authentication or drug sensitive culture medium |
Non-Patent Citations (1)
| Title |
|---|
| 徐修礼等.结核分枝杆菌新型改良罗氏快速培养基的研究.《中国感染控制杂志》.2007,第6卷(第1期),38-40. * |
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