CN101168766A - Diphase Roe's authentication or drug sensitive culture medium - Google Patents
Diphase Roe's authentication or drug sensitive culture medium Download PDFInfo
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Abstract
This invention relates to biphase Roche identification or drug sensitive culture medium and the preparation method thereof, and belongs to the medical detection technical field. The culture medium of the invention consists of solid culture medium and liquid culture medium, and simultaneously includes the nutrient contents of the solid culture medium and the liquid culture medium; the solid part includes basic nutrients for the growing of bacteria, and the colony features thereof can be observed; the liquid part includes growth promoter, bacteriostat, identification reagent or am antituberculotic medicine, thereby significantly promoting the growth of mycobacteria. Compared with conventional solid identification culture medium, the invention can identify mycobacterium tuberculosis or make the drug sensitive culture of the mycobacterium tuberculosis faster, simplier, and more exact, and solves the problems that the identification and the drug sensitive test of the mycobacterium tuberculosis take too long time and have poor accuracy. The culture medium can also obtain single colonies, thereby being favorable for further bacteriological research.
Description
Technical field
The invention belongs to technical field of medical detection, relate to diphase Roe's and differentiate or drug sensitive culture medium.Be specifically related to a kind of differential medium that can carry out the Rapid identification mycobacterium tuberculosis and maybe can carry out mycobacterium tuberculosis drug sensitive culture medium of quick medicine-sensitive test and preparation method thereof.
Background technology
Tuberculosis is the serious infectious diseases of serious harm human health.Have and close the statistics report, the whole world has 1/3 population to infect tubercule bacillus approximately at present, and present tuberculous several people are about 2,000 ten thousand, annual neopathy number 8,000,000～1,000 ten thousand, and the death number is about 2,000,000, is the summation of other all transmissible disease death tolls.The tuberculosis epidemic situation of China is very severe, and the whole nation has 5.5 hundred million populations to infect tubercule bacillus, and existing active tuberculosis patient is about 4,500,000, wherein has communicable bacterium yang disease people 1,960,000, occupies the second in the world.Relevant studies show that, China is again the high resistance of tuberculosis country, the tuberculosis patient resistant rate occupies the whole world second.Mycobacterium tuberculosis is a pathogenic bacteria lungy.Because rapid detection, evaluation and the drug sensitive test of mycobacterium tuberculosis have important directive function to lungy making a definite diagnosis with medicine for treatment, therefore press for and carry out mycobacterium tuberculosis evaluation fast and accurately and drug sensitive test.
Yet, China adopts solid Russell medium method to carry out the separation and Culture of mycobacterium tuberculosis always for a long time, identify and drug sensitive test, obtain assay take 1～February (see the tuberculosis bacteriological analysis rules that Chinese anti-tuberculosis association edits and publishes---Chinese education culture press, in January, 2006 first version, 46～49 and 52～56 pages), present detection method seriously lags behind the needs of clinical diagnosis and treatment, and, relevant identification reagent and medicine cause the accuracy of Bacteria Identification and susceptibility detected result to be affected because of destroyed because of being heated in preparation substratum process.At present, although also have suggestion use the liquid culture based method carry out the Rapid identification of mycobacterium tuberculosis and drug sensitive test (see the tuberculosis bacteriological analysis rules that Chinese anti-tuberculosis association edits and publishes---Chinese education culture press, in January, 2006 first version, 37～44 pages and 89～93 pages), but whether because of not forming bacterium colony in the mycobacterium tuberculosis bacterium liquid medium within, therefore can't make according to colony characteristics is the preliminary evaluation of mycobacterium.Therefore must get nutrient solution earlier carries out smear, acid-fast stain, observes at microscopically again, can draw just whether the bacterium that grows in the liquid nutrient medium is acid-fast stain male mycobacterium, and the result is not only time-consuming but also require great effort, and does not still reach fast and convenient purpose.Therefore, clinical and research practice press for novel quick, easy, mycobacterium tuberculosis is differentiated and drug sensitive culture medium accurately.
Summary of the invention
The objective of the invention is provides a kind of diphase Roe's to differentiate or drug sensitive culture medium at the various solids of above-mentioned prior art and the defective that liquid is differentiated and drug sensitive culture medium exists.Substratum of the present invention can differentiate mycobacterium tuberculosis quick, easy, accurately or carry out the mycobacterium tuberculosis susceptibility and cultivate, and solved that mycobacterium tuberculosis is identified and drug sensitive test takes for a long time, the problem of poor accuracy.
Another object of the present invention provides the preparation method of above-mentioned diphase Roe's discriminating or drug sensitive culture medium.
The present invention realizes above-mentioned purpose by the following technical solutions:
Preparing a kind of diphase Roe's that had not only contained solid but also contained the nutritious mycobacterium special use of liquid differentiates or drug sensitive culture medium, this substratum contains the nutritive ingredient of solid and two kinds of substratum of liquid simultaneously, can provide competent nutriment for the quick growth of mycobacterium, reach the purpose that shortens incubation time, reports ahead of time; Simultaneously, because bacterium can form bacterium colony at solid surface when growing in this substratum, therefore by observing colony characteristics, whether the bacterium of growth is made is the preliminary evaluation of mycobacterium tuberculosis or non-tuberculous mycobacteria; In addition, but also the single bacterium colony of picking further identify.
Diphase Roe's of the present invention is differentiated or drug sensitive culture medium is made up of solid medium and liquid nutrient medium, and wherein, the solid culture base section is made up of the component of following weight proportion:
Copper sulfate 0.01～0.02g, zinc sulfate 0.01～0.02g, calcium chloride 0.01～0.02g, sal epsom 0.01～0.02g, Ferric Ammonium Citrate 0.05～0.1g, Sodium Citrate 0.1～0.2g, ammonium sulfate 0.2～0.4g, Sodium.alpha.-ketopropionate 0.5～1g, asparagine 1～2g, potassium primary phosphate 1～2g, SODIUM PHOSPHATE, MONOBASIC 2～4g, neutral glycerine 8～12ml, 1% malachite green solution, 10～20ml, ovum gallinaceum liquid 1000ml, sterile distilled water 600ml;
The liquid culture base section comprises basal liquid, growth promoter, fungistat and identification reagent or antitubercular agent, wherein,
(1) basal liquid is made up of the component of following weight proportion:
Copper sulfate 0.005～0.01g, zinc sulfate 0.005～0.01g calcium chloride 0.005～0.01g, sal epsom 0.005～0.01g, oleic acid 0.01～0.02g, Ferric Ammonium Citrate 0.01～0.02g, Sodium Citrate 0.05～0.1g, ammonium sulfate 0.1～0.2g, Sodium.alpha.-ketopropionate 0.25～0.5g, tween-80 0.5～1g, asparagine 0.5～1g, potassium primary phosphate 0.5～1g, SODIUM PHOSPHATE, MONOBASIC 1～2g use dissolved in distilled water;
(2) growth promoter is made up of the component of following weight proportion:
Pyridoxine hydrochloride 0.01～0.02g, vitamin H 0.01～0.02g, coenzyme A 0.1～0.2g, Triphosaden 0.1～0.2g, catalase 0.1～0.2g, α-Nai Yisuan 0.1～0.2g, peptone 1～2g, casein hydrolysate 1～2g, glucose 10～20g, the bovine serum albumin v5 factor 100～200g, mycobacterium culture supernatant 5～10ml;
(3) fungistat is selected from penbritin, amphotericin B, PXB, the distilled water solution mixture of pyridine ketone acid and azlocillin 0.05～0.1mg/ml how;
(4) identification reagent is selected from the thiophene-2-carboxylic acid hydrazine, and concentration is 0.1～0.2mg/ml; Or p-nitrobenzoic acid, concentration is 0.5～1mg/ml;
(5) antitubercular agent is selected from vazadrine, Rifampin, Streptomycin sulphate and Tibutol, and the working concentration of described four kinds of medicines is respectively 0.005～0.01mg/ml, 0.02～0.04mg/ml, 0.01～0.02mg/ml and 0.01～0.02mg/ml.
Prepare the diphase Roe's differential medium by following method and step:
1) prepares solid and liquid nutrient medium respectively;
2) imbitition substratum 3～4ml adds in the solid medium;
3) in the liquid of above-mentioned substratum, add thiophene-2-carboxylic acid hydrazine and p-nitrobenzoic acid 0.1～0.2ml respectively, make the diphase Roe's differential medium; Or,
4) in the liquid of above-mentioned substratum, add each 0.1～0.2ml of antitubercular agent vazadrine, Rifampin, Streptomycin sulphate and Tibutol respectively, make the diphase Roe's drug sensitive culture medium.
Use the method for described diphase Roe's differential medium as follows:
1) bacterial strain to be identified is configured to 10
-2Mg/ml;
2) drawing 0.1ml bacterium liquid to be checked respectively is inoculated in thiophene-2-carboxylic acid hydrazine, p-nitrobenzoic acid differential medium and does not contain in the liquid of contrast culture pipe of identification reagent;
3) above-mentioned culture tube is tilting, 35～37 ℃ of 1～2 weeks of incubation;
4) every day observations once, be the bacterial growth positive as bacteria particles in the liquid of substratum, occurring and/or bacterium colony occurring at solid surface;
5) bacterial strain to be checked is not grown in the p-nitrobenzoic acid differential medium as growing in thiophene-2-carboxylic acid hydrazine differential medium, then is mycobacterium tuberculosis; As all growths in thiophene-2-carboxylic acid hydrazine and two kinds of differential mediums of p-nitrobenzoic acid, then be non-tuberculous mycobacteria.
Use described diphase Roe's drug sensitive culture medium method as follows:
1) bacterial strain to be checked is mixed with 10
-2Mg/ml;
2) drawing 0.1ml bacterium liquid to be checked respectively is inoculated in diphase Roe's drug sensitive culture medium that contains different antitubercular agents and the liquid of the control medium that does not contain medicine;
3) above-mentioned culture tube is tilting, 35～37 ℃ of 1～2 weeks of incubation;
4) every day observations once, be the bacterial growth positive as occurring bacteria particles in the liquid medium within and/or bacterium colony occurring at the solid culture primary surface;
5) grow in the control medium of pastille not as bacterial strain to be checked, do not grow in the substratum of pastille, then this bacterial strain is the medicaments insensitive strain; As bacterium all growths in the control medium of pastille not and the substratum that contains, then this bacterial strain is a persister.
Adopt differential medium of the present invention to detect 200 strain mycobacterium tuberculosis and 200 strain non-tuberculous mycobacterias, and compare with solid differential medium that routine is used.The result shows, the more conventional substratum qualification time of substratum of the present invention 17～23 days ahead of time; The differential medium qualification result is identical with the conventional solid differential medium qualification result of using, coincidence rate 100%; Pollution rate is lower than conventional differential medium;
Adopt drug sensitive culture medium of the present invention that the susceptibility that 200 strain mycobacterium tuberculosis clinical separation strains carry out vazadrine, Rifampin, Streptomycin sulphate and four medicines of Tibutol is detected, and compare with solid medicine sensitive culture medium result that routine is used.The result shows, 14～16 days ahead of time more conventional substratum susceptibility time of substratum of the present invention; The solid medicine sensitive culture medium detected result coincidence rate of using with routine is 95～99%; The drug sensitive test of medicine coincidence rate as a result is respectively: the vazadrine is 95%, and Rifampin is 99%, and Streptomycin sulphate is 98%, and Tibutol is 96%; This drug sensitive culture medium detected result pollution rate is lower than conventional solid medicine sensitive culture medium pollution rate.
Embodiment
Embodiment 1 preparation diphase Roe's is differentiated or drug sensitive culture medium
1) preparation solid medium
(1) accurately weighs following each component of solid medium: copper sulfate 0.01～0.02g, zinc sulfate 0.01～0.02g, calcium chloride 0.01～0.02g, sal epsom 0.01～0.02g, Ferric Ammonium Citrate 0.05～0.1g, Sodium Citrate 0.1～0.2g, ammonium sulfate 0.2～0.4g, Sodium.alpha.-ketopropionate 0.5～1g, asparagine 1～2g, potassium primary phosphate 1～2g, SODIUM PHOSPHATE, MONOBASIC 2～4g, distilled water 600ml, 110～120 ℃ were heated 15～20 minutes, cooling;
(2) add neutral glycerine 8～12ml, 1% malachite green solution, 10～20ml, ovum gallinaceum liquid 1000ml, fully mixing is sub-packed in the sterile culture pipe every pipe 7～10ml;
(3) culture tube is inclined in the well heater, 80～90 ℃ were heated 10～20 minutes, and the rearmounted 4 ℃ of preservations of cooling are standby;
2) preparation liquid nutrient medium
(1) accurately weigh each component of following basal liquid in the liquid nutrient medium: copper sulfate 0.005～0.01g, zinc sulfate 0.005～0.01g calcium chloride 0.005～0.01g, sal epsom 0.005～0.01g, oleic acid 0.01～0.02g, Ferric Ammonium Citrate 0.01～0.02g, Sodium Citrate 0.05～0.1g, ammonium sulfate 0.1～0.2g, Sodium.alpha.-ketopropionate 0.25～0.5g, tween-80 0.5～1g, asparagine 0.5～1g, potassium primary phosphate 0.5～1g, SODIUM PHOSPHATE, MONOBASIC 1～2g, use dissolved in distilled water;
(2) add neutral glycerine 8～12ml, mixing, adding distil water be to 1000ml, 110～120 ℃ of heat sterilizations 10～20 minutes;
(3) the cooling back adds the growth promoter of 1～2ml filtration sterilization: pyridoxine hydrochloride 0.01～0.02g, vitamin H 0.01～0.02g, coenzyme A 0.1～0.2g, Triphosaden 0.1～0.2g, catalase 0.1～0.2g, α-Nai Yisuan 0.1～0.2g, peptone 1～2g, casein hydrolysate 1～2g, glucose 10～20g, the bovine serum albumin v5 factor 100～200g, the fungistat of mycobacterium culture supernatant 5～10ml and 0.1～0.2ml: penbritin, amphotericin B, PXB, how the abundant mixing of distilled water solution of pyridine ketone acid and azlocillin 50～100 μ g/ml;
3) preparation had not only contained solid but also had contained the quick growth medium of mycobacterium tuberculosis of liquid
(1) in above-mentioned solid medium, the aseptic adding 3～4ml of every pipe liquid nutrient medium is made the quick growth medium of mycobacterium tuberculosis that not only contains solid but also contain liquid;
(2) with the quick growth medium of the above-mentioned mycobacterium tuberculosis for preparing, place 37 ℃ and cultivate and to carry out sterility test in 24 hours, standby if no bacterial growth can be put 4 ℃ of preservations;
4) in the liquid of the above-mentioned substratum for preparing, add the identification reagent thiophene-2-carboxylic acid hydrazine and the p-nitrobenzoic acid of mycobacterium tuberculosis respectively, make mycobacterium tuberculosis diphase Roe's differential medium by following step:
(1) accurately take by weighing thiophene-2-carboxylic acid hydrazine and p-nitrobenzoic acid, with the dimethyl formamide dissolving, again with sterile purified water dilution thiophene-2-carboxylic acid hydrazine concentration to 0.1～0.2mg/ml, dilution p-nitrobenzoic acid concentration to 0.5～1mg/ml, standby behind the aseptic suction filtration;
(2) draw 0.1～0.2ml thiophene-2-carboxylic acid hydrazine and p-nitrobenzoic acid solution respectively, in the aseptic liquid that joins the above-mentioned substratum that makes, make the diphase Roe's differential medium.
5) differentiate the cultivation application test:
The differential medium that employing makes detects 200 strain mycobacterium tuberculosis and 200 strain non-tuberculous mycobacterias, and the solid differential medium of using with routine compares.The result shows that differential medium qualification time of the present invention is 3～7 days, and the conventional solid differential medium of using takes 21～30 days, the more conventional substratum qualification time of substratum of the present invention 17～23 days ahead of time; Qualification result is identical, coincidence rate 100%; Differential medium detected result pollution rate of the present invention is 1.2%, and the conventional solid differential medium pollution rate of using is 2.0%.
Mycobacterium tuberculosis diphase Roe's differential medium of the present invention can carry out the quick strain identification of mycobacterium tuberculosis and non-tuberculous mycobacteria to the bacterium of growth.In the differential medium of the present invention,, therefore can avoid causing the inaccurate defective of qualification result, can reduce the identification reagent consumption simultaneously, reduce cost because of adding the heat collapse identification reagent owing to adopted aseptic adding identification reagent to the liquid of the substratum that makes.Compare with the Luo Shi differential medium of prior art, in its preparation process, adopt to add identification reagent earlier behind substratum, carry out heat sterilization again, the identification reagent that causes being added is subjected to destruction and identification reagent large usage quantity and the low problem of qualification result accuracy in various degree.
Table 1 is that this diphase Roe's differential medium and conventional Luo Shi differential medium detected result compare.
Table 1
| Substratum | The bacterial strain number | Identify fate | Pollution rate (%) | Qualification result | |
| The mycobacterium tuberculosis number | Non-tuberculosis branch bar | ||||
| The bacterium number | |||||
| The diphase Roe's differential medium | 400 | 3～7 | 1.2 | 200 | 200 |
| Conventional Luo Shi differential medium | 400 | 20～30 | 2.0 | 200 | 200 |
6) above-mentioned 2) in the liquid of the substratum that makes, add antitubercular agent respectively, prepare the diphase Roe's drug sensitive culture medium by following step:
(1) accurately take by weighing antitubercular agent vazadrine, Rifampin, Streptomycin sulphate and Tibutol medicinal powder, except that Rifampin dimethyl formamide dissolving, all the other use dissolved in distilled water, and with distilled water diluting to using liquid, standby behind the aseptic suction filtration.The working concentration of each medicine is as follows: vazadrine 0.005～0.01mg/ml, Rifampin 0.02～0.04mg/ml, Streptomycin sulphate 0.01～0.02mg/ml, Tibutol 0.01～0.02mg/ml;
(2) draw the antitubercular agent 0.1～0.2ml configure respectively, in the aseptic liquid that joins the described substratum that makes, make the mycobacterium tuberculosis rapid drug sensitive substratum.
7) drug sensitive culture medium application test
Adopt drug sensitive culture medium of the present invention that the susceptibility that 200 strain mycobacterium tuberculosis clinical separation strains carry out vazadrine, Rifampin, Streptomycin sulphate and four medicines of Tibutol is detected, and compare with solid medicine sensitive culture medium result that routine is used.The result shows, drug sensitive culture medium of the present invention detection time is 7～14 days, and the conventional solid medicine sensitive culture medium of using takes 21～30 days, 14～16 days ahead of time more conventional substratum susceptibility time of substratum of the present invention; This drug sensitive culture medium detected result is 95～99% with the conventional solid medicine sensitive culture medium detected result coincidence rate of using; The drug sensitive test of 4 medicines coincidence rate as a result is: the vazadrine is 95%, and Rifampin is 99%, and Streptomycin sulphate is 98%, and Tibutol is 96%; This drug sensitive culture medium detected result pollution rate is 1.1%, and conventional solid medicine sensitive culture medium pollution rate is 2.1%.
Drug sensitive culture medium of the present invention can carry out chemical sproof rapid detection to the mycobacterium tuberculosis in the growth.In this drug sensitive culture medium, adopt aseptic adding antitubercular agent to the liquid of the substratum that makes, can avoid medicine to cause drug level to reduce the inaccurate possibility of drug sensitivity tests that is caused, reduce drug dose simultaneously, reduce the drug sensitive test cost because of adding heat collapse.And the conventional at present solid susceptibility differential medium that uses in its preparation process, joins identification reagent in the substratum earlier, carry out heat sterilization again, therefore medicine is subjected to destruction in various degree, so drug use concentration is bigger, and the accuracy of susceptibility detected result is affected.
Table 2 is that diphase Roe's drug sensitive culture medium and conventional Luo Shi drug sensitive culture medium detected result compare.
Table 2
| Substratum | The bacterial strain number | The susceptibility fate | Pollution rate (%) | Two kinds of drug sensitivity tests coincidence rates (%) | |||
| The vazadrine | Rifampin | Streptomycin sulphate | Tibutol | ||||
| The diphase Roe's drug sensitive culture medium | 200 | 7～14 | 1.1 | 95 | 99 | 98 | 96 |
| Conventional Luo Shi drug sensitive culture medium | 200 | 21～30 | 2.1 | ||||
Embodiment 2: the preparation of diphase Roe's differential medium and application
1. take by weighing thiophene-2-carboxylic acid hydrazine 1～2mg, p-nitrobenzoic acid 5～10mg, with the dimethyl formamide dissolving, with the sterile purified water dilution, thiophene-2-carboxylic acid hydrazine working concentration is 0.01～0.02mg/ml more respectively, the p-nitrobenzoic acid working concentration is 0.05～0.1mg/ml, aseptic suction filtration; Draw 0.1～0.2ml thiophene-2-carboxylic acid hydrazine and p-nitrobenzoic acid solution respectively, in the aseptic liquid that joins the quick growth medium of mycobacterium tuberculosis for preparing, make the diphase Roe's differential medium.
2. preparation had not only contained solid but also had contained the quick growth medium of mycobacterium tuberculosis of liquid: the solid ingredient that weighs substratum: copper sulfate 0.01～0.02g, zinc sulfate 0.01～0.02g, calcium chloride 0.01～0.02g, sal epsom 0.01～0.02g, Ferric Ammonium Citrate 0.05～0.1g, Sodium Citrate 0.1～0.2g, ammonium sulfate 0.2～0.4g, Sodium.alpha.-ketopropionate 0.5～1g, asparagine 1～2g, potassium primary phosphate 1～2g, SODIUM PHOSPHATE, MONOBASIC 2～4g, use the 600ml dissolved in distilled water, 110～120 ℃ were heated 10～20 minutes; Add neutral glycerine 8～12ml, 1% malachite green solution, 10～20ml, ovum gallinaceum liquid 1000ml, fully mixing is sub-packed in the sterile culture pipe every pipe 7～10ml; Be inclined in the well heater, 80～90 ℃ were heated 10～20 minutes;
Weigh the liquid ingredient of substratum: copper sulfate 0.005～0.01g, zinc sulfate 0.005～0.01g calcium chloride 0.005～0.01g, sal epsom 0.005～0.01g, oleic acid 0.01～0.02g, Ferric Ammonium Citrate 0.01～0.02g, Sodium Citrate 0.05～0.1g, ammonium sulfate 0.1～0.2g, Sodium.alpha.-ketopropionate 0.25～0.5g, tween-80 0.5～1g, asparagine 0.5～1g, potassium primary phosphate 0.5～1g, SODIUM PHOSPHATE, MONOBASIC 1～2g, neutral glycerine 8～12ml, distilled water 1000ml; Dissolving, mixing, 110～120 ℃ of heat sterilizations 10～20 minutes; The cooling back adds the growth promoter of 1～2ml filtration sterilization: pyridoxine hydrochloride 0.01～0.02g, vitamin H 0.01～0.02g, coenzyme A 0.1～0.2g, Triphosaden 0.1～0.2g, catalase 0.1～0.2g, α-Nai Yisuan 0.1～0.2g, peptone 1～2g, casein hydrolysate 1～2g, glucose 10～20g, the bovine serum albumin v5 factor 100～200g, fungistat (the penbritin of mycobacterium culture supernatant 5～10ml and 0.1～0.2ml, amphotericin B, PXB, how the distilled water solution of pyridine ketone acid and azlocillin 50 μ g～100 μ g/ml), draw 3～4ml liquid nutrient medium and join in every pipe solid medium, make the quick growth medium of mycobacterium tuberculosis that not only contains solid but also contain liquid.
3. bacterial strain to be identified is mixed with 10
-2Mg/ml draws 0.1ml respectively and is inoculated in thiophene-2-carboxylic acid hydrazine, p-nitrobenzoic acid diphase Roe's differential medium and does not contain in the liquid of control medium of identification reagent, is inclined in 35～37 ℃ of incubators, hatches for 1～2 week, every day observations once; Bacteria particles in the liquid of substratum, occurs and/or bacterium colony occurs being bacterial growth at the solid culture primary surface; Growing in thiophene-2-carboxylic acid hydrazine differential medium as bacterial strain to be identified, do not grow in the p-nitrobenzoic acid differential medium, then is mycobacterium tuberculosis; As all growths in thiophene-2-carboxylic acid hydrazine and two kinds of differential mediums of p-nitrobenzoic acid, then be non-tuberculous mycobacteria.
Embodiment 3: the preparation of diphase Roe's drug sensitive culture medium and application
1. weigh vazadrine 0.5～1mg/ml, Rifampin 2～4mg/ml, Streptomycin sulphate 1～2mg/ml, Tibutol 1～2mg is with sterile distilled water dissolving (Rifampin dissolves with dimethyl formamide), adding distil water is to 100ml again, the working concentration that makes each medicine is vazadrine 0.005～0.01mg/ml, Rifampin 0.02～0.04mg/ml, Streptomycin sulphate 0.01～0.02mg/ml, Tibutol 0.01～0.02mg/ml, aseptic suction filtration.Draw the antitubercular agent of 0.1～0.2ml respectively, in the aseptic liquid that joins the quick growth medium of mycobacterium tuberculosis for preparing, be the diphase Roe's drug sensitive culture medium.
2. the quick growth medium preparation method of mycobacterium tuberculosis who had not only contained solid but also contained liquid is with embodiment 2.
3. bacterial strain to be checked is mixed with 10
-2Mg/ml draws 0.1ml respectively and is inoculated in the diphase Roe's drug sensitive culture medium and does not contain in the liquid of control medium of medicine, is inclined in 35～37 ℃ of incubators, hatches for 1～2 week, every day observations once; Bacteria particles in the liquid of substratum, occurs and/or bacterium colony occurs being bacterial growth at the solid culture primary surface; Grow in the control medium of pastille not as bacterial strain to be checked, do not grow in the substratum of pastille, then this bacterial strain is the medicaments insensitive strain; As bacterial strain to be checked all growths in the substratum of the control medium of pastille not and pastille, then this bacterial strain is a persister.
Claims (10)
1. diphase Roe's is differentiated or drug sensitive culture medium, and it is characterized in that: this substratum is made up of solid medium and liquid nutrient medium.
2. differentiate or drug sensitive culture medium by the described diphase Roe's of claim 1, it is characterized in that: described diphase Roe's is differentiated or the solid medium of drug sensitive culture medium is made up of the component of following weight proportion:
Copper sulfate 0.01～0.02g, zinc sulfate 0.01～0.02g, calcium chloride 0.01～0.02g, sal epsom 0.01～0.02g, Ferric Ammonium Citrate 0.05～0.1g, Sodium Citrate 0.1～0.2g, ammonium sulfate 0.2～0.4g, Sodium.alpha.-ketopropionate 0.5～1g, asparagine 1～2g, potassium primary phosphate 1～2g, SODIUM PHOSPHATE, MONOBASIC 2～4g, neutral glycerine 8～12ml, 1% malachite green solution, 10～20ml, ovum gallinaceum liquid 1000ml, sterile distilled water 600ml;
Described liquid nutrient medium comprises basal liquid, growth promoter, fungistat and identification reagent or antitubercular agent, wherein,
(1) basal liquid is made up of the component of following weight proportion:
Copper sulfate 0.005～0.01g, zinc sulfate 0.005～0.01g calcium chloride 0.005～0.01g, sal epsom 0.005～0.01g, oleic acid 0.01～0.02g, Ferric Ammonium Citrate 0.01～0.02g, Sodium Citrate 0.05～0.1g, ammonium sulfate 0.1～0.2g, Sodium.alpha.-ketopropionate 0.25～0.5g, tween-80 0.5～1g, asparagine 0.5～1g, potassium primary phosphate 0.5～1g, SODIUM PHOSPHATE, MONOBASIC 1～2g use dissolved in distilled water;
(2) growth promoter is made up of following weight proportion component:
Pyridoxine hydrochloride 0.01～0.02g, vitamin H 0.01～0.02g, coenzyme A 0.1～0.2g, Triphosaden 0.1～0.2g, catalase 0.1～0.2g, α-Nai Yisuan 0.1～0.2g, peptone 1～2g, casein hydrolysate 1～2g, glucose 10～20g, the bovine serum albumin v5 factor 100～200g, mycobacterium culture supernatant 5～10ml;
(3) fungistat is selected from penbritin, amphotericin B, PXB, the distilled water solution mixture of pyridine ketone acid and azlocillin 0.05～0.1mg/ml how;
(4) identification reagent is selected from thiophene-2-carboxylic acid hydrazine or p-nitrobenzoic acid;
(5) antitubercular agent is selected from vazadrine, Rifampin, Streptomycin sulphate and Tibutol.
3. differentiate or drug sensitive culture medium that by the described diphase Roe's of claim 2 it is characterized in that: the concentration of described identification reagent thiophene-2-carboxylic acid hydrazine is 0.1～0.2mg/ml; The concentration of p-nitrobenzoic acid is 0.5～1mg/ml.
4. differentiate or drug sensitive culture medium by the described diphase Roe's of claim 2, it is characterized in that: the working concentration of described antitubercular agent is respectively the vazadrine: 0.005～0.01mg/ml, Rifampin: 0.02～0.04mg/ml, Streptomycin sulphate: 0.01～0.02mg/ml, Tibutol: 0.01～0.02mg/ml.
5. the described diphase Roe's of claim 1 is differentiated or the preparation method of drug sensitive culture medium, it is characterized in that by following method and step preparation:
1) component of getting described weight proportion prepares solid and liquid nutrient medium respectively;
2) draw the liquid nutrient medium that makes, add in the solid medium;
3) above-mentioned 2) the liquid of substratum in add thiophene-2-carboxylic acid hydrazine and p-nitrobenzoic acid respectively, make the diphase Roe's differential medium;
Or,
4) above-mentioned 2) the liquid of substratum in add antitubercular agent vazadrine, Rifampin, Streptomycin sulphate and Tibutol respectively, make the diphase Roe's drug sensitive culture medium.
6. by the method for claim 5, it is characterized in that, draw step 2) liquid nutrient medium 3～4ml, add in the solid medium.
7. by the method for claim 5, it is characterized in that the thiophene-2-carboxylic acid hydrazine of step 3) and the add-on of p-nitrobenzoic acid are respectively 0.1～0.2ml.
8. by the method for claim 5, it is characterized in that the add-on of the antitubercular agent of step 4) is respectively 0.1～0.2ml.
9. the diphase Roe's of claim 1 is differentiated or drug sensitive culture medium is being differentiated the purposes that detects in the mycobacterium tuberculosis, and wherein, described purposes is by following step:
1) bacterial strain to be identified is configured to 10
-2Mg/ml;
2) drawing 0.1ml bacterium liquid to be checked respectively is inoculated in thiophene-2-carboxylic acid hydrazine, p-nitrobenzoic acid differential medium and does not contain in the liquid of contrast culture pipe of identification reagent;
3) above-mentioned culture tube is tilting, 35～37 ℃ of 1～2 weeks of incubation;
4) observations bacteria particles occurs and/or bacterium colony occurs at solid surface being the bacterial growth positive in the liquid of substratum;
5) bacterial strain to be checked is grown in thiophene-2-carboxylic acid hydrazine differential medium, in the p-nitrobenzoic acid differential medium, do not grow, and then be mycobacterium tuberculosis; All growths then are non-tuberculous mycobacteria in thiophene-2-carboxylic acid hydrazine and two kinds of differential mediums of p-nitrobenzoic acid.
10. the diphase Roe's of claim 1 is differentiated or the purposes of drug sensitive culture medium in detecting the mycobacterium tuberculosis susceptibility, and wherein, described purposes is by following step:
1) bacterial strain to be checked is mixed with 10
-2Mg/ml;
2) drawing 0.1ml bacterium liquid to be checked respectively is inoculated in diphase Roe's drug sensitive culture medium that contains different antitubercular agents and the liquid of the control medium that does not contain medicine;
3) above-mentioned culture tube is tilting, 35～37 ℃ of 1～2 weeks of incubation;
4) observations bacteria particles occurs and/or bacterium colony occurs at the solid culture primary surface being the bacterial growth positive in the liquid medium within;
5) grow in the control medium of pastille not as bacterial strain to be checked, do not grow in the substratum of pastille, then this bacterial strain is the medicaments insensitive strain; As bacterium all growths in the control medium of pastille not and the substratum that contains, then this bacterial strain is a persister.
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| CNA2007100481764A CN101168766A (en) | 2007-11-13 | 2007-11-13 | Diphase Roe's authentication or drug sensitive culture medium |
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| CN102191196A (en) * | 2011-03-24 | 2011-09-21 | 湖南省天骑医学新技术有限公司 | Mycobacterium tuberculosis solid/liquid culture medium and use method thereof |
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