CN1103893A - Dry substratum for Mycobacterium and the prodn. method - Google Patents
Dry substratum for Mycobacterium and the prodn. method Download PDFInfo
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- CN1103893A CN1103893A CN 93112623 CN93112623A CN1103893A CN 1103893 A CN1103893 A CN 1103893A CN 93112623 CN93112623 CN 93112623 CN 93112623 A CN93112623 A CN 93112623A CN 1103893 A CN1103893 A CN 1103893A
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Abstract
This invented mycobacteria dry culture medium relates to selected culture medium for determining microbe existence, and is prepd. by compounding of phosphate, citrate, amino acid, gluconate, glucose, magnesium salt, sodium acetonate, pyrimidine, zinc salt, sodium selenate, calcium pantothenate, malachite green, DNA, calcium succinate, yeast powder, starch and agar, and by large scale production technique of dry breeding, through dehydration, adsorption, drying, and ball milling. It has the advantages of simple operation, quick detecting, high positive detection rate, low price, easy standardization production, and convenient transport and storing.
Description
Dry substratum for Mycobacterium of the present invention and production method thereof relate to microorganism and have the selective medium of measuring.
Tuberculosis is a kind ofly to be present in one of very high communicable disease of world wide sickness rate, though be that a kind of cause of disease is clear and definite, has controlled way, the anti-disease kind that measure is arranged, and crucial problem is still wants early discovery, and in time treatment could not have anxiety.Therefore, differentiating as early as possible and diagnose is this sick first step of control.
In traditional X radioactive rays radiodiagnosis, OT test and microbial culture three big diagnosis of tuberculosis technology, find that with microbial culture the tubercule bacillus growth is the most direct especially.And microbial culture detection technique itself, what need to solve is the supply problem of high-quality, inexpensive mycobacterium growth substratum.At present, existing substratum is most because reasons such as " raw material sources difficulty ", " being difficult to obtain pure bacterium liquid ", " complex operation ", " observing inconvenience ", " it is low to detect positive rate " can not get popularization.With Luo Shi egg base substratum and nutrient agar is the solid medium of representative, though be that current mycobacterium separation and Culture and susceptibility detect topmost means, limitation is arranged all.It is the substratum of vehicle and nutritive ingredient with the egg that Russell medium, modified Russell medium, sour Russell medium, Sodium.alpha.-ketopropionate substratum, Xiao Chuanshi substratum are tradition, process for preparation is loaded down with trivial details, can only prepare in a small amount, mass discrepancy is big, medicine easily takes place add defectives such as thermal failure and protein adsorption consumption, be difficult to guarantee the accuracy that detects, and need with serum coagulator etc., be unfavorable for that basic unit and non-special hospital apply; The Michaelis substratum is to be the substratum of vehicle with agar, owing to need bovine serum albumin V part (Fv) and peroxidase, cost an arm and a leg, and need CO2gas incubator and special bacteriological filtration equipment, only can use in developed country, the vast third world can't promote.In recent years, because the vital role of pyrazinoic acid amide (PZA) in SCC caused scholars' pay attention to day by day, drug sensitive test has difficulties to PZA in view of above-mentioned traditional substratum, therefore, seeking a kind of easy, reliable method, to detect susceptibility extremely urgent, in view of the best sterilization acidity of PZA requires at pH5.6, this characteristic gives that a kind of strain growth of development is good, the result accurately substratum increased difficulty.
The objective of the invention is to provide a kind of and have easy and simple to handlely, detect fast, the positive rate height, cheap, be convenient to transport dry substratum for Mycobacterium with preservation.
The present invention realizes like this, substratum is a vehicle with agar and starch, add the required carbon source of mycobacterium growth, nitrogenous source, phosphoric acid salt, vitriol and and micro-stimulating growth factor set is got on well with others and other assistant agents constitute, the weight proportion of its each main ingredient is: potassium primary phosphate 68-88, Sodium phosphate dibasic 10-15, Triammonium citrate 10.5-14, trisodium citrate 9-11, ferric ammonium citrate 0.5-1.1, L-L-glutamic acid or aspartic acid 10.5-14.5, Sunmorl N 60S or Potassium Gluconate 10.5-14.5, glucose 45-48, sal epsom or magnesium chloride 1.2-2.5, Sodium.alpha.-ketopropionate 10.5-14.5, uridylic is or/and thymus pyrimidine 0.8-1.0,-adenosine phosphate (AMP) or guanylic acid (GMP) 0.4-0.6, zinc sulfate or zinc chloride 0.2-0.68, Sodium Metabisulfite 2-3, sodium selenate 0.05-0.16, calcium pantothenate 0.04-0.05, Victoria Green WPB 0.05-0.16, DNA0.10-0.50, calcium succinate 3-4.5, yeast powder 4.6-10.0, starch 150-270, agar 487-664.
Its preparation method is: take by weighing each component raw material of the present invention respectively by above-mentioned weight ratio, prepare substratum of the present invention with the following step:
1, inorganic salt such as phosphoric acid salt, vitriol are put respectively in the baking box and are toasted down at 100 ℃, slough crystal water;
2, Victoria Green WPB is adsorbed in above-mentioned phosphoric acid salt, grind evenly, receive the end with 10-50 part starch;
3, with above-mentioned Citrate trianion, L-L-glutamic acid, gluconate, glucose, magnesium salts, Sodium.alpha.-ketopropionate is put to grind in the pulverizer and is spared, and 80-90 ℃ was dried by the fire 1-2 hour in baking box, added starch 10-50 part and mixed;
4, DNA with minimum distilled water heating for dissolving, with minimum agar absorption, puts in the baking box and dried by the fire 24 hours under 80-90 ℃, adds the extractum carnis identical with the agar amount, an amount of ball milling of peptone and yeast powder, 300 rev/mins, mixing 2 hours;
5, with above-mentioned pyrimidine, Nucleotide, zinc salt, Sodium Metabisulfite, sodium selenate, calcium pantothenate and calcium succinate grind evenly, put 37 ℃ of incubators and spend the night;
6, get each composition after the above-mentioned processing, supply agar and starch, behind the yeast powder aequum, Place grinding machine mixing, 300 rev/mins * 1 hour, left alone without help half an hour, ball milling again, 4 times repeatedly, take out and put 80 ℃ of baking oven bakings 1 hour, promptly get the dehydrated medium of the present invention of pale yellow powder shape.Product is packed with the Al-plastic sealing bag, wouldn't pack and then put the Plastic Bottle sealing, places the dry place of lucifuge and preserves.
When using basal culture medium, only need to add an amount of glycerine and distilled water, mixing, sterilization is 15-20 minute under High Temperature High Pressure, add calf serum after the cooling again, do not need with specific equipments such as serum coagulators, then, can carry out the separation and Culture of mycobacterium, the work of evaluation with reference to Chinese anti-tuberculosis association bacteriological standard working specification.Owing to used phosphate buffered saline buffer, the pH of substratum is stabilized in 5.6 ± 0.10, for detecting, the PZA susceptibility provides optimum acidity, and can scale operation, and eliminate mass discrepancy, thereby provide reliable assurance for the accuracy of detected result.Separation and Culture through being used for mycobacterium proves that strain growth is rapid, and six weeks can go out report, were used for the PZA drug sensitive test, and most of bacterial strains can report in three weeks that then needed for 10 weeks with sour Russell medium, the present invention also can react the true resistance of bacterial strain to medicine.So substratum of the present invention, and production method, have easy and simple to handlely, detect fast, the positive rate height, cheap, be easy to standardized production, be convenient to transport the advantage with preservation, for instructing routine administration and PZA clinical application to be significant.
Following examples will be further described technical characterictic of the present invention.
Embodiment 1, and with potassium primary phosphate 68 gram, SODIUM PHOSPHATE, MONOBASIC 10 restrains in baking box 100 ℃ of bakings 4 hours, sloughs crystal water; Victoria Green WPB 0.05 gram is adsorbed in above-mentioned phosphoric acid salt, grinds evenly, receive the end with 50 gram starch; Take by weighing Triammonium citrate 10.5 grams, trisodium citrate 9 grams, ferric ammonium citrate 0.5 gram, L-L-glutamic acid 10.5 grams, Sunmorl N 60S 10.5 grams, glucose 45 grams, sal epsom 1.2 grams, Sodium.alpha.-ketopropionate 10.5 grams, put to grind in the pulverizer and spare, 80-90 ℃ was dried by the fire 1 hour in the baking box, added starch 50 and restrained mixing; Take by weighing the DNA0.10 gram, with minimum distilled water heating for dissolving, be adsorbed in minimum agar, 80-90 ℃ of baking box dried by the fire 24 hours, the extractum carnis of adding and agar equivalent, peptone and 1-2 gram yeast powder, ball milling, 300 rev/mins, mixing 2 hours; Take by weighing uridylic 0.8 gram, adenylic acid 0.4 gram, zinc sulfate 0.2 gram, Sodium Metabisulfite 2 grams, sodium selenate 0.05 gram, calcium pantothenate 0.04 gram and calcium succinate 3 grams grind evenly, put 37 ℃ of incubators and spend the night; Get the composition after the above-mentioned processing, supply agar and starch aequum, Place grinding machine mixing, 300 rev/mins * 1 hour, every half an hour, 4 times repeatedly, take out and put 80 ℃ of baking oven bakings 1 hour, pack with the Al-plastic sealing bag, wouldn't pack and then put 500 gram Plastic Bottle sealings, place the dry place of lucifuge and preserve.
Embodiment 2, and with potassium primary phosphate 68 gram, SODIUM PHOSPHATE, MONOBASIC 10 restrains in baking box 100 ℃ of bakings 4 hours, sloughs crystal water; Victoria Green WPB 0.05 gram is adsorbed in above-mentioned phosphoric acid salt, grinds evenly, receive the end with 50 gram starch; Take by weighing Triammonium citrate 10.5 grams, trisodium citrate 9 grams, ferric ammonium citrate 0.5 gram, aspartic acid 10.5 grams, Potassium Gluconate 10.5 grams, glucose 45 grams, magnesium chloride 1.2 grams, Sodium.alpha.-ketopropionate 10.5 grams, put to grind in the pulverizer and spare, 80-90 ℃ was dried by the fire 1 hour in the baking box, added starch 50 and restrained mixing; Take by weighing the DNA0.10 gram, with minimum distilled water heating for dissolving, be adsorbed in minimum agar, 80-90 ℃ of baking box dried by the fire 24 hours, the extractum carnis of adding and agar equivalent, peptone and 1-2 gram yeast powder, ball milling, 300 rev/mins, mixing 2 hours; Take by weighing thymus pyrimidine 0.8 gram, guanylic acid 0.4 gram, zinc chloride 0.2 gram, Sodium Metabisulfite 2 grams, sodium selenate 0.05 gram, calcium pantothenate 0.04 gram and calcium succinate 3 grams grind evenly, put 37 ℃ of incubators and spend the night; Get the composition after the above-mentioned processing, supply agar and starch to full dose, Place grinding machine mixing, 300 rev/mins * 1 hour, every half an hour, 4 times repeatedly, take out and put 80 ℃ of baking oven bakings 1 hour, pack with the Al-plastic sealing bag, wouldn't pack and then put 500 gram Plastic Bottle sealings, place the dry place of lucifuge and preserve.
Embodiment 3-15, operating procedure are with embodiment 1, and each components contents of substratum is:
Embodiment 13, and the present invention is used for the mycobacterium separation and Culture,
One, bacterium source: ox type, mycobacterium tuberculosis avium reference culture derive from the U.S..All the other are provided by tuberculosis research department, this Qing of Ri Seto city.Clinical samples derives from Shanghai City and national various big hospital is accepted the patient for medical treatment.
Two, medium preparation and use
1, take by weighing 10 gram packing dehydrated medium pulvis, add 3～4 milliliters of glycerine, 250 milliliters of distilled water shake up, 115 ℃ of autoclavings 10 minutes;
2, to be cooled to 60-65 ℃, add 10 milliliters of calf serums, mixing is aseptic subpackaged, places the inclined-plane, solidifies back 37 ℃ and increases bacterium and spend the night stand-by.Room temperature or 4 ℃ of casees are preserved, and use in 4 weeks and finish;
3, handle sample with 2%NaOH, vibration digestion sputum is to transparent or semitransparent, and inoculation is finished in half an hour, and every 0.1 milliliter of 37 ℃ of incubator is cultivated, and observes once the 6th week report result weekly;
Three, colonial morphology
Keep humanoid tubercule bacillus essential characteristic, relatively bacterium colony is tiny slightly, moistening, smooth with traditional Russell medium, is surface growth.
Four, the result
1, reference culture is at the growth test of dehydrated medium
Human-like mycobacterium (H
37R
v) ox type bacterial strain and 9 strain atypia reference cultures at dehydrated medium and Russell medium relatively, big inoculum size (10 no matter
-3Mg/ml) and little inoculum size (10
-5Mg/ml) speed of growth and final bacterium are measured equal indifference:
2, the test of clinical samples separation and Culture
Use the 2%NaOH pre-treatment, with acid Luo Shi contrast, add up 1133 routine clinical samples, the dehydrated medium positive is 121 examples, and sour Luo Shi is 103 examples, statistical analysis P〉0.05, there was no significant difference:
Dehydrated medium amounts to 2068 routine samples through 6 tame hospitals, and relatively 5 kinds of substratum growth test results prove that its positive rate all is higher than Luo Shi and Xiao Chuanshi, only a little less than the Sodium.alpha.-ketopropionate substratum:
Show from above-listed three kinds of substratum growth curve comparison diagrams, the speed of growth of dehydrated medium, faster than egg base substratum, 6 weeks can go out report, and 3 Zhousheng reach the peak.And sour Luo Shi, Sodium.alpha.-ketopropionate substratum growth curve are similar, still have a small amount of positive after 8 weeks, and 4 Zhou Houcai peak.Dehydrated medium can shift to an earlier date two weeks to go out to report:
Embodiment 14, and dehydrated medium of the present invention is used for mycobacterium pyrazinoic acid amide (PZA) drug sensitive test:
1, bacterium source: ox type, mycobacterium tuberculosis avium derive from the U.S. in the type strain.All the other are provided by tuberculosis research department, this Qing of Ri Seto city.Clinical just, control bacterium source in the Shanghai City various big hospital again.
2, medium preparation
Basal liquid A: get 10 gram packing dehydrated medium pulvis, add 3 milliliters of glycerine, 250 milliliters of distilled water, heating for dissolving was sterilized 15 minutes for 10 pounds; Basal liquid B: the fresh calf serum 10ml that quarrels.
Treat that A liquid is cooled to 50-65 ℃, pour B liquid into mixing, add PZA medicinal powder respectively, get four groups of ultimate density 0,25,50,250 μ g/ml, the inclined-plane is placed in packing while hot.Solidify rearmounted 37 ℃ of incubators and increase bacterium and spend the night, room temperature preservation used up in 4 weeks.
3, working method
With reference to Chinese anti-tuberculosis association bacteriological standard working specification, to do each group of mycobacterium reference culture growth test and clinically just control the indirect drug sensitive test of bacterium again, the final inoculum size of drug sensitive test is 10
-3Mg/ml.
4, the result observes
Each is observed once in 2,3 weeks of inoculation back, and the 4th week went out report.Grow to more than "+" with blank pipe in principle, can report drug sensitivity tests.
158 strain clinical isolates PZA drug sensitive test results are by the aforesaid operations gained:
The result shows the first most sensitivities of bacterium of controlling, and controls resistant strains again and produces soon, can reach 48%, 1 year person's 90% resistance of medication in three months.
Each concentration pipe growing state of bacterium is just controlled in 118 strains
Claims (2)
1, dry substratum for Mycobacterium, it is characterized in that, substratum comprises: potassium primary phosphate 68-88 (weight part), Sodium phosphate dibasic 10-15 (weight part), Triammonium citrate 10.5-14 (weight part), trisodium citrate 9-11 (weight part), ferric ammonium citrate 0.5-1.1 (weight part), L-L-glutamic acid 10.5-14.5 (weight part), Sunmorl N 60S 10.5-14.5 (weight part), glucose 45-48 (weight part), sal epsom 1.2-2.5 (weight part), Sodium.alpha.-ketopropionate 10.5-14.5 (weight part), uridylic 0.8-1.0 (weight part), adenylic acid 0.4-0.6 (weight part), zinc sulfate 0.2-0.28 (weight part), Sodium Metabisulfite 2-3 (weight part), sodium selenate 0.05-0.16 (weight part), calcium pantothenate 0.04-0.05 (weight part), Victoria Green WPB 0.05-0.16 (weight part), DNA0.10-0.27 (weight part), calcium succinate 3-4.5 (weight part), yeast powder 4.6-10.0 (weight part), starch 150-270 (weight part), agar 487-664 (weight part).
2, the production method of dry substratum for Mycobacterium as claimed in claim 2: it is characterized in that: press the nutrient media components ratio requirement, take by weighing raw material respectively, prepare substratum of the present invention by following steps:
(1), inorganic salt such as phosphoric acid salt, vitriol are put respectively in the baking box and are toasted down at 100 ℃, slough crystal water and surface adsorption water;
(2), Victoria Green WPB is adsorbed in above-mentioned phosphoric acid salt, grinds evenly, receive the end with appropriate amount of starch;
(3), with Citrate trianion, amino acid, gluconate, glucose, magnesium salts, Sodium.alpha.-ketopropionate is put to grind in the pulverizer and is spared, and 80-90 ℃ was dried by the fire 1-2 hour in baking box, added appropriate amount of starch and mixed;
(4), DNA with minimum distilled water heating for dissolving, with minimum agar absorption, puts in the baking box and dried by the fire 24 hours under 80-90 ℃, adds the extractum carnis identical with the agar amount, an amount of ball milling of peptone and yeast powder, 300 rev/mins, mixing 2 hours;
(5), with pyrimidine, Nucleotide, zinc salt, Sodium Metabisulfite, sodium selenate, calcium pantothenate and calcium succinate grind evenly, put 37 ℃ of incubators and spend the night;
(6), get each composition after the above-mentioned processing, supply agar, starch, yeast to full dose, mix Place grinding machine mixing, 300 rev/mins * 1 hour, left alone without help half an hour, ball milling again, 4 times repeatedly, take out and put 80 ℃ of baking ovens bakings 1 hour, promptly get cultured product of the present invention;
(7) the dry place of lucifuge being put in above-mentioned substratum product sealing preserves.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93112623A CN1038851C (en) | 1993-12-14 | 1993-12-14 | Dry substratum for Mycobacterium and the prodn. method |
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| CN93112623A CN1038851C (en) | 1993-12-14 | 1993-12-14 | Dry substratum for Mycobacterium and the prodn. method |
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| CN1103893A true CN1103893A (en) | 1995-06-21 |
| CN1038851C CN1038851C (en) | 1998-06-24 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002046360A2 (en) * | 2000-11-03 | 2002-06-13 | Vladimir Vasiljevich Vlasenko | Methods for producing tuberculosis causative agent on a culture medium, culture medium for producing the tuberculosis causative agent, method for producing said medium and growth stimulator for the tuberculosis causative agent |
| CN100352936C (en) * | 2002-11-08 | 2007-12-05 | 林黎明 | Quick detecting method of maltphile olige unicellular bacteria and its culture medium composition used for detection |
| CN103215338A (en) * | 2013-04-25 | 2013-07-24 | 王洪生 | Mycobacterium culture medium and preparation method thereof |
| CN102449137B (en) * | 2008-12-05 | 2013-12-25 | 地中海(埃克斯-马赛第二)大学 | Mycobacteria culture medium and method including mycobacteria of mycobacterium tuberculosis complex |
| CN106854622A (en) * | 2016-12-27 | 2017-06-16 | 广东环凯生物科技有限公司 | The culture medium flat plate of pH stabilizations after a kind of irradiation sterilization |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100392096C (en) * | 2005-04-29 | 2008-06-04 | 胡忠义 | Two phase Roe's culture medium and preparation method thereof |
-
1993
- 1993-12-14 CN CN93112623A patent/CN1038851C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002046360A2 (en) * | 2000-11-03 | 2002-06-13 | Vladimir Vasiljevich Vlasenko | Methods for producing tuberculosis causative agent on a culture medium, culture medium for producing the tuberculosis causative agent, method for producing said medium and growth stimulator for the tuberculosis causative agent |
| WO2002046360A3 (en) * | 2000-11-03 | 2002-09-26 | Vladimir Vasiljevich Vlasenko | Methods for producing tuberculosis causative agent on a culture medium, culture medium for producing the tuberculosis causative agent, method for producing said medium and growth stimulator for the tuberculosis causative agent |
| CN100352936C (en) * | 2002-11-08 | 2007-12-05 | 林黎明 | Quick detecting method of maltphile olige unicellular bacteria and its culture medium composition used for detection |
| CN102449137B (en) * | 2008-12-05 | 2013-12-25 | 地中海(埃克斯-马赛第二)大学 | Mycobacteria culture medium and method including mycobacteria of mycobacterium tuberculosis complex |
| CN103215338A (en) * | 2013-04-25 | 2013-07-24 | 王洪生 | Mycobacterium culture medium and preparation method thereof |
| CN103215338B (en) * | 2013-04-25 | 2015-02-18 | 王洪生 | Mycobacterium culture medium and preparation method thereof |
| CN106854622A (en) * | 2016-12-27 | 2017-06-16 | 广东环凯生物科技有限公司 | The culture medium flat plate of pH stabilizations after a kind of irradiation sterilization |
| CN106854622B (en) * | 2016-12-27 | 2019-07-23 | 广东环凯生物科技有限公司 | PH stable culture medium flat plate after a kind of irradiation sterilization |
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| CN1038851C (en) | 1998-06-24 |
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