CN1226602A - Fast culture media for mycobaterium tuberculosis and preparation thereof - Google Patents

Abstract

A quick culture medium of bacillus tubercle for testing the existence of bacillus tubercle is prepared from potassium dihydrogen phosphate, magnesium sulfate, magnesium citrate, ammonium irion citrate, asparagusamide, neutral glycerin, potato starch, fresh egg liquid, 2% aqueous solution of malachite green, water, animal's serum, coenzyme A, adhenosine triphosphate, cytochrome B and alpha-naphthaleneacetic acid through mixing, making paste, conditioning, packing, fusion sealing and sterilization. It features long storage period, standardized production, quick test and high correctness.

Description

Fast culture media for mycobaterium tuberculosis and preparation method thereof

The invention provides a kind of fast culture media for mycobaterium tuberculosis and preparation method thereof, it belongs to tubercule bacillus and has the special culture medium technical field of measuring.

China generally uses the solid tubercule bacillus substratum of a kind of being referred to as " modified Russell medium " to come tubercule bacillus is carried out the vitro human work point from cultivation at present, and its composition is: " asparagine 0.72 gram, potassium primary phosphate 0.96 gram, citrate of magnesia 0.12 gram, sal epsom (MgSO ₄7H ₂O) 0.048 gram, 2.4 milliliters of neutral glycerines, 120 milliliters in water, potato powder 6 grams, new fresh hen egg 6-8 only, 8 milliliters of 1% malachite green solutions "; Its method for making is: " after asparagine, potassium primary phosphate, citrate of magnesia and glycerine added water, place the boiling water bath heating for dissolving.Add potato powder again, continued in boiling water bath heating 30 minutes, the time add stirring, make into even pasty state.When waiting to be chilled to 65 ℃, add 8 milliliters of 200 milliliters of fresh liquid whole eggs and 1% Victoria Green WPBs, fully stir evenly the back and filter with double gauze, be sub-packed in then in the Boiling tube, 10 milliliters of every pipes are used the soft rubber ball jam-pack, are set to long inclined-plane.With 85 ℃, 150 minutes mobile vapor sterilizations ".On the 454th page of " clinical medical inspection " that these contents are documented in that army hospital general, Foochow compiles, Zhu Zhongyong Chen Zhihang chief editor, Shanghai science tech publishing house publish in April, 1978.Owing to existing solid tubercule bacillus substratum all is not have off-the-shelf by each hospital's deallocation system can purchase, this just needs school to acquire a cover corresponding production equipment separately for this reason, both strengthened the equipment expense of hospital, the corresponding again preparation workload that increased, by the above as can be known, the comparatively complicated and trouble of the existing process for preparation of solid tubercule bacillus substratum, some are standby so hospital can only give preparation earlier according to the roughly consumption of solid tubercule bacillus substratum at no distant date, but, the whole nation several thousand tame hospitals disperse the solid tubercule bacillus substratum prepared separately, and it is inconsistent all to exist quality standard, yield rate is not high, length is lost because of moisture content slightly to be difficult for long storage and storage period, medicine decomposes and makes the inaccurate defective of detected result.Find in the clinical detection that there is following defective in existing solid tubercule bacillus substratum: 1, be deposited in the Boiling tube owing to existing solid tubercule bacillus substratum, mouth of pipe soft rubber ball jam-pack, so degree sealed during this nutrient media storage 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 produce in enormous quantities by the suitability for industrialized production means, and can only prepare voluntarily by hospital, therefore, hospital's preparation is only arranged and there is no industrialization product at China's solid tubercule bacillus substratum at present.2, as everyone knows, the required condition of the growth and breeding of tubercule bacillus is higher, in its substratum the multiple nutrients material need be 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 to accelerate the tubercule bacillus metabolic activity in addition in it to improve the energy matter and the plant growth stimulating material of tubercule bacillus growth and breeding speed yet, make tubercule bacillus in existing solid tubercule bacillus substratum, can not carry out growth and breeding well like this, mitotic cycle reaches 12-16 hour, want 4-6 week can see bacterium colony, therefore, use existing solid tubercule bacillus substratum to come tubercule bacillus is carried out the vitro human work point when cultivating, its detection speed is quite slow, and positive rate is low, is unfavorable for early diagnosis and therapy lungy.

The object of the present invention is to provide a kind of fast culture media for mycobaterium tuberculosis and preparation method thereof, it is to adopt basic substance, energy matter, plant growth stimulating material mixed preparing to form, and it is long to have a storage period, is easy to standardized production, detect fast the superiority that positive rate is high.

Fast culture media for mycobaterium tuberculosis provided by the present invention, it is to be formed by basic substance, energy matter, plant growth stimulating material mixed preparing, and potassium primary phosphate, sal epsom (MgSO are arranged in the basic substance ₄7H ₂O), citrate of magnesia, ferric ammonium citrate, asparagine, neutral glycerine, potato powder, fresh egg liquid, 2% malachite green solution, water, animal serum, coenzyme A is arranged in the energy matter, Triphosaden, Nitrosylferricytochrome C, in the plant growth stimulating material α-Nai Yisuan is arranged, fresh egg liquid 560-600 gram sal epsom 0.136-0.14 restrains 2% malachite green solution 11-13 gram citrate of magnesia 0.3-0.4 gram water 300-340 gram ferric ammonium citrate 0.028-0.03 gram animal serum 56-60 gram asparagine 2.06-2.1 gram coenzyme A 0.4-0.8 gram neutral glycerine 6-8 gram Triphosaden 0.04-0.08 gram potato powder 17-17.5 gram Nitrosylferricytochrome C 0.045-0.075 gram α-Nai Yisuan 0.05-0.1 milligram to each components contents for potassium primary phosphate 1.36-1.4 restrains in per 1000 gram fast culture media for mycobaterium tuberculosis.Described animal serum can be any one in sheep blood serum, bovine serum, porcine blood serum, the human serum.

Described water can replace with the beef of equivalent weight or ox, Pigs Hearts Digestive system, also bentiamine 2 can be arranged in the described energy matter, also can have 2 in the described plant growth stimulating material, 4-fenac and 6-furfuryl group chloro-purine, the content of said components is in per 1000 gram fast culture media for mycobaterium tuberculosis: bentiamine 2 0.25-0.75 gram 2,4 dichloro benzene acetate 0.05-0.15 gram 6-furfuryl group chloro-purine 5-15 milligram.

Prepare the method for per 1000 gram fast culture media for mycobaterium tuberculosis, it in turn includes the following steps:

(a), paste producing:

After the potato powder of 17-17.5 gram added 150-170 gram water, place boiling water bath to heat, the time add stirring, make into even pasty state, reduce to after the room temperature standby naturally;

(b), mixing preparation:

Potassium primary phosphate with the 1.36-1.4 gram, 0.136-0.14 the sal epsom of gram, 0.3-0.4 the citrate of magnesia of gram, 0.028-0.03 the ferric ammonium citrate of gram, 2.06-2.1 the asparagine of gram, after the neutral glycerine of 6-8 gram 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 the 560-600 gram, the animal serum of 56-60 gram, 0.4-0.8 the coenzyme A of gram, 0.04-0.08 the Triphosaden of gram, 0.045-0.075 the Nitrosylferricytochrome C of gram, 0.05-0.1 the α-Nai Yisuan of milligram, after fully stirring evenly, 2% malachite green solution that potato is stuck with paste and 11-13 restrains that adds the 167-187.5 gram again, 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 day, 80 ℃, 30 minutes.

Above-mentioned (a) and (b) two steps also can use (e), (f) two steps to replace simultaneously:

(e), paste producing:

After the Ma Lingzhu powder of 17-17.5 gram added 150-170 gram beef or ox, Pigs Hearts Digestive system, place boiling water bath to heat, the time add stirring, make into even pasty state, reduce to after the room temperature standby naturally

(f), mixing preparation:

Potassium primary phosphate with the 1.36-1.4 gram, 0.136-0.14 the sal epsom of gram, 0.3-0.4 the citrate of magnesia of gram, 0.028-0.03 the ferric ammonium citrate of gram, 2.06-2.1 the asparagine of gram, the neutral glycerine of 6-8 gram adds 150-170 gram beef or ox, behind the Pigs Hearts Digestive system, place the boiling water bath heating for dissolving, when waiting to be chilled to below 50 ℃, the fresh egg liquid that adds the 560-600 gram, the animal serum of 56-60 gram, 0.4-0.8 the coenzyme A of gram, 0.04-0.08 the Triphosaden of gram, 0.045-0.075 the Nitrosylferricytochrome C of gram, 0.25-0.75 the bentiamine 2 of gram, 0.05-0.1 the α-Nai Yisuan of milligram, 0.05-0.15 2 of gram, the 4-fenac, the 6-furfuryl group chloro-purine of 5-15 milligram, after fully stirring evenly, 2% malachite green solution that potato is stuck with paste and 11-13 restrains that adds the 167-187.5 gram again, and after fully stirring evenly, get 1000 gram mixtures.

Described animal serum can be any one in sheep blood serum, bovine serum, porcine blood serum, the human serum.

When preparing fast culture media for mycobaterium tuberculosis of the present invention, used vessel, test tube must use behind autoclaving.

Used egg is cleaned eggshell with clear water earlier when making fresh egg liquid, with 75% alcohol immersion 30 minutes, squeezes in the sterilising vessel after drying with sterile gauze.

In the ox or on the composition of ox, Pigs Hearts Digestive system and method for making are documented in that army hospital general, Foochow compiles, Zhu Zhongyong Chen Zhihang chief editor, Shanghai science tech publishing house publish in April, 1978 " clinical medical inspection " the 440th page.

Get the fast culture media for mycobaterium tuberculosis of the inventive method preparation by the surveyor and can implement the tubercule bacillus detection patient, need not rely on each hospital preparation chamber at all and give first deallocation system, also need not remove the special equipment of preparing a cover in order to the preparation fast culture media for mycobaterium tuberculosis, so reduced the equipment expense of hospital, order should alleviate the preparation workload again; In addition, the fast culture media for mycobaterium tuberculosis of the inventive method preparation can now be opened existing usefulness, so wasting phenomenon can not occur, it is long slightly because of moisture content scatters and disappears, medicine decomposes the phenomenon that makes detected result inaccurate storage period can not occur, therefore, guarantee the accurate and effective that detects to a certain extent, improved quality of medical care.

Fast culture media for mycobaterium tuberculosis of the present invention and preparation method thereof has following advantage:

(1), fast culture media for mycobaterium tuberculosis of the present invention is to adopt basic substance, energy matter, plant growth stimulating material, by extensive mixing production technology, formulated through paste producing, mixing preparation, packing and sealing by fusing, sterilization steps, like this, the invention provides a kind of method that adopts the suitability for industrialized production means to prepare fast culture media for mycobaterium tuberculosis, make fast culture media for mycobaterium tuberculosis be easy to standardized production, filled up a blank of China's medicine industry; Fast culture media for mycobaterium tuberculosis of the present invention as mentioned above is to adopt the sealing by fusing method to be deposited in the Boiling tube, and degree sealed during this nutrient media storage is fairly good, makes that the moisture content in this substratum is difficult for scattering and disappearing, so prolonged the shelf-life of this substratum greatly.

(2), as mentioned above, contain basic substance in the fast culture media for mycobaterium tuberculosis of the present invention, comprise 9 kinds of all components in the existing solid tubercule bacillus substratum in the basic substance, they are: phosphoric acid dichloro potassium, sal epsom (MgSO ₄7H ₂O), citrate of magnesia, asparagine, neutral glycerine, potato powder, fresh egg liquid, 2% malachite green solution, water, preceding 8 kinds of component roles are respectively: potato powder, fresh egg liquid is a nutrition, contain phosphorus in the yolk, phosphatide and some salts, in the egg albumen energy and the toxicity of lipid acid and shock absorption is arranged, citrate of magnesia, the neutral glycerine supplementary carbon source, salts such as sal epsom are supplied with magnesium, elements such as potassium, the effect of regulating osmotic pressure is arranged, make the cytolemma of tubercule bacillus that good permeability be arranged, asparagine is a nitrogenous source, can promote growth and the breeding of tubercule bacillus, potassium primary phosphate is a buffer reagent, regulate the substratum pH value, 2% malachite green solution can suppress the growth of the assorted bacterium of part in the sample, in addition, also have ferric ammonium citrate and animal serum in the basic substance, and the water in the basic substance is the beef or the ox of available equivalent weight also, the Pigs Hearts Digestive system replaces, ferric ammonium citrate can provide certain irony required to satisfy the tubercule bacillus growth and breeding, the X somatomedin is arranged in the animal serum, V somatomedin and a large amount of high-protein nutrient materials, beef or ox, the Pigs Hearts Digestive system can increase bacterium as tubercule bacillus and cultivate usefulness, therefore, fast culture media for mycobaterium tuberculosis of the present invention is compared with existing solid tubercule bacillus substratum, kind of nutritive substance is many in it, and the growth and breeding that it can be tubercule bacillus provides the sufficient nutrient material;

Also contain energy matter and plant growth stimulating material in the fast culture media for mycobaterium tuberculosis of the present invention, energy matter can quicken the metabolic activity of tubercule bacillus, can improve the growth and breeding speed of tubercule bacillus, the plant growth stimulating material can promote the growth of plant, and tubercule bacillus is the same with vegetable cell, the plant growth stimulating material cell walls all arranged, so can improve the growth and breeding speed of tubercule bacillus equally;

In sum, existingly in the fast culture media for mycobaterium tuberculosis of the present invention satisfy the required multiple nutrients material of tubercule bacillus growth and breeding, have again and can accelerate energy matter and the plant growth stimulating material of tubercule bacillus metabolic activity to improve its growth and breeding speed, make the structure bacterium in fast culture media for mycobaterium tuberculosis of the present invention, can carry out growth and breeding well like this, and mitotic cycle shortens dramatically, therefore, use fast culture media for mycobaterium tuberculosis of the present invention to come tubercule bacillus is carried out the vitro human work point when cultivating, its detection speed is quite fast, the positive rate height helps early diagnosis and therapy lungy.

Detect through the fast culture media for mycobaterium tuberculosis to the inventive method preparation, its steady quality is reliable, and preservation period was more than 1 year, was easy to standardized production, was convenient to circulation, storage and clinical use; From using the rough estimates of this fast culture media for mycobaterium tuberculosis clinically, the detection speed that detects tubercule bacillus with this fast culture media for mycobaterium tuberculosis is 1-2 week, and the positive rate height.

The invention will be further described below in conjunction with three embodiment of the present invention:

Embodiment one:

Prepare the method for per 1000 gram fast culture media for mycobaterium tuberculosis, it in turn includes the following steps:

Paste producing:

After the potato powders of 17 grams are added 167.5 gram beef or oxen, Pigs Hearts Digestive system, place boiling water bath to heat, the time add stirring, make into even pasty state, reduce to after the room temperature standby naturally;

Mixing preparation:

Potassium primary phosphate with 1.36 grams, 0.136 the sal epsom of gram, 0.3 the citrate of magnesia of gram, 0.028 the ferric ammonium citrate of gram, 2.06 the asparagine of gram, the neutral glycerine of 6 grams adds 167.5 gram beef or oxen, behind the Pigs Hearts Digestive system, place the boiling water bath heating for dissolving, when waiting to be chilled to 40 ℃, the fresh egg liquid that adds 570.32595 grams, the sheep blood serum of 56 grams, 0.4 the coenzyme A of gram, 0.04 the Triphosaden of gram, 0.045 the Nitrosylferricytochrome C of gram, 0.25 the bentiamine 2 of gram, 0.05 the α-Nai Yisuan of milligram, 0.05 2 of gram, the 4-fenac, 5 milligrams 6-furfuryl group chloro-purine, after fully stirring evenly, the potato that adds 184.5 grams is again stuck with paste and 11 2% malachite green solutions that restrain, and gets 1000 gram mixtures after fully stirring evenly.

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;

Sterilization:

Adopt serum coagulator that Boiling tube is sterilized, sterilising conditions is: head day, 85 ℃, 30 minutes; Next day, 80 ℃, 30 minutes; The 3rd, 80 ℃, 30 minutes, get product.

Embodiment two:

Prepare the method for per 1000 gram fast culture media for mycobaterium tuberculosis, it in turn includes the following steps:

Paste producing:

After the potato powders of 17.25 grams are added 155 gram water, place boiling water bath to heat, the time add stirring, make into even pasty state, reduce to after the room temperature standby naturally;

Mixing preparation:

Potassium primary phosphate with 1.38 grams, 0.138 the sal epsom of gram, 0.35 the citrate of magnesia of gram, 0.029 the ferric ammonium citrate of gram, 2.08 the asparagine of gram, after the neutral glycerine of 7 grams adds 155 gram water, place the boiling water bath heating for dissolving, when waiting to be chilled to 45 ℃, the fresh egg liquid that adds 591.052925 grams, the bovine serum of 58 grams, 0.6 the coenzyme A of gram, 0.06 the Triphosaden of gram, 0.06 the Nitrosylferricytochrome C of gram, 0.075 the α-Nai Yisuan of milligram, after fully stirring evenly, the potato that adds 172.25 grams is again stuck with paste and 12 2% malachite green solutions that restrain, and gets 1000 gram mixtures after fully stirring evenly;

Packing and sealing: (summary);

Sterilization: (summary).

The processing step of packing and sealing, sterilization and processing condition thereof are identical with embodiment one.

Embodiment three:

Prepare the method for per 1000 gram fast culture media for mycobaterium tuberculosis, it in turn includes the following steps:

Paste producing:

After the potato powders of 17.5 grams are added 150 gram water, place boiling water bath to heat, the time add stirring, make into even pasty state, reduce to after the room temperature standby naturally;

Mixing preparation:

Potassium primary phosphate with 1.4 grams, 0.14 the sal epsom of gram, 0.4 the citrate of magnesia of gram, 0.03 the ferric ammonium citrate of gram, 2.1 the asparagine of gram, after the neutral glycerine of 8 grams adds 150 gram water, place the boiling water bath heating for dissolving, when waiting to be chilled to 48 ℃, the fresh egg liquid that adds 596.4749 grams, the porcine blood serum of 60 grams, 0.8 the coenzyme A of gram, 0.08 the Triphosaden of gram, 0.075 the Nitrosylferricytochrome C of gram, 0.1 the α-Nai Yisuan of milligram, after fully stirring evenly, the potato that adds 167.5 grams is again stuck with paste and 13 2% malachite green solutions that restrain, and gets 1000 gram mixtures after fully stirring evenly;

Packing and sealing: (summary);

Sterilization: (summary).

The processing step of packing and sealing, sterilization and processing condition thereof are identical with embodiment one.

Claims (4)

1, fast culture media for mycobaterium tuberculosis is characterized in that: it is to be formed by basic substance, energy matter, plant growth stimulating material mixed preparing, and potassium primary phosphate, sal epsom (MgSO are arranged in the basic substance ₄7H ₂O), citrate of magnesia, ferric ammonium citrate, asparagine, neutral glycerine, potato powder, fresh egg liquid, 2% malachite green solution, water, animal serum, coenzyme A is arranged in the energy matter, Triphosaden, Nitrosylferricytochrome C, in the plant growth stimulating material α-Nai Yisuan is arranged, fresh egg liquid 560-600 gram sal epsom 0.136-0.14 restrains 2% malachite green solution 11-13 gram citrate of magnesia 0.3-0.4 gram water 300-340 gram ferric ammonium citrate 0.028-0.03 gram animal serum 56-60 gram asparagine 2.06-2.1 gram coenzyme A 0.4-0.8 gram neutral glycerine 6-8 gram Triphosaden 0.04-0.08 gram potato powder 17-17.5 gram Nitrosylferricytochrome C 0.045-0.075 gram α-Nai Yisuan 0.05-0.1 milligram to each components contents for potassium primary phosphate 1.36-1.4 restrains in per 1000 gram fast culture media for mycobaterium tuberculosis.

2, a kind of method for preparing fast culture media for mycobaterium tuberculosis is characterized in that: prepare the method for per 1000 gram fast culture media for mycobaterium tuberculosis, it in turn includes the following steps: (a), paste producing:

After the potato powder of 17-17.5 gram added 150-170 gram water, place boiling water bath to heat, the time add stirring, make into even pasty state, reduce to after the room temperature standby naturally; (b), mixing preparation:

Potassium primary phosphate with the 1.36-1.4 gram, 0.136-0.14 the sal epsom of gram, 0.3-0.4 the citrate of magnesia of gram, 0.028-0.03 the ferric ammonium citrate of gram, 2.06-2.1 the asparagine of gram, after the neutral glycerine of 6-8 gram 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 the 560-600 gram, the animal serum of 56-60 gram, 0.4-0.8 the coenzyme A of gram, 0.04-0.08 the Triphosaden of gram, 0.045-0.075 the Nitrosylferricytochrome C of gram, 0.05-0.1 the α-Nai Yisuan of milligram, after fully stirring evenly, 2% malachite green solution that potato is stuck with paste and 11-13 restrains that adds the 167-187.5 gram again, 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 day, 80 ℃, 30 minutes.

3, fast culture media for mycobaterium tuberculosis according to claim 1, it is characterized in that: the beef of described water usefulness equivalent weight or ox, Pigs Hearts Digestive system replace, bentiamine 2 is arranged in the described energy matter, have 2 in the described plant growth stimulating material, 4-fenac and 6-furfuryl group chloro-purine, the content of said components is in per 1000 gram fast culture media for mycobaterium tuberculosis: bentiamine 2 0.25-0.75 gram 2,4 dichloro benzene acetate 0.05-0.15 gram 6-furfuryl group chloro-purine 5-15 milligram.

4, a kind of method for preparing fast culture media for mycobaterium tuberculosis according to claim 2 is characterized in that: (a) and (b) use (e), (f) to replace simultaneously in 2: (e), paste producing:

After the potato powder of 17-17.5 gram added 150-170 gram beef or ox, Pigs Hearts Digestive system, place boiling water bath to heat, the time add stirring, make into even pasty state, reduce to standby (f), mixing preparation after the room temperature naturally:

Potassium primary phosphate with the 1.36-1.4 gram, 0.136-0.14 the sal epsom of gram, 0.3-0.4 the citrate of magnesia of gram, 0.028-0.03 the ferric ammonium citrate of gram, 2.06-2.1 the asparagine of gram, the neutral glycerine of 6-8 gram adds 150-170 gram beef or ox, behind the Pigs Hearts Digestive system, place the boiling water bath heating for dissolving, when waiting to be chilled to below 50 ℃, the fresh egg liquid that adds the 560-600 gram, the animal serum of 56-60 gram, 0.4-0.8 the coenzyme A of gram, 0.04-0.08 the Triphosaden of gram, 0.045-0.075 the Nitrosylferricytochrome C of gram, 0.25-0.75 the bentiamine 2 of gram, 0.05-0.1 the α-Nai Yisuan of milligram, 0.05-0.15 2 of gram, the 4-fenac, the 6-furfuryl group chloro-purine of 5-15 milligram, after fully stirring evenly, 2% malachite green solution that potato is stuck with paste and 11-13 restrains that adds the 167-187.5 gram again, and after fully stirring evenly, get 1000 gram mixtures.