CN101271117A - Method for measuring content of ayfivin and its preparations by nephelometry - Google Patents
Method for measuring content of ayfivin and its preparations by nephelometry Download PDFInfo
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Abstract
The invention discloses a method for measuring bacitracin and the content of the preparation thereof by the turbidity method, and the method contains the following steps: the preparation of standard solution, the preparation of test solution, the preparation of test organism suspension, the content measurement, etc. The antibiotics-microbial test and multi-dose turbidity method of the invention are adopted for measuring the bacitracin and the content of the preparation thereof, thus the test method is accurate and effective and has low cost, and test results can truly reflect antibacterial activity of the bacitracin and the preparation thereof; the method has stronger practicality and operability in the production and the quality control of the drug and the preparation thereof.
Description
Technical field
The present invention relates to a kind of assay method of antibiotic content, especially a kind of nephelometry is measured the method for bacitracin and formulation content thereof.
Background technology
The generation bacterium of bacitracin (Bacitracin) is bacillus subtilis (Bacilus subtilis) and liver moss sample bacillus (Bacilus Licheniformis), is the polypeptide class polycomponent microbiotic that contains thiazole ring.The bacillus Toplink suppresses the formation of principal ingredient linear peptides polysaccharide chains in the bacteria cell wall, also influence bioplast, destroy serous coat etc., the antibacterial activity that gram-positive bacteria is had height, bacterium seldom produces acquired resistance to this product, with neomycin (Neomycin), polymyxin B associating topical application such as (Polymyxin B) synergy is arranged.At present, bacitracin bulk drug and preparation thereof are by " Chinese pharmacopoeia two ones of versions in 2005 are recorded, its content assaying method is the cylinder-plate method of antibiotic-microbial assay, its ultimate principle is to utilize the diffusion of microbiotic in the agar medium of stand Bu Teding test organisms, form the certain density antibiotic ball-type district that contains, suppress the breeding of test organisms and presented transparent inhibition zone, known standard solution of tiring and unknown need testing solution of tiring are cultivated under similarity condition, compare both inhibition zone sizes, in certain antibiotic concentration scope, logarithm concentration is directly proportional with antibacterial circle diameter.
Cylinder-plate method is wanted strict control incubation time, prevents the influence of unsuitable incubation time to the sharpness of inhibition zone, because some microbiotic is bacteriostasis, flora at the inhibition zone edge just is suppressed, after prolonging incubation time, bacteria total amount increases, and the antibiotic sensitive degree is descended.It is irregular with the edge that the flora continued growth at inhibition zone edge causes inhibition zone to diminish, as when adopting bacillus subtilis as test organism, incubation time is long, and mycelia grows in circle, cause the edge irregular, but when adopting micrococcus luteus, after the temperature and time of regulation is cultivated, can put room temperature again and continue and cultivate 2-3 hour as test organism, can make inhibition zone clear, this may be the flora continued growth of inhibition zone edge, and chromogenesis, makes the inhibition zone edge fine and close and apparent clear.Just caused cylinder-plate method when measuring antibiotic content, the control of time to be had relatively high expectations, and consuming time longer like this.So the content that adopts cylinder-plate method to measure microbiotic such as bacitracin has following shortcoming: operation is difficult for controlling, the measurement result error is big, finding speed is slower, makes it have very big application limitation in commercial production.
Summary of the invention
The technical problem to be solved in the present invention provides the method that a kind of nephelometry is measured bacitracin and formulation content thereof, to measure bacitracin and formulation content thereof simply, fast and accurately.
For solving the problems of the technologies described above, the present invention adopts following method step: the preparation of (1), standard solution: take by weighing bacitracin standard items 2.5mg, be settled to 25ml with the 0.1M hydrochloric acid solution, make the standard items stock solution; Get above-mentioned standard items stock solution 5ml, be settled to 50ml, make the standard items intermediate solution with the sterilization damping fluid; With the sterilization damping fluid standard items intermediate solution is diluted to the 3-5 group solution of bacitracin standard items concentration between 0.1ug/ml-0.8ug/ml, make standard items series dosage solution, wherein contain the solution of 0.4ug/ml in the concentration of standard items series dosage solution, other solution concentration is dispersed in the 0.4ug/ml both sides;
(2), the preparation of need testing solution: take by weighing bacitracin sample 2.5mg, water is settled in the 25ml measuring bottle, makes the sample stock solution; Get above-mentioned sample stock solution 5ml, be settled to 50ml, make the sample intermediate solution with the sterilization damping fluid; Sample thief intermediate solution 4.0ml is settled to 100ml with the sterilization damping fluid, makes need testing solution;
(3), the preparation of test organisms suspension: get the Staphylococcus epidermidis slant culture, in bacteria culture media, cultivate, make the test organisms suspension after the dilution;
(4), assay: above-mentioned standard items series dosage solution and need testing solution are formed 4-6 concentration group, get the developmental tube of corresponding group number, in each developmental tube, all add test organisms suspension 9.0ml, add standard items or each 1.0ml of need testing solution of each concentration more respectively; Cultivate 3h-4h at 37 ℃, with the absorbance of each pipe of turbidimetry measuring instrument on-line determination at the 530nm place; Get 2 developmental tubes, wherein the phosphate buffer 1 .0ml of an adding pH=6.0 and antibiotic microorganism are examined and determine and are used medium liquid 9.0ml, as the blank of the solution of cultivating with condition; Another adds phosphate buffer 1 .0ml and the test organisms suspension 9.0ml of pH=6.0, as the positive control of cultivating with condition; To repeat above operation, make the 3-5 group and measure;
(5), on-line measurement and result of calculation promptly get the content of bacitracin and preparation thereof on microbiotic turbidimetry measuring instrument.
The present invention also can adopt following method step: the preparation of (1), standard solution: take by weighing bacitracin standard items 2.5mg, be settled to 25ml with 0.1M hydrochloric acid, make the standard items stock solution; Measure above-mentioned standard items stock solution 5ml, damping fluid is rare to be settled in the 25ml measuring bottle with sterilizing, and makes the standard items intermediate solution; With the sterilization damping fluid standard items intermediate solution is diluted to the 2 group solution of bacitracin standard items concentration between 0.1ug/ml-0.8ug/ml, makes standard items high dose solution and standard items low dosage solution;
(2), take by weighing bacitracin sample 2.5mg, water is settled to 25ml, makes the stock solution of sample; Measure above-mentioned stock solution 5ml, be settled to 25ml, make the sample intermediate solution; With sterilization damping fluid dilute sample intermediate solution, make test sample high dose solution identical and test sample low dosage solution with above-mentioned standard solution concentration;
(3), the preparation of test organisms suspension: get the Staphylococcus epidermidis slant culture, in bacteria culture media, cultivate, make the test organisms suspension after the dilution;
(4), assay: above-mentioned standard items series dosage solution and need testing solution are formed 4 concentration groups, get 6 developmental tubes, all add test organisms suspension 9.0ml in each developmental tube, add standard items or each 1.0ml of need testing solution of each concentration more respectively; Cultivate 3h-4h at 37 ℃, with the absorbance of each pipe of turbidimetry measuring instrument on-line determination at the 530nm place; Get 2 developmental tubes, wherein the phosphate buffer 1 .0ml of an adding pH=6.0 and antibiotic microorganism are examined and determine and are used medium liquid 9.0ml, as the blank of the solution of cultivating with condition; Another adds phosphate buffer 1 .0ml and the test organisms suspension 9.0ml of pH=6.0, as the positive control of cultivating with condition; To repeat above operation, make the 3-5 group and measure;
(5), on-line measurement and result of calculation promptly get the content of bacitracin and preparation thereof on microbiotic turbidimetry measuring instrument.
The present invention also can adopt following method step: the preparation of standard solution: take by weighing bacitracin standard items 2.5mg, be settled to 25ml with the 0.1M hydrochloric acid solution, make the standard items stock solution; Measure above-mentioned standard items stock solution 5ml, be settled to 50ml, make the standard items intermediate solution with the sterilization damping fluid; With the sterilization damping fluid standard items intermediate solution is diluted to the 3 group solution of bacitracin standard items concentration between 0.1ug/ml-0.8ug/ml, makes dosage solution and standard items low dosage solution in standard items high dose solution, the standard items;
(2), take by weighing bacitracin sample 2.5mg, add water and be settled to 25ml, make the sample stock solution; Measure sample stock solution 5ml, be settled to 50ml, make the sample intermediate solution with the sterilization damping fluid; With sterilization damping fluid dilute sample intermediate solution, make dosage solution and test sample low dosage solution in the test sample high dose solution consistent, the test sample with above-mentioned standard solution concentration;
(3), the preparation of test organisms suspension: get the Staphylococcus epidermidis slant culture, in bacteria culture media, cultivate, make the test organisms suspension after the dilution;
(4), assay: above-mentioned standard items series dosage solution and need testing solution are formed 6 concentration groups, get 6 developmental tubes, all add test organisms suspension 9.0ml in each developmental tube, add standard items or each 1.0ml of need testing solution of each concentration more respectively; Cultivate 3h-4h at 37 ℃, with the absorbance of each pipe of turbidimetry measuring instrument on-line determination at the 530nm place; Get 2 developmental tubes, wherein the phosphate buffer 1 .0ml of an adding pH=6.0 and antibiotic microorganism are examined and determine and are used medium liquid 9.0ml, as the blank of the solution of cultivating with condition; Another adds phosphate buffer 1 .0ml and the test organisms suspension 9.0ml of pH=6.0, as the positive control of cultivating with condition; To repeat above operation, make the 3-5 group and measure;
(5), on-line measurement and result of calculation promptly get the content of bacitracin and preparation thereof on microbiotic turbidimetry measuring instrument.
The present invention adopts nephelometry to measure the content of bacitracin and preparation thereof, and nephelometry is called turbidimetry again, and it determines the method for suspended material concentration for measuring the light intensity that sees through the particle in suspension medium, and this is a kind of light scattering measurement technology.When light beam contains the medium of particle in suspension by one, because particle in suspension weakens the transmission light intensity to scattering of light effect and optionally absorption.In nephelometry, the relation object of penetrability and suspended material concentration is similar to Lambert-Beer's law:
S is turbid luminosity (or turbid rate) in the formula, the light intensity decays that expression causes owing to the scattering process of suspension; I
0Be respectively incident intensity (passing through neat solvent) and transmitted intensity (by muddy sample) with I; B is an optical path length; C is the concentration of particle in suspension; K is a constant, is turbidity coefficient sometimes again, and its value is relevant with the refractive index of size, shape, incident light wavelength, suspension and the medium of particle.
Principle of the present invention is a certain amount of microbiotic to be added to inoculation have in the nutritious fluid nutrient medium of the clarification of testing microorganism, behind the mixing, at a certain temperature, Short-term Culture, it is muddy that nutrient culture media becomes, the increase of its muddy degree and bacterial population, there is direct relation between the increase of the increase of bacterial colonies quality and bacterial colonies cell volume, when certain light beam irradiates nutrient culture media, just know the situation of bacterial growth by measuring its transmitance, its absorbance and antibiotic concentration relation character composition and division in a proportion Er Dinglv, promptly in certain antibiotic concentration scope, dose response is a straight line.Therefore, in the linear extent of dose response response curve, can design and use the turbidimetry for Determination antibiotic content.Nephelometry is divided into a dosimetry, two dosimetries and three-dose method, and the present invention selects for use these three kinds of methods to measure the content of bacitracin and preparation thereof respectively.
The nephelometry that the present invention adopts adopts jolting to cultivate, to increase the supply of oxygen, promote bacterial growth, so the inventive method will shorten incubation time as far as possible, avoid because of leaving standstill in the incubation, bacterium slowly is settled down to the bottom, produces the growth of microorganism district of gradient, the surface is little oxytropism, and the bottom is an anaerobic.This method has just shortened the time of cultivating greatly like this, thereby has accelerated the speed of measuring.This assay method is simple, accurate, quick, in application widely and commercial production and the laboratory.
Antibiotic-microbial assay of the present invention-multiple dose nephelometry is to utilize the inhibiting effect of in the microbiotic liquid medium within test organisms being grown, by measuring the size of cultivating back bacterial turbidity value, standard of comparison product and test sample are to the growth inhibiting degree of test organisms, to measure a kind of method that test sample is tired.When adopting cylinder-plate method to measure content, be subjected to all multifactor impacts such as coefficient of diffusion, nutrient culture media viscosity, inhibition zone size, so error at measurment is bigger; And bacitracin system contains the polypeptide class polycomponent microbiotic of thiazole ring, form and divide complexity, and each component coefficient of diffusion difference in solid medium, inhibition zone very easily produces secondary loops, measures thereby influence, and result precision is descended greatly.
The employing nephelometry is measured, and is to carry out in the liquid medium within, can avoid the influence of above-mentioned factor; Measure the absorbance of bacterium liquid with fixed wave length, its precision is better than in the cylinder-plate method mensuration to inhibition zone.The training method of the inventive method adopts jolting to cultivate, to increase the supply of oxygen, promote bacterial growth, so the inventive method will shorten incubation time as far as possible, avoid because of leaving standstill in the incubation, bacterium slowly is settled down to the bottom, produces the growth of microorganism district of gradient, the surface is little oxytropism, and the bottom is an anaerobic.The present invention measures process 3h～4h, adds other operations, can obtain the result on the same day; And adopt cylinder-plate method to need second day ability to obtain the result.Therefore assay method of the present invention is more quick, accurate, when running into the dispute sample, adopts nephelometry as referee method.
Adopt a dosimetry and two dosimetries fast measuring product in a large number, be applicable to the fast detecting of manufacturing enterprise's intermediate, directive significance is arranged for the feeding intake of product, product synthetic etc.; The fast detecting that also can be used as departments such as hospital, customs is used.Adopting three-dose method to measure product, is the best and the highest detection method of the accuracy of product content, can be used as referee method.
Employing the inventive method is measured, and its result can truly reflect the antibacterial activity in vitro of test sample.Relevant manufacturing enterprise can optimize best synthesis and production process according to measurement result, therefore, antibiotic-microbial assay-multiple dose nephelometry instructs relevant manufacturing enterprise and produces, and further improves production technology, improves drug quality and has irreplaceable directive function.Not only test method is accurate and effective, with low cost for the content of employing antibiotic-microbial assay-multiple dose nephelometry mensuration bacitracin and preparation thereof, and test findings can truly reflect the antibacterial activity of bacitracin and preparation thereof, and this method has stronger practicality and operability in the production of this medicine and preparation thereof and quality control.
Below the present invention is further detailed explanation.
Embodiment
Experiment material: test organism adopts Staphylococcus epidermidis (Staph.Epidermidis) ATCC12228 of American Type Culture Collection public offering, and bacterial concentration is 6.0%-8.0%; Nutrient culture media adopts " the microbiotic calibrating nutrient culture media by the III pH=7.0-7.2 of Nat'l Pharmaceutical ﹠ Biological Products Control Institute's public offering of two regulations of Chinese pharmacopoeia version in 2005, the sterilization damping fluid is the phosphate buffer of pH=6.0, its compound method is: get dipotassium hydrogen phosphate 2g and potassium dihydrogen phosphate 8g, add water and make into 1000ml, filter, 115 ℃ of sterilizations 30 minutes, standby;
Process of the test:
(1) preparation 1 of standard solution (dosimetry): precision takes by weighing bacitracin standard items 2.5mg, puts in the 25ml measuring bottle, adds the 0.1M dissolve with hydrochloric acid solution and is diluted to scale, shakes up, and makes the stock solution that every 1ml contains the solution of bacitracin 100ug; Precision is measured above-mentioned stock solution 5ml, puts in the 50ml measuring bottle, is diluted to scale with the sterilization damping fluid, makes the intermediate solution that every 1ml contains the solution of bacitracin 10ug.Get this solution 1.0ml, 2.0ml, 4.0ml, 6.0ml and 8.0ml respectively, put and add the sterilization damping fluid in the 100ml volumetric flask and be diluted to scale as standard items series dosage solution; Making bacitracin standard items concentration is 0.1ug/ml, 0.2ug/ml, 0.4ug/ml, 0.6ug/ml, 0.8ug/ml totally 5 groups of standard items series dosage solution.Preferably bacitracin standard items concentration is made for 0.256ug/ml, 0.32ug/ml, 0.40ug/ml, 0.50ug/ml, 0.625ug/ml totally 5 groups of standard items series dosage solution, to obtain better testing result.
The preparation 2 of standard solution (two dosimetries): precision takes by weighing bacitracin standard items 2.5mg, puts in the 25ml measuring bottle, adds the 0.1M dissolve with hydrochloric acid solution and is diluted to scale, shakes up, and makes the stock solution that every 1ml contains the solution of bacitracin 100ug; Precision is measured above-mentioned stock solution 5ml, puts in the 25ml measuring bottle, is diluted to scale with the sterilization damping fluid, makes the intermediate solution that every 1ml contains the solution of bacitracin 20ug.Get this solution 2.0ml respectively, put add in the 100ml volumetric flask sterilization damping fluid be diluted to scale as standard items high dose solution; Put add in the 200ml volumetric flask sterilization damping fluid be diluted to scale as standard items low dosage solution; Making bacitracin standard items concentration is the standard items low dosage solution of 0.40ug/ml standard items high dose solution and 0.20ug/ml.
The preparation 3 (three-dose method) of standard solution: precision takes by weighing bacitracin standard items 2.5mg, puts in the 25ml measuring bottle, adds the 0.1M dissolve with hydrochloric acid solution and is diluted to scale, shakes up, and makes the stock solution that every 1ml contains the solution of bacitracin 100ug; Precision is measured above-mentioned stock solution 5ml, puts in the 50ml measuring bottle, is diluted to scale with the sterilization damping fluid, makes the intermediate solution that every 1ml contains the solution of bacitracin 10ug.Get this solution 3.6ml, 3.0ml, 2.4ml respectively, split add in the 100ml volumetric flask sterilization damping fluid be diluted to scale respectively as standard items high dose solution, middle dosage solution, low dosage solution; Make dosage solution and standard items high dose solution in standard items low dosage solution that bacitracin standard items concentration is respectively 0.24ug/ml, 0.30ug/ml, 0.36ug/ml, the standard items
(2) preparation 1 of need testing solution (dosimetry): precision takes by weighing bacitracin sample 2.5mg, puts in the 25ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, and makes the stock solution that every 1ml contains the solution of bacitracin 100ug; Precision is measured above-mentioned stock solution 5ml, puts in the 50ml measuring bottle, is diluted to scale with the sterilization damping fluid, makes the intermediate solution that every 1ml contains the solution of bacitracin 10ug.Get this solution 4.0ml respectively, put add in the 100ml volumetric flask sterilization damping fluid be diluted to scale as need testing solution;
The preparation 2 of need testing solution (two dosimetries): precision takes by weighing bacitracin sample 2.5mg, puts in the 25ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, and makes the stock solution that every 1ml contains the solution of bacitracin 100ug; Precision is measured above-mentioned stock solution 5ml, puts in the 25ml measuring bottle, is diluted to scale with the sterilization damping fluid, makes the intermediate solution that every 1ml contains the solution of bacitracin 10ug.Get this solution 2.0ml respectively, put add in the 100ml volumetric flask sterilization damping fluid be diluted to scale as test sample high dose solution; Put add in the 200ml volumetric flask sterilization damping fluid be diluted to scale as test sample low dosage solution;
The preparation 3 (three-dose method) of need testing solution: precision takes by weighing bacitracin sample 2.5mg, puts in the 25ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, and makes the stock solution that every 1ml contains the solution of bacitracin 100ug; Precision is measured above-mentioned stock solution 5ml, puts in the 50ml measuring bottle, is diluted to scale with the sterilization damping fluid, makes the intermediate solution that every 1ml contains the solution of bacitracin 10ug.Get this solution 3.6ml, 3.0ml, 2.4ml respectively, split add in the 100ml volumetric flask sterilization damping fluid be diluted to scale respectively as standard items high dose solution, middle dosage solution, low dosage solution;
(3) preparation of test organisms suspension: get test organisms Staphylococcus epidermidis slant culture, be inoculated in the nutrient agar, putting 35-37 ℃ cultivated after 16-18 hour, wash lawn with aseptic 0.9% sodium chloride solution, and add aseptic 0.9% sodium chloride solution dilution, make that its absorbance at 580nm wavelength place is 1.0.
(4) method of operating: 6 concentration groups of a dosimetry, 4 concentration groups of two dosimetries, 6 concentration groups of three-dose method, each concentration group are made the 3-5 group and are measured, and preferably make 5 groups of mensuration, to reduce error.In every group was measured, respectively contained Staphylococcus epidermidis (580nm A=1.0, test organisms suspension 9.0ml 6.0%-8.0%), accurate respectively again standard items or each 1.0ml of need testing solution that adds each concentration accurate the adding in each developmental tube.Cultivated 3-4 hour at 37 ℃,, measure the absorbance of each pipe at the 530nm place with turbidimetry measuring instrument on-line determination.After getting simultaneously the phosphate buffer 1 .0ml that 2 developmental tubes respectively add pH6.0 in addition, one adds liquid medium 9.0ml, as the blank of the solution of cultivating with condition; Another adds test organisms suspension 9.0ml, as the positive control of cultivating with condition.
(5) on-line measurement and result of calculation promptly get the content of bacitracin and preparation thereof on microbiotic turbidimetry measuring instrument.
Claims (10)
1, a kind of nephelometry is measured the method for bacitracin and formulation content thereof, it is characterized in that this method step is:
(1), the preparation of standard solution: take by weighing bacitracin standard items 2.5mg, be settled to 25ml, make the standard items stock solution with the 0.1M hydrochloric acid solution; Get above-mentioned standard items stock solution 5ml, be settled to 50ml, make the standard items intermediate solution with the sterilization damping fluid; With the sterilization damping fluid standard items intermediate solution is diluted to the 3-5 group solution of bacitracin standard items concentration between 0.1ug/ml-0.8ug/ml, make standard items series dosage solution, wherein contain the solution of 0.4ug/ml in the concentration of standard items series dosage solution, other solution concentration is dispersed in the 0.4ug/ml both sides;
(2), the preparation of need testing solution: take by weighing bacitracin sample 2.5mg, water is settled in the 25ml measuring bottle, makes the sample stock solution; Get above-mentioned sample stock solution 5ml, be settled to 50ml, make the sample intermediate solution with the sterilization damping fluid; Sample thief intermediate solution 4.0ml is settled to 100ml with the sterilization damping fluid, makes need testing solution;
(3), the preparation of test organisms suspension: get the Staphylococcus epidermidis slant culture, in bacteria culture media, cultivate, make the test organisms suspension after the dilution;
(4), assay: above-mentioned standard items series dosage solution and need testing solution are formed 4-6 concentration group, get the developmental tube of corresponding group number, in each developmental tube, all add test organisms suspension 9.0ml, add standard items or each 1.0ml of need testing solution of each concentration more respectively; Cultivate 3h-4h at 37 ℃, with the absorbance of each pipe of turbidimetry measuring instrument on-line determination at the 530nm place; Get 2 developmental tubes, wherein the phosphate buffer 1 .0ml of an adding pH=6.0 and antibiotic microorganism are examined and determine and are used medium liquid 9.0ml, as the blank of the solution of cultivating with condition; Another adds phosphate buffer 1 .0ml and the test organisms suspension 9.0ml of pH=6.0, as the positive control of cultivating with condition; To repeat above operation, make the 3-5 group and measure;
(5), on-line measurement and result of calculation promptly get the content of bacitracin and preparation thereof on microbiotic turbidimetry measuring instrument.
2, nephelometry according to claim 1 is measured the method for bacitracin and formulation content thereof, it is characterized in that the test organisms suspension prepares with following method: get test organisms Staphylococcus epidermidis slant culture, be inoculated in the nutrient agar, after putting 35 ℃-37 ℃ cultivation 16h-18h, wash lawn with aseptic 0.9% sodium chloride solution, and add aseptic 0.9% sodium chloride solution dilution, make that its absorbance at 580nm wavelength place is 1.0.
3, nephelometry according to claim 1 and 2 is measured the method for bacitracin and formulation content thereof, and it is 0.1ug/ml, 0.2ug/ml, 0.4ug/ml, 0.6ug/ml, 0.8ug/ml totally 5 groups of standard items series dosage solution that its feature is diluted to bacitracin standard items concentration with the sterilization damping fluid with the standard items intermediate solution.
4, nephelometry according to claim 1 and 2 is measured the method for bacitracin and formulation content thereof, and it is 0.256ug/ml, 0.32ug/ml, 0.40ug/ml, 0.50ug/ml, 0.625ug/ml totally 5 groups of standard items series dosage solution that its feature is diluted to bacitracin standard items concentration with the sterilization damping fluid with the standard items intermediate solution.
5, a kind of nephelometry is measured the method for bacitracin and formulation content thereof, it is characterized in that this method step is:
(1), the preparation of standard solution: take by weighing bacitracin standard items 2.5mg, be settled to 25ml, make the standard items stock solution with 0.1M hydrochloric acid; Measure above-mentioned standard items stock solution 5ml, damping fluid is rare to be settled in the 25ml measuring bottle with sterilizing, and makes the standard items intermediate solution; With the sterilization damping fluid standard items intermediate solution is diluted to the 2 group solution of bacitracin standard items concentration between 0.1ug/ml-0.8ug/ml, makes standard items high dose solution and standard items low dosage solution;
(2), the preparation of need testing solution: take by weighing bacitracin sample 2.5mg, water is settled to 25ml, makes the stock solution of sample; Measure above-mentioned stock solution 5ml, be settled to 25ml, make the sample intermediate solution; With sterilization damping fluid dilute sample intermediate solution, make test sample high dose solution consistent and test sample low dosage solution with above-mentioned standard solution concentration;
(3), the preparation of test organisms suspension: get the Staphylococcus epidermidis slant culture, in bacteria culture media, cultivate, make the test organisms suspension after the dilution;
(4), assay: above-mentioned standard items series dosage solution and need testing solution are formed 4 concentration groups, get 6 developmental tubes, all add test organisms suspension 9.0ml in each developmental tube, add standard items or each 1.0ml of need testing solution of each concentration more respectively; Cultivate 3h-4h at 37 ℃, with the absorbance of each pipe of turbidimetry measuring instrument on-line determination at the 530nm place; Get 2 developmental tubes, wherein the phosphate buffer 1 .0ml of an adding pH=6.0 and antibiotic microorganism are examined and determine and are used medium liquid 9.0ml, as the blank of the solution of cultivating with condition; Another adds phosphate buffer 1 .0ml and the test organisms suspension 9.0ml of pH=6.0, as the positive control of cultivating with condition; To repeat above operation, make the 3-5 group and measure;
(5), on-line measurement and result of calculation promptly get the content of bacitracin and preparation thereof on microbiotic turbidimetry measuring instrument.
6, nephelometry according to claim 5 is measured the method for bacitracin and formulation content thereof, it is characterized in that the test organisms suspension prepares with following method: get test organisms Staphylococcus epidermidis slant culture, be inoculated in the nutrient agar, after putting 35 ℃-37 ℃ cultivation 16h-18h, wash lawn with aseptic 0.9% sodium chloride solution, and add aseptic 0.9% sodium chloride solution dilution, make that its absorbance at 580nm wavelength place is 1.0.
7, measure the method for bacitracins and formulation content thereof according to claim 5 or 6 described nephelometries, it is characterized in that in the preparation process of standard solution, the standard items intermediate solution is diluted to the standard items low dosage solution that bacitracin standard items concentration is 0.40ug/ml standard items high dose solution and 0.2ug/ml with the sterilization damping fluid; The concentration of need testing solution high and low dose solution is identical with the concentration of the high low dosage solution of standard items.
8, a kind of nephelometry is measured the method for bacitracin and formulation content thereof, it is characterized in that this method step is:
(1), the preparation of standard solution: take by weighing bacitracin standard items 2.5mg, be settled to 25ml, make the standard items stock solution with the 0.1M hydrochloric acid solution; Measure above-mentioned standard items stock solution 5ml, be settled to 50ml, make the standard items intermediate solution with the sterilization damping fluid; With the sterilization damping fluid standard items intermediate solution is diluted to the 3 group solution of bacitracin standard items concentration between 0.1ug/ml-0.8ug/ml, makes dosage solution and standard items low dosage solution in standard items high dose solution, the standard items;
(2), the preparation of need testing solution: take by weighing bacitracin sample 2.5mg, add water and be settled to 25ml, make the sample stock solution; Measure sample stock solution 5ml, be settled to 50ml, make the sample intermediate solution with the sterilization damping fluid; With sterilization damping fluid dilute sample intermediate solution, make dosage solution and test sample low dosage solution in the test sample high dose solution identical, the test sample with above-mentioned standard solution concentration;
(3), the preparation of test organisms suspension: get the Staphylococcus epidermidis slant culture, in bacteria culture media, cultivate, make the test organisms suspension after the dilution;
(4), assay: above-mentioned standard items series dosage solution and need testing solution are formed 6 concentration groups, get 6 developmental tubes, all add test organisms suspension 9.0ml in each developmental tube, add standard items or each 1.0ml of need testing solution of each concentration more respectively; Cultivate 3h-4h at 37 ℃, with the absorbance of each pipe of turbidimetry measuring instrument on-line determination at the 530nm place; Get 2 developmental tubes, wherein the phosphate buffer 1 .0ml of an adding pH=6.0 and antibiotic microorganism are examined and determine and are used medium liquid 9.0ml, as the blank of the solution of cultivating with condition; Another adds phosphate buffer 1 .0ml and the test organisms suspension 9.0ml of pH=6.0, as the positive control of cultivating with condition; To repeat above operation, make the 3-5 group and measure;
(5), on-line measurement and result of calculation promptly get the content of bacitracin and preparation thereof on microbiotic turbidimetry measuring instrument.
9, nephelometry according to claim 8 is measured the method for bacitracin and formulation content thereof, it is characterized in that the test organisms suspension prepares with following method: get test organisms Staphylococcus epidermidis slant culture, be inoculated in the nutrient agar, after putting 35 ℃-37 ℃ cultivation 16h-18h, wash lawn with aseptic 0.9% sodium chloride solution, and add aseptic 0.9% sodium chloride solution dilution, make that its absorbance at 580nm wavelength place is 1.0.
10, according to Claim 8 or 9 described nephelometries measure the method for bacitracins and formulation content thereof, it is characterized in that in the preparation process of standard solution, the standard items intermediate solution is diluted to dosage solution and standard items high dose solution in standard items low dosage solution that bacitracin standard items concentration is respectively 0.24ug/ml, 0.30ug/ml, 0.36ug/ml, the standard items with the sterilization damping fluid; The concentration of dosage solution and test sample low dosage solution is identical with the concentration of the low solution of dosage solution and standard items dosage in standard items high dose solution, the standard items respectively in test sample high dose solution, the test sample.
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| CNA2008100549390A CN101271117A (en) | 2008-05-09 | 2008-05-09 | Method for measuring content of ayfivin and its preparations by nephelometry |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102333854A (en) * | 2009-02-25 | 2012-01-25 | 亚历法克斯控股有限公司 | Method for the bacteriological investigation of a biological sample and relative device |
| CN103913426A (en) * | 2013-01-08 | 2014-07-09 | 中国科学院生态环境研究中心 | Method for evaluation of residual biopotency of antibiotics in waste water |
| CN104250292A (en) * | 2013-06-26 | 2014-12-31 | 北京勤邦生物技术有限公司 | Preparation method and application of bacitracin antigen |
| CN107764754A (en) * | 2017-08-31 | 2018-03-06 | 厦门博露环保科技有限公司 | A kind of online test method of microbes biomass |
-
2008
- 2008-05-09 CN CNA2008100549390A patent/CN101271117A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102333854A (en) * | 2009-02-25 | 2012-01-25 | 亚历法克斯控股有限公司 | Method for the bacteriological investigation of a biological sample and relative device |
| CN102333854B (en) * | 2009-02-25 | 2013-11-27 | 亚历法克斯控股有限公司 | Method for bacteriological investigation of biological sample and relative device |
| CN103913426A (en) * | 2013-01-08 | 2014-07-09 | 中国科学院生态环境研究中心 | Method for evaluation of residual biopotency of antibiotics in waste water |
| CN103913426B (en) * | 2013-01-08 | 2016-03-02 | 中国科学院生态环境研究中心 | A kind of method assessing antibiotic relict usefulness in waste water |
| CN104250292A (en) * | 2013-06-26 | 2014-12-31 | 北京勤邦生物技术有限公司 | Preparation method and application of bacitracin antigen |
| CN107764754A (en) * | 2017-08-31 | 2018-03-06 | 厦门博露环保科技有限公司 | A kind of online test method of microbes biomass |
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