CN110903961A - Improved device and method for determining biological potency of a large number of antibiotic samples - Google Patents
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Abstract
The invention relates to an improved device and a method for measuring biological potency of a large number of antibiotic samples. When the biological potency is measured, the culture medium is placed into an open container to be solidified to prepare a flat plate, an oxford cup is placed on the flat plate and aligned to the intersection of lines, then an antibiotic standard substance solution and an antibiotic sample solution are dripped into the oxford cup, the diameter of an inhibition zone is measured after constant temperature culture, a standard curve is drawn according to the diameter of the inhibition zone, and the biological potency is calculated according to the standard curve. Compared with the prior art, the method saves the operation time, improves the working efficiency, ensures that the temperature of the square glass wooden plate culture dish is more stable, ensures that the observation of the measured biological antibacterial zone is clearer, and is very suitable for simultaneously measuring the biological potency of a large amount of antibiotic samples.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a device and a method for measuring biological potency of a large number of antibiotic samples.
Background
The microorganism detection method is a universal and classical antibiotic potency determination method in the world, and the antibiotic microorganism detection method is mainly a tube-disk method and a turbidity method specified in Chinese pharmacopoeia, wherein the tube-disk method mainly comprises a one-dose method, a two-dose method, a three-dose method and the like, and the two-dose method is most commonly used.
The tube-disc method for measuring potency is characterized by that it utilizes the diffusion action of antibiotic in agar culture medium, and makes the standard solution with known potency and sample solution with unknown potency be cultured on the culture medium on which the high-sensitive specific test bacteria are spread under the same condition, after a certain period of time, the antibiotic can be diffused in the culture medium and reached to proper range so as to produce transparent bacteriostatic zone for inhibiting growth of test bacteria. The titer of the sample can be calculated by comparing the diameters or the areas of the inhibition zones generated by the standard solution and the sample solution according to the calculation principles of different design methods.
The two-dose method is to dilute the antibiotic standard substance and the sample into two solutions with a certain concentration ratio (2: 1 or 4: 1), compare the drug resistance activities of the two solutions on the same culture dish, and calculate the biological potency of the sample according to the principle that the logarithm of the antibiotic concentration and the diameter of the zone of inhibition are in a linear relationship under a certain condition. The specific operation is that a certain amount of solid culture medium is poured on a culture dish after being melted on a water bath, a layer of melted culture medium mixed with high-sensitivity test bacteria is poured on the solid culture medium after the solid culture medium is solidified, and double dishes are prepared, wherein at least 4-6 double dishes are needed for each sample. After the upper layer culture medium is solidified, 4 Oxford cups are uniformly placed on the surface, solutions with high and low concentrations of a standard substance and a sample are dripped by diagonal lines, the double dishes are placed in an incubator after the dripping is finished, and after the culture medium is cultured for a proper time at a proper temperature, a transparent bacteriostatic ring appears on the surface of the culture medium. And measuring the biological potency of the sample by measuring the bacteriostatic circle diameters of the standard substance and the sample solution.
In the case of small samples, the above two-dose method was used with a small number of petri dishes to complete the assay. However, when a large number of antibiotic samples are measured, since at least 4 to 6 double dishes are required for each sample, the amount of the culture dish used is greatly increased, the operation efficiency is low, and it is difficult to measure the biological potency of a plurality of samples in the same culture dish. Therefore, in order to solve the problem of measuring the biological potency of a large number of samples, improve the working efficiency and shorten the working time, the method for simultaneously measuring the biological potency of a large number of samples is significant.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an improved device for measuring the biological potency of a large amount of antibiotic samples and a method for measuring by using the device.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the utility model provides a modified survey device of a large amount of antibiotic sample biological valence, is used for uncovered confined apron including uncovered container and lid on opening the container, and uncovered container includes bottom plate and the curb plate of setting on the bottom plate, and its characterized in that has the priming paint on the bottom plate, has the square of size with the line drawing on the priming paint.
The bottom plate is a wood plate, and the side plates and the cover plate are made of glass.
The upper surface of the wood board is provided with a groove which is enclosed into a rectangle, and the side plates are vertically arranged in the rectangular groove.
The primer is black paint, and the lines are white solid lines.
The invention also provides a method for measuring the biological potency of a large number of antibiotic samples by using the device, which comprises the following steps:
1) preparing an antibiotic standard solution and an antibiotic sample solution;
2) adding culture medium into an open container to prepare a flat plate;
3) placing the oxford cup on a flat plate and at the intersection of lines, after the oxford cup is stable, sequentially dripping the antibiotic standard substance solution and the antibiotic sample solution obtained in the step 1), and covering a cover plate;
4) carrying out constant temperature culture;
5) measuring the diameter of the bacteriostatic circle, calculating the average value of the diameter of the bacteriostatic circle, judging the quality of the prepared plate bacteriostatic circle, obtaining a standard curve according to the average value of the diameter of the bacteriostatic circle of the standard solution, and calculating the biological value of the sample according to the standard curve.
In the step 1), preparing an antibiotic standard solution includes: accurately weighing antibiotic standard substance, dissolving and diluting to a certain concentration gradient; preparing the antibiotic sample solution comprises: accurately weighing an antibiotic sample, and dissolving and diluting.
The concentration gradient of the antibiotic standard substance is at least 3-4 concentrations, and the antibiotic sample is dissolved and diluted into two dilution times; the solvent used for dissolving and diluting is deionized water, distilled water or 0.05-1.0 mol/L phosphate buffer solution.
In step 2), adding a culture medium into an open container to prepare a flat plate comprises the following steps: sucking a certain amount of hot-melt lower culture medium, adding the lower culture medium into an open container, quickly paving, solidifying, sucking a certain amount of upper culture medium mixed with indicator bacteria, adding the upper culture medium into the open container, quickly paving, and fully solidifying to obtain the flat plate.
In the step 3), the culture medium is ensured to be completely solidified before the oxford cup is placed; each oxford cup added with the standard solution is crossed and uniformly placed at the line intersection.
In the step 4), the constant-temperature culture refers to placing the open container covered with the cover plate into a constant-temperature incubator for culture, wherein the culture temperature is 20-40 ℃, and the culture time is 15-48 h.
Compared with the prior art, the invention has the beneficial effects that:
1) compared with the traditional method, the bottom surface of the square glass wood tray is made of wood, so that the heat preservation effect is better, and the condition that the culture medium is not spread smoothly due to rapid cooling can be avoided.
2) The wood bottom surface of the square glass-wood tray is sprayed with the black paint, so that the size of the antibacterial zone can be observed more conveniently.
3) According to the invention, the square glass-wood plate is engraved with the grids which are designed according to the inner size of the container and are separated by the thin white solid line and the thin white dotted line, so that the placement position of the Oxford cup is more accurate, the inhibition zone is easier to measure, and the numerical error is smaller.
4) By adopting the device and the method for measuring the biological potency of a large amount of antibiotic samples, the large amount of antibiotic samples can be measured, the operation time is effectively saved, and the operation efficiency is improved.
Drawings
The invention is further described below with reference to the accompanying drawings:
FIG. 1 is a schematic side view of the apparatus of the present invention;
FIG. 2 is a schematic top view of the apparatus of the present invention.
Detailed Description
In order to clearly understand the objects, technical solutions and technical effects of the present invention, the present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
The present invention provides, in a first aspect, an improved apparatus for determining the bio-potency of a plurality of antibiotic samples, as shown in fig. 1-2, which comprises an open container 1 and a cover 5 covering the open container 1 for closing the open container. Uncovered container 1 includes bottom plate 2 and sets up curb plate 4 on bottom plate 2, is provided with on bottom plate 2 to enclose into the recess 3 of rectangle, installs curb plate 4 in the recess 3, and the highly uniform of curb plate 4 is covered with rectangular apron 5 on the curb plate 4, and the distance that apron 5 outwards stretched out from every curb plate 4 is the same.
The bottom plate 2 is preferably made of wood, but other similar materials, such as teflon plate, solid wood, steel wood, marble, fiber composite board, metal plate, etc.;
preferably, the cover plate 5 and the side plate 4 are made of glass;
the base plate 2 is sprayed with the primer, so that the upper surface of the base plate is smooth and uniform, preferably black paint, or other colors which enable the bacteriostatic zone to be observed more clearly, such as red, white, orange and the like;
the bottom plate 2 is also sprayed with squares arranged according to the internal size of the open container, the squares are separated by thin white solid lines and thin white dotted lines, for example, 6 × 5 squares can be sprayed, but the invention is not limited thereto; the thin white dotted lines are used to subdivide the squares for ease of measurement.
The invention also provides a using method of the device, which comprises the following steps:
1) preparing an antibiotic standard solution and an antibiotic sample solution;
2) adding culture medium into an open container to prepare a flat plate;
3) placing the oxford cup on a flat plate at the intersection of the white solid lines, and after the oxford cup is stabilized, sequentially dripping the antibiotic standard substance solution and the antibiotic sample solution obtained in the step 1); covering the cover plate;
4) carrying out constant temperature culture;
5) measuring the diameter of the bacteriostatic circle, calculating the average value of the diameter of the bacteriostatic circle, judging the quality of the prepared plate bacteriostatic circle, obtaining a standard curve according to the average value of the diameter of the bacteriostatic circle of the standard solution, and calculating the biological value of the sample according to the standard curve.
Specifically, in the step 1), the preparation of the antibiotic standard solution comprises accurately weighing the antibiotic standard, and dissolving and diluting the antibiotic standard solution into a certain concentration gradient; preparing an antibiotic sample solution comprises accurately weighing an antibiotic sample, and dissolving and diluting;
wherein the gradient concentration of the standard substance is at least 3-4 concentrations, and the antibiotic sample is dissolved and diluted into two dilution times;
the solvent for dissolving and diluting is deionized water, distilled water or 0.05-1.0 mol/L phosphate buffer solution.
Specifically, the preparation of the flat plate in the step 2) comprises the following steps: firstly, sucking a certain amount of hot-melt lower layer culture medium, adding the hot-melt lower layer culture medium into an open container 1, quickly paving, after solidification, sucking a certain amount of upper layer culture medium mixed with indicator bacteria, adding the upper layer culture medium into the open container 1, quickly paving, and fully solidifying to obtain a flat plate;
specifically, in the step 3), the oxford cups are uniformly and stably placed on the flat plate obtained in the step 2), and the culture medium needs to be completely solidified before the oxford cups are placed; accurately placing the oxford cup at the intersection of the white solid lines on the wood board sprayed with the black paint, dropwise adding the antibiotic solution obtained in the step 2) in sequence after the oxford cup slightly sinks and is stable in the agar, and carefully covering a glass cover plate after dropwise adding;
specifically, in the step 4), the constant-temperature culture is carried out in a constant-temperature incubator; the temperature of constant temperature culture is set to be 20-40 ℃, and the culture time is 15-48 h.
Example 1
11 kasugamycin samples were diluted to sample solutions and standards were dissolved and diluted to concentration gradient standard solutions of 100U/mL, 50U/mL, 25U/mL, and 12.5U/mL. The alternaria alternate is used as an indicator bacterium. Pouring the culture medium on the upper layer and the lower layer into open containers with squares of 6 × 5 respectively, after the plate is fully solidified, accurately placing oxford cups at the intersection of the white solid lines of the squares, and adding standard solution parallel samples (4 × 2= 8) and 11 sample solution parallel samples (11 × 2= 22) into the oxford cups respectively for 30 samples. Culturing at 28 + -1 deg.C for 16-18 hr, measuring the diameter of each zone, calculating the average value of the diameters of the zones, calculating the standard curve of the plate, and calculating the biological potency of each sample according to the diameter of the zone.
Example 2
The standard was weighed out to dissolve and dilute into 100U/mL, 50U/mL, 25U/mL and 12.5U/mL concentration gradient standard solutions. Bacillus subtilis is used as indicator bacteria. And (3) respectively pouring upper and lower layers of culture medium into open containers with squares of 6 × 7, accurately placing oxford cups at the intersection of white squares and solid lines after the plates are fully solidified, and respectively adding 42 standard solution parallel samples (4 × 2= 8) and 17 zhongshengmycin sample solution parallel samples (17 × 2= 34) into the oxford cups. Culturing at 37 + -1 deg.C for 16-18 hr, measuring the diameter of each zone, calculating the standard curve of the plate, and calculating the biological potency of each sample.
Example 3
Bacillus subtilis is used as indicator bacteria. And (3) respectively pouring upper and lower layers of culture medium into open containers with squares of 6 multiplied by 7, accurately placing oxford cups at the intersection of white solid lines and solid lines of the squares after the plates are fully solidified, and carefully placing 42 kasugamycin single colony agar blocks at the center of each square of each plate. Culturing at 37 +/-1 ℃ for 16-18 hours, measuring the diameter of each inhibition zone, and observing and comparing by naked eyes to obtain a single colony with a larger inhibition zone diameter, namely the high-yield strain obtained by primary screening.
Example 4
Using alternaria leaf spot as an indicator bacterium. Weighing standard substance, dissolving and diluting to 100U/mL, 50U/mL, 25U/mL and 12.5U/mL concentration gradient standard solution, diluting 22 polyoxins to sample solution, respectively pouring upper and lower layer culture medium into two open containers with squares of 6 × 5, accurately placing an Oxford cup at the intersection of white solid lines of the squares after the flat plate is fully solidified, and respectively adding standard solution parallel samples (4 × 2 × 2= 16) and 22 sample solution parallel samples (11 × 2 × 2= 44) into the Oxford cup for 60 in total. Culturing at 31 + -1 deg.C for 20-22 hr, measuring the diameter of each zone, calculating the standard curve of the plate, and calculating the biological potency of each sample.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The utility model provides a modified survey device of a large amount of antibiotic sample biological valence, is used for uncovered confined apron including uncovered container and lid on opening the container, and uncovered container includes bottom plate and the curb plate of setting on the bottom plate, and its characterized in that has the priming paint on the bottom plate, has the square of size with the line drawing on the priming paint.
2. The improved apparatus for determining the biological potency of a plurality of antibiotic samples according to claim 1, wherein said bottom plate is a wooden plate and said side and cover plates are glass.
3. The improved apparatus for determining the biological potency of a plurality of antibiotic samples according to claim 2, wherein the upper surface of the wood board is provided with a groove surrounding a rectangle, and the side plate is vertically arranged in the rectangular groove.
4. The improved apparatus for determining the bio-potency of a plurality of antibiotic samples according to claim 2, wherein said primer is black paint and said line is a solid white line.
5. A method for determining the biological potency of a plurality of antibiotic samples using the device of claim 1, comprising the steps of:
1) preparing an antibiotic standard solution and an antibiotic sample solution;
2) adding culture medium into an open container to prepare a flat plate;
3) placing the oxford cup on a flat plate and at the intersection of lines, after the oxford cup is stable, sequentially dripping the antibiotic standard substance solution and the antibiotic sample solution obtained in the step 1), and covering a cover plate;
4) carrying out constant temperature culture;
5) measuring the diameter of the bacteriostatic circle, calculating the average value of the diameter of the bacteriostatic circle, judging the quality of the prepared plate bacteriostatic circle, obtaining a standard curve according to the average value of the diameter of the bacteriostatic circle of the standard solution, and calculating the biological value of the sample according to the standard curve.
6. The method of claim 5, wherein the step 1) of preparing the antibiotic standard solution comprises: accurately weighing antibiotic standard substance, dissolving and diluting to a certain concentration gradient; preparing the antibiotic sample solution comprises: accurately weighing an antibiotic sample, and dissolving and diluting.
7. The method of claim 6, wherein the antibiotic standard has a concentration gradient of at least 3-4 concentrations, and the antibiotic sample is solubilized and diluted at two dilution times; the solvent used for dissolving and diluting is deionized water, distilled water or 0.05-1.0 mol/L phosphate buffer solution.
8. The method of claim 5, wherein the step 2) of adding medium to the open container to form a plate comprises: sucking a certain amount of hot-melt lower culture medium, adding the lower culture medium into an open container, quickly paving, solidifying, sucking a certain amount of upper culture medium mixed with indicator bacteria, adding the upper culture medium into the open container, quickly paving, and fully solidifying to obtain the flat plate.
9. The method as claimed in claim 5, wherein in step 3), the culture medium must be completely solidified before the Oxford cup is placed; each oxford cup added with the standard solution is crossed and uniformly placed at the line intersection.
10. The method according to claim 5, wherein the constant temperature culture in step 4) is performed by placing the open container covered with the cover plate into a constant temperature incubator at 20-40 ℃ for 15-48 h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115343402A (en) * | 2022-07-28 | 2022-11-15 | 广州金域医学检验中心有限公司 | Method for detecting free testosterone |
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