CN106467924B - Test method for evaluating in-vitro antibacterial activity of antibacterial substance - Google Patents
Test method for evaluating in-vitro antibacterial activity of antibacterial substance Download PDFInfo
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Abstract
The invention relates to the technical field of biological control, and particularly discloses a test method for evaluating in-vitro antibacterial activity of an antibacterial substance, which is characterized in that a methylene blue agar plate containing bacteria is used for in-vitro antibacterial activity test of an agar plate diffusion method. The method is visual and simple, and overcomes the defect that the diameter of the bacteriostatic circle is difficult to accurately measure due to deep color turbidity and complex components when the traditional agar plate diffusion method is used for in-vitro antibacterial tests of traditional Chinese medicines or other substances to be tested with similar properties by observing the colored bacteriostatic circle.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to a test method for evaluating in-vitro antibacterial activity of an antibacterial substance.
Background
In vitro antibacterial tests are tests for determining the sensitivity of microorganisms to certain substances in vitro, are commonly used for evaluating the activity of inhibiting and killing pathogenic bacteria, and are widely applied to scientific research, production and clinic, such as antibacterial drug screening, tracking in the extraction process, antibacterial spectrum determination, drug sensitivity tests for guiding clinical medication and the like. Agar plate diffusion is one of the most commonly used methods.
The traditional agar plate diffusion method is to coat the indicator bacteria on the surface of agar or uniformly mix the indicator bacteria into the agar, and then add the object to be detected by adopting a paper method, an oxford cup method, a punching method and other methods to diffuse the object to be detected in the agar. The agar is colorless and transparent, and the bacteria are colorless and translucent, so that the agar area where the bacteria do not grow is colorless and transparent. If the object to be tested has antibacterial activity, the growth of microorganisms such as bacteria and the like in the object to be tested is inhibited or killed, so that a transparent ring a1 (shown in figure 1) in which the microorganisms do not grow is formed in the range of effective concentration of the medicament, and the diameter of the transparent ring a1 is distinguished and measured in a better light environment to evaluate whether the object to be tested has the antibacterial activity or not and preliminarily evaluate the activity of the object to be tested. The transparent zone is called as a bacteriostatic zone, and the diameter of the bacteriostatic zone is directly related to the concentration of the antibacterial substance and the concentration of the indicating bacteria.
A large number of clinical application researches show that the traditional Chinese medicine has a non-negligible antibacterial effect. With the increasing severity of the problem of drug-resistant bacteria in clinical antibiotic application, the research on antibacterial traditional Chinese medicines has a wide application prospect. At present, no standardized method exists for the in vitro antibacterial activity research of traditional Chinese medicines, and an agar plate diffusion method is taken as one of international universal antibiotic antibacterial activity evaluation methods and is also widely introduced into the antibacterial activity measurement of other materials including traditional Chinese medicines. However, the Chinese medicine extract has special properties, the diffusion phenomenon in the agar plate is different from that of antibiotics, and the Chinese medicine in-vitro antibacterial test cannot completely follow the antibiotic in-vitro antibacterial test. The traditional Chinese medicine extracting solution has the main characteristics that: (1) color depth turbidity: the dark and turbid drug diffusion ring appears in the agar after the drug diffusion, and the ring is superposed with the transparent ring in which bacteria do not grow, so that the measurement of the diameter of the antibacterial ring is influenced, and the size of the antibacterial activity is difficult to visually evaluate. As shown in fig. 2: the diffusion antibacterial phenomenon of the deep-colored scutellaria baicalensis water extract is found in two holes of an agar plate (01) (02), wherein b1 is a dark brown traditional Chinese medicine diffusion ring (inner ring), and b2 is a light brown traditional Chinese medicine diffusion ring (outer ring); (03) (04) the antibiotic ceftriaxone sodium diffusion antibacterial phenomenon with colorless and clear two holes is adopted, wherein b3 is a transparent antibacterial ring, and the following results are shown: when the traditional flat agar diffusion method is adopted for carrying out an antibacterial test, the antibiotic is colorless and clear, the transparent bacteriostatic zone is not covered by a medicine diffusion zone, the measurement is easy, the transparent bacteriostatic zone is covered by the Chinese medicine with deep turbidity, and the bacteriostatic zone is difficult to distinguish and measure due to the fact that the bacteria non-growth area needs to be carefully or even cannot be observed; (2) the components are complex: antibiotics are generally single in composition, and the drugs are different only in concentration and the same in composition at a distance from the diffusion center. However, the traditional Chinese medicine extract and the like are complex in components, the diffusion speed of each component is different, and the concentration and the components of the medicine which are different at the far and near positions from the diffusion center are different, so that the size of the bacteria non-growth ring is not only related to the medicine concentration but also related to the complex components of the medicine.
In conclusion, the traditional agar plate diffusion test has the defects in the evaluation of the in vitro antibacterial activity of the traditional Chinese medicine and the tested object with similar properties, deep turbidity and complex components, and the in vitro antibacterial method which can more intuitively and effectively indicate the antibacterial activity of the traditional Chinese medicine has important application value for the development of antibacterial substances.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a test method for evaluating the in vitro antibacterial activity of an antibacterial substance, which can display the in vitro antibacterial activity of traditional Chinese medicine extracts with deep turbidity and complex components and antibacterial substances with similar properties in a layered manner, and is simple, convenient, intuitive and effective.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a test method for evaluating the in vitro antibacterial activity of antibacterial substance is to use the agar plate containing antibacterial methylene blue in the in vitro antibacterial activity test of agar plate diffusion method.
Preferably, the in vitro antibacterial activity test comprises the following specific steps: adding the solution of the object to be tested to a freshly prepared agar plate containing methylene blue, diffusing the solution of the object to be tested in the agar, culturing at constant temperature, observing and measuring the diameter of a blue circle around the object to be tested or a mixed colored circle of the color of the object to be tested and the blue color as the diameter of an antibacterial circle after the indicating bacteria and the object to be tested act, observing the color depth of the antibacterial circle, and evaluating the external antibacterial activity of the object to be tested.
Preferably, the preparation method of the agar plate containing the mycomethylene blue comprises the following steps: preparing a strain which is activated and grows for 6-8h by a slant culture medium into an indicator bacterium suspension, then quickly and uniformly mixing the indicator bacterium suspension with drug sensitive agar which is sterilized, cooled to 45 ℃ and melted and contains methylene blue, and cooling and solidifying to obtain the indicator bacterium.
Preferably, the content of methylene blue in the agar plate containing the mycomethylene blue is as follows: each 100mL of the mycomethylene blue-containing agar contained 2mL of a 1% methylene blue aqueous solution.
Preferably, the drug sensitive agar is M-H agar medium or other drug sensitive agar suitable for bacterial growth.
Preferably, the method for adding the solution to be detected into the agar plate containing the methylene blue adopts any one of a punching method, a paper sheet method and an oxford cup method.
Preferably, the action time of the indicator bacteria and the substance to be detected is 18 h.
The test method for evaluating the in vitro antibacterial activity of the antibacterial substance is applied to the colorless and clear antibacterial substance with single component.
The test method for evaluating the in vitro antibacterial activity of the antibacterial substance is applied to the antibacterial substance with the characteristics of traditional Chinese medicine, similar color depth turbidity and complex components.
The main principle of the invention is as follows: the substance to be tested diffuses in the agar and acts on bacteria in the agar, and the survival state of the bacteria influences the color of the agar plate containing the methylene blue. The oxidized and reduced states of methylene blue are blue and colorless, respectively. Methylene blue in agar before bacterial culture is usually in a blue-appearing oxidation state, so methylene blue containing plates are blue. After the object to be tested is added into a methylene blue bacterium-containing plate for constant temperature culture, the methylene blue enters bacterial cells along with the culture of bacteria, the reductase of the growing bacteria enables the methylene blue to discolor from blue to colorless in a reduced state, and the reductase which is killed or inhibited to be in a resting state is inactivated without generating the discoloring effect, so that the methylene blue is still blue. It can be seen that the colorless area in the bactromethene blue-containing agar plate is the bacterial growth area, the blue area is the area where the bacteria die or are inhibited (and the shade of blue is related to the activity of the bacteria). Comprehensively considering the color of the object to be detected diffused into the agar and the colored ring area of the blue color and the mixed color of the blue color and the color of the object to be detected as an antibacterial area, namely a bacteriostatic ring; the colorless area and the area which is only the color of the object to be tested are the antibacterial-free area.
Compared with the traditional agar plate diffusion method, the method has the following beneficial effects:
1. the influence of color depth turbidity on observation of a transparent bacteriostasis zone is improved: according to the invention, the inhibition and killing effects of the to-be-detected object on bacteria are visually displayed in blue, the colored area is observed by combining the color of the to-be-detected object solution, the size of the antibacterial effect is comprehensively evaluated, the influence of the color and turbidity of the to-be-detected object solution on the observation of the transparent antibacterial zone in the traditional agar plate diffusion method is effectively overcome, and the antibacterial effect can be visually and rapidly evaluated;
2. improving the influence of complex components on the diffusion of agar to be tested: the invention visually displays the antibacterial action of all components of the object to be tested through blue, and hierarchically displays a plurality of blue antibacterial rings or blue and self-color mixed antibacterial rings for the object to be tested with a plurality of antibacterial components, thereby overcoming the influence of different diffusion positions of different substances due to the complex components of the object to be tested such as traditional Chinese medicines in the traditional agar plate diffusion method and comprehensively evaluating the overall antibacterial activity of the object to be tested with complex components in a layered and objective way.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a transparent zone of inhibition of traditional agar plate diffusion antibiotics: a 1-transparent bacteriostatic circle;
FIG. 2 shows the traditional agar plate diffusion method for inhibiting bacteria by traditional Chinese medicine and antibiotics: b 1-dark brown traditional Chinese medicine diffusion ring (inner ring), b 2-light brown traditional Chinese medicine diffusion ring (outer ring), b 3-transparent bacteriostatic ring;
FIG. 3 shows the comparison of the diffusion results of the conventional agar plate method for antibiotics and the methylene blue agar plate method: FIG. 3-a, traditional agar plate diffusion method, d 1-transparent zone of inhibition; FIG. 3-b, methylene blue agar plate diffusion method, d 2-blue zone of inhibition;
FIG. 4 shows the comparison of the results of the traditional plate agar diffusion method and methylene blue agar diffusion method for aqueous extract of Chinese medicine: FIG. 4-a, diffusion of aqueous extract of mugwort leaf on agar plate, e 1-dark brown diffusion ring (inner ring) of Chinese medicinal materials, e 2-light brown diffusion ring (outer ring); FIG. 4-b, diffusion method of folium Artemisiae Argyi aqueous extract methylene blue agar plate, e 3-dark green Chinese medicinal bacteriostatic circle (inner circle), e 4-light green Chinese medicinal bacteriostatic circle (outer circle); FIG. 4-c, traditional agar plate diffusion of red peony aqueous extract, e 5-light brown Chinese medicine diffusion ring; 4-d, a radix paeoniae rubra water extract methylene blue agar plate diffusion method, e 6-blue-green Chinese medicine bacteriostasis ring;
FIG. 5 shows the results of methylene blue agar diffusion method for the antibacterial tests of the aqueous extracts of Schisandra chinensis and Prunus mume: FIG. 5-a, the results of tests on anti-Salmonella enteritidis by fructus Schisandrae water extractive solution, f 1-dark blue Chinese medicinal bacteriostatic loop (inner circle), and f 2-milky white Chinese medicinal diffusion loop (outer circle); FIG. 5-b shows the result of the test of the dark plum water extract for resisting Salmonella enteritidis, f 3-dark blue Chinese medicinal bacteriostatic ring (inner ring), f 4-grey white Chinese medicinal diffusion ring (outer ring); FIG. 5-c, the test result of anti-Salmonella typhimurium with fructus Schisandrae water extract, f 5-dark blue Chinese medicinal bacteriostatic circle (inner circle), f 6-milky white Chinese medicinal diffusion circle (middle circle), f 7-light blue Chinese medicinal bacteriostatic circle (outer circle); FIG. 5-d, test results of the dark plum water extract against Salmonella typhimurium, f 8-dark blue Chinese medicinal bacteriostatic circle (inner circle), f 9-grey white Chinese medicinal diffusion circle (middle circle), f 10-dark blue Chinese medicinal bacteriostatic circle (outer circle);
FIG. 6 shows the antibacterial tests of the aqueous extract of Scutellariae radix with different concentrations by methylene blue agar plate diffusion method: FIG. 6-a, 0.5g/ml, g 1-brown yellow traditional Chinese medicine diffusion ring (inner ring), g 2-green traditional Chinese medicine bacteriostasis ring (middle ring), g 3-dark blue traditional Chinese medicine bacteriostasis ring (outer ring); FIG. 6-b 0.25g/ml, g 4-brown yellow traditional Chinese medicine diffusion ring (inner ring), g 5-green traditional Chinese medicine bacteriostasis ring (middle ring), g 6-light blue traditional Chinese medicine bacteriostasis ring (outer ring); fig. 6-c 0.125g/ml, g 7-brown yellow traditional Chinese medicine diffusion ring (inner ring), g 8-green traditional Chinese medicine bacteriostasis ring (middle ring), g 9-light blue traditional Chinese medicine bacteriostasis ring (outer ring).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
1. preparation of methylene blue agar plate
1.1 preparing indicator bacteria suspension: inoculating salmonella to a common slant culture medium, activating and growing for 6-8 h. And (3) lightly scraping the lawn on the inclined plane by using an inoculating loop, placing the lawn in sterile physiological saline, fully oscillating by using a vortex oscillator and adjusting the concentration of the bacterial suspension to be 0.5 McLeod turbidity.
1.2 preparation of agar plates containing bacteriomethylene blue: weighing methylene blue and adding distilled water to prepare a 1% methylene blue aqueous solution, and sterilizing the methylene blue aqueous solution for later use. Placing M-H agar in a triangular flask, sterilizing, keeping the temperature of a water bath at 45 ℃, adding 2ml of methylene blue aqueous solution into every 100ml of methylene blue agar containing bacteria, uniformly mixing, and placing in a 45 ℃ constant temperature water bath for later use. Adding 6ml of bacterial liquid and 45 ℃ methylene blue M-H agar into 100ml of agar of 0.5 McLeod turbidity bacterial suspension, fully mixing, slowly pouring into a sterilization plate (avoiding air bubbles), cooling and solidifying to prepare a plate containing the methylene blue agar for later use (ready for use).
2. Control test: respectively adopting traditional agar plate containing bacteria and methylene blue agar plate to make in vitro antibacterial test
2.1 antibiotic in vitro antibacterial control test
The test steps are as follows: respectively punching holes on the traditional bacteria-containing agar plate and the methylene blue bacteria-containing agar plate by using a puncher with the diameter of 6mm, and adding an antibiotic aqueous solution into the holes for diffusion, wherein the antibiotic aqueous solution is a 5mg/ml ceftriaxone sodium aqueous solution.
In vitro antibacterial effect evaluation: and (3) putting the bacteria-containing plate into a constant temperature of 37 ℃ for culturing for 18h, measuring the diameter of a bacteriostatic ring on the traditional bacteria-containing agar plate and the diameter of a blue ring or a mixed color ring of the self color of the medicine and the blue color on the methylene blue bacteria-containing agar plate, determining the diameter of the bacteriostatic ring, and comprehensively evaluating the antibacterial activity.
As shown in FIG. 3, the antibiotic was colorless and transparent, and was colorless when diffused into agar, and the concentration decreased as the distance from the diffusion center was increased, but the composition was unchanged. The area where bacteria did not grow in the conventional agar plate diffusion method antibacterial test shown in FIG. 3-a was transparent, and the antibacterial effect was evaluated by measuring the size of the transparent circle d 1. In order to distinguish the transparent area from the semitransparent area around the transparent area, an environment with better light needs to be selected when the diameter of the transparent ring is observed, the boundary of the transparent ring is not very clear, and the transparent ring needs to be carefully observed and defined when in measurement; the area where bacteria do not grow in the methylene blue agar plate diffusion method antibacterial test shown in the figure 3-b is blue, the antibacterial effect is evaluated by measuring the size of a blue circle d2, as shown in the figure, the methylene blue developed antibacterial circle is uniformly colored, the boundary is clear and easy to observe, and the measurement is simple, convenient and visual.
Comparing fig. 3-a and 3-b, it can be seen that the diameter of the inhibition zone shown by the two methods in the 5mg/ml ceftriaxone sodium antibiotic antibacterial test is the same, the methylene blue agar plate diffusion method does not affect the inhibition result, and the result display is more visual and clear and is easy to measure.
2.2 control of in vitro antibacterial test of Chinese medicinal extractive solution
The test steps are as follows: punching holes on the traditional bacteria-containing agar plate and methylene blue bacteria-containing agar plate respectively by using a puncher with the diameter of 6mm, adding the water extract of the traditional Chinese medicine into the holes for diffusion, and respectively taking the water extract of the folium artemisiae argyi and the water extract of the radix paeoniae rubra from the water extract of the traditional Chinese medicine, wherein the concentrations of the water extracts are 1 g/mL.
In vitro antibacterial effect evaluation: after the plate is placed at the constant temperature of 37 ℃ for culturing for 18h, the diameter of an inhibition zone on the traditional bacteria-containing agar plate and the diameter of a blue zone on the methylene blue bacteria-containing agar plate or a mixed color zone of the self color of the medicine and the blue color are measured, the diameter of the inhibition zone is determined, and the antibacterial activity is comprehensively evaluated.
And (3) test results: compared with antibiotics, the Chinese medicinal extract has the characteristic of deep turbidity, and the diffusion ring in agar has deep turbidity. In the traditional bacteria-containing agar plate, brown traditional Chinese medicine diffusion rings with different depths are formed after brown turbid traditional Chinese medicines are diffused, so that transparent bacteriostatic rings are covered, the size of the bacteriostatic rings is difficult to measure, and the antibacterial activity of the traditional Chinese medicine diffusion rings cannot be accurately evaluated, as shown in figures 4-a and 4-c: dark brown traditional Chinese medicine diffusion ring (inner ring) e1 formed by folium Artemisiae Argyi water extractive solution, light brown traditional Chinese medicine diffusion ring (outer ring) e2 and light brown traditional Chinese medicine diffusion ring e5 formed by radix Paeoniae Rubra water extractive solution in traditional method; the Chinese medicinal liquid extract is diffused in a methylene blue bacterium-containing agar plate to act on bacteria, the inhibited bacteria display the methylene blue in the agar as blue, the blue is mixed with brown yellow Chinese medicinal diffusion rings with different shades to form green or blue-green bacteria inhibition rings, and the green or blue-green bacteria inhibition rings are observed and measured to evaluate the antibacterial activity of the bacteria. As shown in fig. 4-b and 4-d, the aqueous extract of mugwort leaf in methylene blue bacteria-containing agar plate produces dark green Chinese medicine inhibition zone (inner circle) e3 and light green Chinese medicine inhibition zone (outer circle) e4, and the aqueous extract of red peony root produces blue-green Chinese medicine inhibition zone e 6.
The test results show that the methylene blue agar plate diffusion method adopts the colored bacteriostatic zone to clearly display the bacteriostatic area, and effectively overcomes the defect that the diffusion zone of the traditional Chinese medicine isochromatic and deep turbid to-be-detected object shields the transparent bacteriostatic zone in the traditional plate agar diffusion method.
Example 2:
application of methylene blue bacteria-containing agar plate diffusion method in-vitro antibacterial test of traditional Chinese medicine extracting solution
The test steps are as follows: 1. the preparation method of methylene blue containing bacterium agar plate is the same as that of example 1, and the strains are 2 kinds of salmonella enteritidis and salmonella typhimurium respectively;
2. punching on methylene blue bacteria-containing agar plate with a puncher having diameter of 6mm, adding Chinese medicinal water extractive solution into the hole for diffusion, and collecting fructus Schisandrae water extractive solution and mume fructus extractive solution with concentration of 1g/mL respectively.
In vitro antibacterial effect evaluation: and (3) placing the plate into a constant temperature of 37 ℃ for culturing for 18h, measuring the diameter of a blue color ring or a mixed color ring of the self color of the medicament and the blue color on the methylene blue bacteria-containing agar plate, determining the diameter of an antibacterial ring, and comprehensively evaluating the antibacterial activity.
And (3) test results: as shown in fig. 5-a and 5-b, in the anti-salmonella enteritidis test, the water extract of schisandra fruit and the water extract of smoked plum both have blue bacteriostatic circles with darker central color, the water extract of schisandra fruit has dark blue traditional Chinese medicine bacteriostatic circle (inner circle) f1 and milky white traditional Chinese medicine diffusion circle (outer circle) f2 for anti-salmonella enteritidis; dark blue traditional Chinese medicine bacteriostasis ring (inner ring) f3 and gray white traditional Chinese medicine diffusion ring (outer ring) f4 for resisting salmonella enteritidis are generated by the dark plum water extract; however, in the anti-salmonella typhimurium test shown in fig. 5-c, the water extract of schisandra chinensis not only has a dark blue inhibition zone f5 with a darker color in the center, but also has a light blue inhibition zone (outer ring) f7 with a lighter color in the second layer outside the milky white traditional Chinese medicine diffusion zone (middle ring) f 6; in fig. 5-d, dark blue traditional Chinese medicine inhibition zone (inner circle) f8 with darker color appears in the center of the dark plum water extract in the anti-salmonella typhimurium test, the diameter is smaller than the schisandra inner circle inhibition zone f5, but a dark blue traditional Chinese medicine inhibition zone (outer circle) f10 with the color of the second layer similar to the center depth appears outside the grey white traditional Chinese medicine diffusion zone (middle circle) f9, and the color is darker than the second layer inhibition zone f6 of the schisandra. It can be seen that the two traditional Chinese medicines have different components and activities of antibacterial substances against two kinds of bacteria. This is related to the complexity of the ingredients of the traditional Chinese medicine and the inconsistent diffusion degree of different ingredients in the agar. The antibacterial substance components can be further separated and identified according to the needs.
The test results show that the traditional plate agar diffusion method is mainly suitable for single-component medicines, the farther the traditional plate agar diffusion method is from the center, the lower the concentration of the traditional plate agar diffusion method is until the traditional plate agar diffusion method can not inhibit the growth of bacteria and presents transparent rings (bacteriostatic rings) with different diameters, and the size of the antibacterial activity of the medicines is evaluated by measuring the size of the transparent rings. However, the traditional Chinese medicine components are complex, the distance from the center is different, the possible components of the medicine concentration are different, and the size cannot be determined only by the diameter of the unique inhibition zone. The methylene blue agar diffusion method displays the bacteriostasis zone as blue or the mixed color of the self color of the object to be detected and the blue, the color is uniform, the boundary is clear, the measurement of the bacteriostasis zone is more direct and simple and convenient than the traditional flat plate agar diffusion method, and the bacteriostasis of different components diffused to different positions of agar can be displayed in a layered mode. Therefore, the methylene blue agar plate diffusion method overcomes the defect that the traditional agar plate diffusion method can not meet the antibacterial test requirements of antibacterial substances with complex components such as traditional Chinese medicines and the like.
Example 3:
methylene blue bacteria-containing agar plate diffusion method antibacterial test of scutellaria baicalensis water extract with different concentrations
The test steps are as follows: 1. the preparation method of methylene blue bacteria-containing agar plate is the same as that of example 1;
2. punching on methylene blue bacteria-containing agar plate with a puncher with diameter of 6mm, adding Scutellariae radix water extract into the hole for diffusion, wherein the Scutellariae radix water extract is 0.5g/mL, 0.25g/mL and 0.125g/mL respectively.
In vitro antibacterial effect evaluation: and (3) placing the plate into a constant temperature of 37 ℃ for culturing for 18h, measuring the diameter of a blue ring on the methylene blue agar plate or a mixed color ring of the self color of the drug and the blue color, determining the diameter of an antibacterial ring, and comprehensively evaluating the antibacterial activity.
And (3) test results: as shown in FIG. 6, the results of the action of the aqueous extracts of Scutellariae radix of FIGS. 6-a, 6-b and 6-c, which were 0.5g/mL, 0.25g/mL and 0.125g/mL, respectively, on the bacteria in the plates after spreading on methylene blue agar plates containing Salmonella. The result shows that the 0.5g/mL scutellaria water extract in fig. 6-a diffuses in the agar and then acts with the bacteria in the agar to influence the bacterial growth, and a brown-yellow traditional Chinese medicine bacteriostasis zone (inner circle) g1, a green traditional Chinese medicine bacteriostasis zone (middle circle) g2 and a dark blue traditional Chinese medicine bacteriostasis zone (outer circle) g3 appear. The green bacteriostasis zone is the mixed color of the brown yellow of the medicament and the blue of methylene blue, and the blue bacteriostasis zone is the bacteriostasis shown by the colorless bacteriostasis component of the scutellaria which diffuses to the outer ring. The scutellaria aqueous extract with different concentrations shows different bacteriostatic zones. The diameter of the brown-yellow traditional Chinese medicine diffusion ring is reduced along with the reduction of the concentration of the liquid medicine, namely: g7 < g4 < g 1; the diameter of the green bacteriostasis zone is reduced, namely: g8 < g5 < g 2; the blue inhibition zone gradually becomes shallow until disappearance, namely: g9 < g6 < g 3. The above all indicate the variety and content of the bacteriostatic component.
Therefore, the methylene blue agar plate diffusion method can be used for visually and effectively indicating the variety and content change of antibacterial ingredients of the traditional Chinese medicine extract with different concentrations in a layering manner.
In conclusion, the test method for evaluating the in-vitro antibacterial activity of the antibacterial substance is intuitive, simple and convenient, and overcomes the defect that the diameter of the antibacterial zone is difficult to accurately measure due to deep color turbidity and complex components when the traditional agar plate diffusion method is used for the in-vitro antibacterial experiment of the traditional Chinese medicine or other substances to be tested with similar properties by observing the colored antibacterial zone.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (4)
1. A test method for evaluating the in vitro antibacterial activity of an antibacterial substance is characterized in that an agar plate containing the antibacterial methylene blue is used for the in vitro antibacterial activity test of the agar plate diffusion method of the antibacterial substance with the color depth turbidity and the complex components similar to the traditional Chinese medicine and the characters;
the preparation method of the agar plate containing the mycomethylene blue comprises the following steps: preparing a strain which is activated and grows for 6-8 hours by a slant culture medium into an indicator bacterium suspension, then quickly and uniformly mixing the indicator bacterium suspension with drug sensitive agar which is sterilized, cooled to 45 ℃ and melted and contains methylene blue, and cooling and solidifying to obtain the indicator bacterium suspension;
the content of methylene blue in the agar plate containing the methylene blue is that every 100mL of the agar containing the methylene blue contains 2mL of 1 percent methylene blue aqueous solution;
the in vitro antibacterial activity test comprises the following specific steps: adding a solution of an object to be tested to a freshly prepared agar plate containing methylene blue to diffuse the solution in the agar, culturing at constant temperature, observing and measuring the diameter of a blue circle around the object to be tested or a mixed colored circle of the self color of an antibacterial drug and the blue color as the diameter of an antibacterial circle after an indicating bacterium and the object to be tested act, observing the color depth of the antibacterial circle, and evaluating the external antibacterial activity of the object to be tested; the diffusion degree of different components in the agar is different in the object to be tested with a plurality of antibacterial components, and the antibacterial activity of the different components diffused to different positions of the agar is displayed in a layered mode.
2. The assay of claim 1 wherein the chemosensitive agar is M-H agar medium or other chemosensitive agar suitable for bacterial growth.
3. The test method for evaluating the in vitro antibacterial activity of an antibacterial substance according to claim 1, wherein the method of adding the solution to be tested to the bactrian methylene blue-containing agar plate is any one of a punch method, a paper sheet method and an oxford cup method.
4. The assay method for evaluating the in vitro antibacterial activity of an antibacterial substance according to claim 1, wherein the time of action of the indicator and the analyte is 18 hours.
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| CN105316389A (en) * | 2014-06-13 | 2016-02-10 | 泰兴瑞泰化工有限公司 | Anti-bacterial test method of composite organic acid preparation and monomer acid |
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| CN105316389A (en) * | 2014-06-13 | 2016-02-10 | 泰兴瑞泰化工有限公司 | Anti-bacterial test method of composite organic acid preparation and monomer acid |
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| Correlation of Etest and Neo-Sensitabs diffusion assays on Mueller-Hinton-methylene blue agar with broth microdilution reference method (CLSI-M27-A2) for testing susceptibilities of Cryptococcus neoformans to amphotericin B and fluconazole;MARIA EUGENIA OCHIUZZI ,等;《Medical Mycology》;20101231;第48卷;摘要，第894页左栏第5段-右栏第6段 * |
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