CN106467924A - A kind of test method evaluating antibiotic substance antibacterial activity in vitro - Google Patents
A kind of test method evaluating antibiotic substance antibacterial activity in vitro Download PDFInfo
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Abstract
The present invention relates to technical field of biological control, specifically disclose a kind of test method evaluating antibiotic substance antibacterial activity in vitro, be the antibacterial activity in vitro test that the agar plate of methylene blue containing bacterium is used for lysoplate assay.The present invention is intuitively easy, by observing coloured inhibition zone, overcome traditional lysoplate assay for Chinese medicine or other similar determinand of character In vitro Bactericidal Experiments when because color depth is muddy and complicated component leads to be difficult to the drawbacks of accurately measure antibacterial circle diameter.
Description
Technical field
The present invention relates to technical field of biological control is and in particular to a kind of test of evaluation antibiotic substance antibacterial activity in vitro
Method.
Background technology
In vitro Bactericidal Experiments are the tests measuring the sensitivity to certain material for the microorganism in vitro, are usually used in evaluating suppression
Kill the activity of pathogen, widely application and scientific research, production and clinic, such as the screening of antibacterials, chasing after in extraction process
Track, the mensure of antimicrobial spectrum, instruct drug sensitive test of clinical application etc..Lysoplate assay be current most common method it
One.
Traditional lysoplate assay is indicator bacteria to be coated agar surface or is uniformly blended in agar, then adopts
The methods such as paper disk method, Odontothrips loti, punch method add determinand, allow determinand to spread in agar.Due to agar water white transparency,
Antibacterial semi-transparent clear, leads to the agar region that antibacterial does not grow to be in water white transparency.If determinand has antibacterial activity, therein thin
The growth of microorganism such as bacterium are suppressed or kill, thus in the range of medicine effective concentration formed microorganism do not grow transparent
Circle a1 (Fig. 1), distinguishes that under the preferable environment of light the diameter of measurement this transparent circle a1 is used for evaluating determinand and has or not antibacterial and lives
Property and its active size of preliminary assessment.This transparent circle is referred to as inhibition zone, antibacterial circle diameter size and antibiotic substance concentration and instruction
Bacteria concentration is directly related.
Substantial amounts of clinical application research shows, Chinese medicine has very important antibacterial action.With clinical antibiotics application
The middle drug-resistant bacteria problem occurring is on the rise, and the research of antibacterial Chinese medicine has broad application prospects.Chinese medicine is external at present
Antibacterial activity research there is no standardized method, and lysoplate assay is as international antibiotic antibacterial activity evaluation methodology
One of, also have been widely cited in the Determination of Antibacterial Activity of the other materials including Chinese medicine.However, Chinese medicine extraction liquid character
Special, its diffusing phenomenon in agar plate is different from antibiotic, and TCM in Vitro antibacterial tests can not indiscriminately imitate antibiosis ferritic completely
Outer antibacterial tests.Chinese medicine extraction liquid main feature has：(1) color depth is muddy：Lead to color occurs in agar deeper after drug diffusion
And the drug diffusion circle of muddiness, this circle overlapped with the transparent circle that antibacterial does not grow, and the measurement of impact antibacterial circle diameter is it is difficult to directly perceived
Evaluate antibacterial activity size.As shown in Figure 2：It is the muddy Radix Scutellariae Aqueous extracts diffusion of color depth in (01) (02) holes on agar plate
Antibacterial phenomenon, wherein b1 are dark-brown Chinese medicine diffusion circle (inner ring), and b2 is light brown Chinese medicine diffusion circle (outer ring)；(03) (04) two
Hole be achromaticity and clarification antibiotic ceftriaxone sodium spread antibacterial phenomenon, wherein b3 be transparent inhibition zone it is seen that：Flat using tradition
When plate agar diffusion method carries out antibacterial tests, antibiotic achromaticity and clarification, its drug diffusion circle is not covered transparent inhibition zone and is easy to survey
Amount, and Chinese medicine color depth muddiness masks transparent inhibition zone, growth district does not lead to be difficult to distinguish carefully even need to cannot to observe antibacterial
Ce Liang not inhibition zone；(2) complicated component：The general composition of antibiotic is single, and it is from dispersal center distance different pharmaceutical only concentration not
Same with forming split-phase.But the often complicated component such as Chinese medicine extract, each composition diffusion velocity is different, from dispersal center distance position
The concentration of different medicines and composition all have a difference, therefore antibacterial do not grow the size of circle not only relevant with drug level also and medicine
The complicated composition of thing is related.
In sum, traditional agar plate diffusion assay is answered in Chinese medicine and the color depth similar to its character muddiness and composition
Remain in the antibacterial activity in vitro evaluation of miscellaneous determinand in drawback, find one kind and more intuitively and effectively indicate its antibacterial activity
In-vitro antibacterial method for the exploitation of antibiotic substance, there is important using value.
Content of the invention
For the deficiencies in the prior art, the invention provides a kind of test side evaluating antibiotic substance antibacterial activity in vitro
Method, can the muddy the antibiotic substance similar with Chinese medicine extract of complicated component and character of Layering manifestation color depth in-vitro antibacterial work
Property, easy, directly perceived, effectively.
For realizing object above, the present invention is achieved by the following technical programs：
A kind of test method evaluating antibiotic substance antibacterial activity in vitro, is that the agar plate of methylene blue containing bacterium is used for fine jade
The antibacterial activity in vitro test of fat Agar diffusion test.
Preferably, what described antibacterial activity in vitro was tested comprises the following steps that：Determinand solution is added to fresh preparation
The agar plate of methylene blue containing bacterium on so as in agar spread, constant temperature culture, bacterium to be instructed and determinand effect after, see
Examine and measure around determinand blue circle or determinand self color and blueness to be mixed with chromosphere diameter straight as inhibition zone
Footpath, and observe inhibition zone shade, evaluate determinand antibacterial activity in vitro.
Preferably, the preparation method of the described agar plate of methylene blue containing bacterium is as follows：By slant medium activated growth 6-
The strain of 8h is prepared into indicator bacteria bacteria suspension, and be then cooled to 45 DEG C of thawings by indicator bacteria bacteria suspension and after sterilizing contains methylene
The blue susceptibility agar of base mixes rapidly, and cooled and solidified obtains final product.
Preferably, the content of the described agar plate Methylene Blue of methylene blue containing bacterium is：Every 100mL methylene blue containing bacterium fine jade
1% aqueous solution of methylene blue containing 2mL in fat.
Preferably, described susceptibility agar is M-H agar culture medium or the susceptibility agar of other appropriate bacterial growth.
Preferably, described determinand solution add method in the agar plate of methylene blue containing bacterium using punch method, paper disk method,
Any one in Odontothrips loti.
Preferably, described indicator bacteria and determinand action time are 18h.
The test method evaluating antibiotic substance antibacterial activity in vitro is applied to achromaticity and clarification and the single antibiotic substance of composition.
The test method evaluating antibiotic substance antibacterial activity in vitro is applied to Chinese medicine and the similar color depth of character is muddy and become
Divide complicated antibiotic substance.
The cardinal principle of the present invention is：The antibacterial that determinand spreads in agar and acts in agar, bacteria live shape
State affects the color of the agar plate of methylene blue containing bacterium.The oxidized and reduced of methylene blue is in blue and colourless respectively.Carefully
Methylene blue in agar before bacterium culture is generally in the oxidation state of aobvious blueness, and therefore methylene blue flat board containing bacterium is in blueness.Treat
After surveying thing addition methylene blue flat board containing bacterium constant temperature culture, with the culture of antibacterial, methylene blue enters in bacterial cell, growth
In the reductase of antibacterial make methylene blue decolour to become the colourless of reduction-state by blue, and be killed or suppress to be in tranquillization shape
The reductase inactivation of the antibacterial of state does not produce decolorization, and therefore methylene blue is still blueness.It can be seen that, the agar of methylene blue containing bacterium
In flat board, colourless region is bacterial growth region, and blue region is bacterial death or repressed region (and the depth of blueness
Shallow related to bacterial activity size).Consider and diffuse to determinand color in agar, blue and blue and determinand color
The coloured collar region of secondary colour is antibacterial region, i.e. inhibition zone；The region of achromatic region and only determinand color is no antibacterial
Region.
The present invention, compared with traditional lysoplate assay, has the advantages that：
1st, improve the muddy impact that transparent inhibition zone is observed of color depth：The present invention passes through blue display determinand directly perceived to thin
The inhibitory or killing effect of bacterium, in conjunction with the color observation colored areas of determinand solution itself, overall merit antibacterial action size, effective gram
Take due to color and the muddy impact that transparent inhibition zone is observed of determinand solution in traditional lysoplate assay, can be straight
See and rapidly evaluate antibacterial action；
2nd, improve the impact to determinand agar diffusion for the complicated component：Chinese medicine and the material of other complicated components, it is in fine jade
When spreading in fat, heterogeneity diffusion velocity is different, and the present invention passes through the blue antibacterial showing determinand all the components directly perceived and makees
With, for the determinand with multiple antipathogenic compositions, the mixing of Layering manifestation multiple blueness inhibition zone or blueness and self color
Color inhibition zone, overcomes in traditional lysoplate assay due to the determinand complicated component such as Chinese medicine, different material diffusion position
The impact differing, can be layered the objectively overall antibacterial activity of overall merit complicated component determinand.
Brief description
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Have technology description in required use accompanying drawing be briefly described it should be apparent that, drawings in the following description be only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, acceptable
Other accompanying drawings are obtained according to these accompanying drawings.
The transparent inhibition zone of Fig. 1 tradition lysoplate assay antibiotic：The transparent inhibition zone of a1-；
Fig. 2 tradition lysoplate assay Chinese medicine and the antibacterial phenomenon of antibiotic：B1- dark-brown Chinese medicine diffusion circle (inner ring),
The transparent inhibition zone of b2- light brown Chinese medicine diffusion circle (outer ring), b3-；
Fig. 3 antibiotic tradition lysoplate assay and methylene blue lysoplate assay results contrast：Fig. 3-a, passes
System lysoplate assay, the transparent inhibition zone of d1-；Fig. 3-b, methylene blue lysoplate assay, d2- blueness inhibition zone；
Fig. 4 Chinese traditional medicine water extract classic flat-plate agar diffusion method and the contrast of methylene blue lysoplate assay methods and resultses：
Fig. 4-a, Folium Artemisiae Argyi Aqueous extracts tradition lysoplate assay, e1- dark-brown Chinese medicine diffusion circle (inner ring), e2- light brown Chinese medicine expands
Dissipate circle (outer ring)；Fig. 4-b, Folium Artemisiae Argyi Aqueous extracts methylene blue lysoplate assay, e3- bottle green Traditional Chinese medicine bacteriostatic circle (inner ring),
E4- light green color Traditional Chinese medicine bacteriostatic circle (outer ring)；Fig. 4-c, Radix Paeoniae Rubra Aqueous extracts tradition lysoplate assay, e5- light brown Chinese medicine expands
Dissipate circle；4-d, Radix Paeoniae Rubra Aqueous extracts methylene blue lysoplate assay, e6- aeruginouss Traditional Chinese medicine bacteriostatic circle；
Fig. 5 Fructus Schisandrae Chinensis and Fructus Mume Aqueous extracts methylene blue agar diffusion method antibacterial tests result：Fig. 5-a, Fructus Schisandrae Chinensis water extraction
Liquid anti-Salmonella enteritidis result of the test, f1- navy blue Traditional Chinese medicine bacteriostatic circle (inner ring), f2- milky Chinese medicine diffusion circle (outer ring)；Figure
5-b, Fructus Mume Aqueous extracts anti-Salmonella enteritidis result of the test, f3- navy blue Traditional Chinese medicine bacteriostatic circle (inner ring), f4- canescence Chinese medicine expands
Dissipate circle (outer ring)；Fig. 5-c, Fructus Schisandrae Chinensis Aqueous extracts anti-Salmonella typhimurtum result of the test, f5- navy blue Traditional Chinese medicine bacteriostatic circle is (interior
Circle), f6- milky Chinese medicine diffusion circle (centre circle), f7- light blue Traditional Chinese medicine bacteriostatic circle (outer ring)；Fig. 5-d, the anti-Mus of Fructus Mume Aqueous extracts
Salmonella typhi result of the test, f8- navy blue Traditional Chinese medicine bacteriostatic circle (inner ring), f9- canescence Chinese medicine diffusion circle (centre circle), f10- is deep
Blue Traditional Chinese medicine bacteriostatic circle (outer ring)；
The Radix Scutellariae Aqueous extracts methylene blue lysoplate assay antibacterial tests of Fig. 6 variable concentrations：Fig. 6-a, 0.5g/ml,
G1- brown color Chinese medicine diffusion circle (inner ring), g2- green Traditional Chinese medicine bacteriostatic circle (centre circle), g3- navy blue Traditional Chinese medicine bacteriostatic circle (outer ring)；
Fig. 6-b 0.25g/ml, g4- brown color Chinese medicine diffusion circle (inner ring), g5- green Traditional Chinese medicine bacteriostatic circle (centre circle), during g6- is light blue
Medicine inhibition zone (outer ring)；Fig. 6-c 0.125g/ml, g7- brown color Chinese medicine diffusion circle (inner ring), g8- green Traditional Chinese medicine bacteriostatic circle (in
Circle), g9- light blue Traditional Chinese medicine bacteriostatic circle (outer ring).
Specific embodiment
Purpose, technical scheme and advantage for making the embodiment of the present invention are clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described it is clear that described embodiment is
The a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art
The every other embodiment being obtained under the premise of not making creative work, broadly falls into the scope of protection of the invention.
Embodiment 1：
1st, the preparation of methylene blue agar plate
1.1 preparation indicator bacteria bacteria suspensions：Salmonella is inoculated common slant medium activated growth 6-8h.Light with inoculating loop
Lawn on light scraping inclined-plane is placed in physiological saline solution, and turbula shaker fully vibrates and adjusts bacteria suspension concentration is 0.5
Maxwell turbidity.
The 1.2 preparation agar plates of methylene blue containing bacterium：Weigh methylene blue to add distilled water to be prepared into 1% methylene blue water-soluble
Liquid, stand-by after sterilizing.M-H agar puts 45 DEG C of water-bath constant temperature after sterilizing in conical flask, by every 100ml methylene blue containing bacterium fine jade
Add the ratio of 2ml aqueous solution of methylene blue to mix after adding 1% aqueous solution of methylene blue sterilizing in fat and put 45 DEG C of constant temperature
Water-bath is stand-by.By 0.5 Maxwell turbidity bacterial suspension according to every 100ml agar addition 6ml bacterium solution and 45 DEG C of methylene blue M-H fine jades
Fat fully mixes, and is slowly poured in sterilizing plates (having avoided bubble), is prepared into the agar of methylene blue containing bacterium after cooled and solidified
Flat board stand-by (now with the current).
2nd, controlled trial：It is respectively adopted traditional flat board containing bacterio-agar and methylene blue flat board containing bacterio-agar carries out in-vitro antibacterial
Test
2.1 Antibiotic in Vitro antibacterial controlled trials
Test procedure：Contain bacterio-agar in traditional flat board containing bacterio-agar and methylene blue respectively with the card punch of diameter 6mm to put down
Punch on plate, aqueous topical antibiotics are added in hole and is diffused, aqueous topical antibiotics are 5mg/ml ceftriaxone sodium water solution.
In-vitro antibacterial effect assessment：Bacterium flat board will be contained and put into 37 DEG C of constant temperature culture 18h, on the traditional flat board containing bacterio-agar of measurement
Antibacterial circle diameter, and methylene blue flat board containing bacterio-agar blueing chromosphere or medicine self color mix the straight of chromosphere with blueness
Footpath, determines antibacterial circle diameter, overall merit antibacterial activity.
Result of the test, as shown in figure 3, the general water white transparency of antibiotic, diffuses to colourless in agar, from dispersal center more away from
Concentration reduces, but components unchanged.The region that in traditional lysoplate assay antibacterial tests shown in Fig. 3-a, antibacterial does not grow is
Light colour, the size of measurement transparent circle d1 evaluates antibacterial effect.In order to distinguish the translucent of transparent region and bacterial growth about
Region, need to select light preferable environment when observing transparent loop diameter, and transparent circle border is not very clear, need to carefully see during measurement
Examine and define；The region that in methylene blue lysoplate assay antibacterial tests shown in Fig. 3-b, antibacterial does not grow is blueness, and measurement is blue
The size of chromosphere d2 evaluates antibacterial effect, as shown in the figure the inhibition zone uniform coloring of methylene blue colour developing and sharpness of border is easy to see
Examine, easy to be directly perceived during measurement.
Comparison diagram 3-a and 3-b it is seen that two methods 5mg/ml ceftriaxone sodium antibiotic antibacterial tests are shown antibacterial
Loop diameter size identical it is seen that methylene blue lysoplate assay do not affect antibacterial result and result show more visual and clear
It is easy to measure.
2.2 Chinese medicine extraction liquid In vitro Bactericidal Experiments comparisons
Test procedure：Contain bacterio-agar in traditional flat board containing bacterio-agar and methylene blue respectively with the card punch of diameter 6mm to put down
Punch on plate, Chinese traditional medicine water extract is added in hole and is diffused, Chinese traditional medicine water extract takes Folium Artemisiae Argyi Aqueous extracts, Radix Paeoniae Rubra water extraction respectively
Liquid, concentration is 1g/mL.
In-vitro antibacterial effect assessment：Flat board is put into after 37 DEG C of constant temperature culture 18h, on the traditional flat board containing bacterio-agar of measurement
Antibacterial circle diameter, and methylene blue flat board containing bacterio-agar blueing chromosphere or medicine self color mix the straight of chromosphere with blueness
Footpath, determines antibacterial circle diameter, overall merit antibacterial activity.
Result of the test：Chinese medicine extraction liquid, compared with antibiotic, has the muddy characteristic of color depth, the diffusion in agar mostly
Circle color depth is muddy.In traditional flat board containing bacterio-agar, form deep mixed brown Chinese medicine after the muddy Chinese medicine diffusion of brown color and expand
Scattered circle has covered transparent inhibition zone it is difficult to measurement inhibition zone size is it is impossible to its antibacterial activity size of accurate evaluation, such as Fig. 4-a, 4-
Shown in c：In Traditional Method Folium Artemisiae Argyi Aqueous extracts formed dark-brown Chinese medicine diffusion circle (inner ring) e1 and light brown Chinese medicine diffusion circle (outward
Circle) the light brown Chinese medicine diffusion circle e5 that formed of e2 and Radix Paeoniae Rubra Aqueous extracts；Chinese traditional medicine water extract is in methylene blue flat board containing bacterio-agar
In antibacterial, the methylene blue in agar is shown as blue to diffusion by repressed antibacterial, with deep mixed brown color
Chinese medicine diffusion circle is mixed to form deep mixed green or aeruginouss inhibition zone, observes and measures green or aeruginouss inhibition zone is commented
Its antibacterial activity size of valency.As shown in Fig. 4-b, 4-d, in methylene blue flat board containing bacterio-agar, Folium Artemisiae Argyi Aqueous extracts produce in bottle green
Medicine inhibition zone (inner ring) e3 and light green color Traditional Chinese medicine bacteriostatic circle (outer ring) e4, Radix Paeoniae Rubra Aqueous extracts produce aeruginouss Traditional Chinese medicine bacteriostatic circle e6.
Above result of the test shows, methylene blue lysoplate assay adopts coloured inhibition zone clearly to show antibacterial area
Domain, the diffusion circle effectively overcoming the muddy determinand of the color depth such as Chinese medicine in classic flat-plate agar diffusion method blocks transparent inhibition zone
The drawbacks of.
Embodiment 2：
Methylene blue Agar diffusion test containing bacterio-agar is applied to Chinese medicine extraction liquid In vitro Bactericidal Experiments
Test procedure：1st, with embodiment 1, strain is respectively Salmonella enteritidis to methylene blue flat board containing bacterio-agar preparation method
With 2 kinds of Salmonella typhimurtum；
2nd, use the card punch of diameter 6mm to punch on methylene blue flat board containing bacterio-agar, Chinese traditional medicine water extract is added in hole
It is diffused, Chinese traditional medicine water extract takes Fructus Schisandrae Chinensis Aqueous extracts and Fructus Mume extracting solution respectively, concentration is 1g/mL.
In-vitro antibacterial effect assessment：Flat board is put into after 37 DEG C of constant temperature culture 18h, measures methylene blue flat board containing bacterio-agar
Blueing chromosphere or medicine self color mix the diameter of chromosphere with blueness, determine antibacterial circle diameter, overall merit antibacterial activity.
Result of the test：As shown in Fig. 5-a, 5-b, in the test of anti-Salmonella enteritidis, Fructus Schisandrae Chinensis Aqueous extracts and Fructus Mume Aqueous extracts are all
The deeper blue inhibition zone of appearance center color, it is (interior that navy blue Traditional Chinese medicine bacteriostatic circle in the anti-Salmonella enteritidis of Fructus Schisandrae Chinensis Aqueous extracts
Circle) f1, milky Chinese medicine diffusion circle (outer ring) f2；It is (interior that navy blue Traditional Chinese medicine bacteriostatic circle in the anti-Salmonella enteritidis of Fructus Mume Aqueous extracts
Circle) f3 and canescence Chinese medicine diffusion circle (outer ring) f4；But Fig. 5-c anti-Salmonella typhimurtum test in, Fructus Schisandrae Chinensis Aqueous extracts except
At center, the deeper navy blue inhibition zone f5 of color occurs, also outside milky Chinese medicine diffusion circle (centre circle) f6, second layer face occurs
Thin light blue Traditional Chinese medicine bacteriostatic circle (outer ring) f7 of color；In Fig. 5-d, Fructus Mume Aqueous extracts also center in anti-Salmonella typhimurtum test
Deeper navy blue Traditional Chinese medicine bacteriostatic circle (inner ring) f8 of color occurs, diameter is less than Fructus Schisandrae Chinensis inner ring inhibition zone f5, but in canescence
Second layer color navy blue Traditional Chinese medicine bacteriostatic circle (outer ring) f10 similar to the center depth occurs outside Chinese medicine diffusion circle (centre circle) f9,
Color is deeper than the second layer inhibition zone f6 of Fructus Schisandrae Chinensis.It can be seen that the composition of antibiotic substance to two kinds of antibacterials of two kinds of Chinese medicine and activity
Of different sizes.This is complicated with Chinese medicine ingredients, the inconsistent correlation of diffusion in agar for the heterogeneity.One can be entered as needed
Step layering carries out isolation identification to its antibiotic substance composition.
Above result of the test shows, classic flat-plate agar diffusion method is mainly suitable for the medicine of single component, and it is in agar
The more remote concentration in diffusion Hou Li center is lower, until being unable to bacteria growing inhibiting and assuming the transparent circle (inhibition zone) that diameter differs,
Evaluate medicine antibacterial activity size by measuring transparent circle size.But Chinese medicine ingredients are complicated, from center distance difference not DO
Concentration is different also may to have difference it is impossible to only to judge with unique antibacterial circle diameter size by composition.Methylene blue agar diffusion
Inhibition zone is shown as blue or determinand self color and blue secondary colour by method, color even sharpness of border, inhibition zone
Measurement is easier compared with classic flat-plate agar diffusion method, and can diffuse to the heterogeneity of agar diverse location by Layering manifestation
Bacteriostasis.Therefore methylene blue lysoplate assay improve traditional lysoplate assay can not meet Chinese medicine etc. become
The drawbacks of divide the antibacterial tests of complicated antibiotic substance to require.
Embodiment 3：
Methylene blue Agar diffusion test containing the bacterio-agar antibacterial tests of variable concentrations Radix Scutellariae Aqueous extracts
Test procedure：1st, methylene blue flat board containing bacterio-agar preparation method is with embodiment 1；
2nd, use the card punch of diameter 6mm to punch on methylene blue flat board containing bacterio-agar, Radix Scutellariae Aqueous extracts are added in hole
It is diffused, Radix Scutellariae Aqueous extracts take 0.5g/mL, 0.25g/mL and 0.125g/mL respectively.
In-vitro antibacterial effect assessment：Flat board is put into after 37 DEG C of constant temperature culture 18h, measures methylene blue agar plate blueing
Chromosphere or medicine self color mix the diameter of chromosphere with blueness, determine antibacterial circle diameter, overall merit antibacterial activity.
Result of the test：As shown in fig. 6, Fig. 6-a, 6-b, 6-c are respectively the Huang of 0.5g/mL, 0.25g/mL, 0.125g/mL
A kind of reed mentioned in ancient books Aqueous extracts spread on the methylene blue agar plate containing salmonella after result with bacterial action in flat board.Result display figure
Affect bacterial growth with after bacterial action in agar after 0.5g/mL Radix Scutellariae Aqueous extracts spread in agar in 6-a, occur in that pale brown
Color Traditional Chinese medicine bacteriostatic circle (inner ring) g1, green Traditional Chinese medicine bacteriostatic circle (centre circle) g2 and navy blue Traditional Chinese medicine bacteriostatic circle (outer ring) g3.Green suppression
Bacterium circle is the secondary colour of the blueness of brown color and the methylene blue of medicine itself, and it is colourless that blue inhibition zone is that Radix Scutellariae diffuses to outer ring
The bacteriostasis that show of antipathogenic composition.The Radix Scutellariae Aqueous extracts of variable concentrations show inhibition zone of different sizes.Dense with medicinal liquid
The Chinese medicine diffusion loop diameter of the brown color of reduction of degree reduces, that is,：G7 ＜ g4 ＜ g1；Green antibacterial circle diameter reduces, that is,：G8 ＜
G5 ＜ g2；Blue inhibition zone gradually becomes shallower as until disappearing, that is,：G9 ＜ g6 ＜ g3.Below all point out antipathogenic composition species and
The change of content.
It can be seen that, methylene blue lysoplate assay can be layered intuitively effectively instruction variable concentrations Chinese medicine extract antibacterial and become
The change of the species and content divided.
In sum, the test method of embodiment of the present invention evaluation antibiotic substance antibacterial activity in vitro is intuitively easy, passes through
Observe coloured inhibition zone, overcome traditional lysoplate assay for the in-vitro antibacterial of Chinese medicine or other similar determinand of character
Because color depth is muddy and complicated component leads to be difficult to the drawbacks of accurately measure antibacterial circle diameter during experiment.
It should be noted that herein, such as first and second or the like relational terms are used merely to a reality
Body or operation are made a distinction with another entity or operation, and not necessarily require or imply these entities or deposit between operating
In any this actual relation or order.And, term " inclusion ", "comprising" or its any other variant are intended to
Comprising of nonexcludability, wants so that including a series of process of key elements, method, article or equipment and not only including those
Element, but also include other key elements being not expressly set out, or also include for this process, method, article or equipment
Intrinsic key element.In the absence of more restrictions, the key element that limited by sentence "including a ..." it is not excluded that
Also there is other identical element including in the process of described key element, method, article or equipment.
Above example only in order to technical scheme to be described, is not intended to limit；Although with reference to the foregoing embodiments
The present invention has been described in detail, it will be understood by those within the art that：It still can be to aforementioned each enforcement
Technical scheme described in example is modified, or carries out equivalent to wherein some technical characteristics；And these modification or
Replace, do not make the essence of appropriate technical solution depart from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (9)
1. a kind of test method evaluating antibiotic substance antibacterial activity in vitro is it is characterised in that be by the agar of methylene blue containing bacterium
Flat board is used for the antibacterial activity in vitro test of lysoplate assay.
2. the test method evaluating antibiotic substance antibacterial activity in vitro as claimed in claim 1 is it is characterised in that described external
What antibacterial activity was tested comprises the following steps that：Determinand solution is added to the agar plate of methylene blue containing bacterium of fresh preparation
In so as to spread in agar, constant temperature culture, after bacterium to be instructed and determinand effect, observe and measure around determinand blue
Circle or determinand self color are mixed with chromosphere diameter as the diameter of inhibition zone with blue, and it is deep to observe inhibition zone color
Shallow, evaluate determinand antibacterial activity in vitro.
3. as claimed in claim 2 evaluate antibiotic substance antibacterial activity in vitro test method it is characterised in that described containing bacterium
The preparation method of methylene blue agar plate is as follows：The strain of slant medium activated growth 6-8h is prepared into indicator bacteria bacterium hang
Liquid, then by indicator bacteria bacteria suspension with sterilizing after be cooled to 45 DEG C thawing the susceptibility agar containing methylene blue mix rapidly,
Cooled and solidified, obtains final product.
4. as claimed in claim 3 evaluate antibiotic substance antibacterial activity in vitro test method it is characterised in that described containing bacterium
The content of methylene blue agar plate Methylene Blue is：1% methylene containing 2mL in every 100mL methylene blue containing bacterium agar
Blue aqueous solution.
5. the test method evaluating antibiotic substance antibacterial activity in vitro as claimed in claim 3 is it is characterised in that described susceptibility
Agar is M-H agar culture medium or the susceptibility agar of other appropriate bacterial growth.
6. the test method evaluating antibiotic substance antibacterial activity in vitro as claimed in claim 2 is it is characterised in that described to be measured
Thing solution adds the method in the agar plate of methylene blue containing bacterium using any one in punch method, paper disk method, Odontothrips loti.
7. the test method evaluating antibiotic substance antibacterial activity in vitro as claimed in claim 2 is it is characterised in that described instruction
Bacterium and determinand action time are 18h.
8. the test method of described evaluation antibiotic substance antibacterial activity in vitro as arbitrary in claim 1-7 is applied to achromaticity and clarification
And the antibiotic substance that composition is single.
9. the test method of described evaluation antibiotic substance antibacterial activity in vitro as arbitrary in claim 1-7 is applied to Chinese medicine and property
The similar color depth muddiness of shape and the antibiotic substance of complicated component.
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| CN114292897A (en) * | 2021-12-30 | 2022-04-08 | 张美英 | Kit for rapidly detecting activity of antibacterial substance and application thereof |
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| CN111304281A (en) * | 2020-03-31 | 2020-06-19 | 广州立白企业集团有限公司 | Evaluation method of corrosion resistance |
| CN111304281B (en) * | 2020-03-31 | 2023-07-04 | 广州立白企业集团有限公司 | Evaluation method of anti-corrosion efficiency |
| CN114292897A (en) * | 2021-12-30 | 2022-04-08 | 张美英 | Kit for rapidly detecting activity of antibacterial substance and application thereof |
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| CN106467924B (en) | 2019-12-20 |
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