CN112300898A - Grid-divided culture medium plate and method for detecting carbapenem-resistant enterobacteriaceae - Google Patents
Grid-divided culture medium plate and method for detecting carbapenem-resistant enterobacteriaceae Download PDFInfo
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- CN112300898A CN112300898A CN202011349355.3A CN202011349355A CN112300898A CN 112300898 A CN112300898 A CN 112300898A CN 202011349355 A CN202011349355 A CN 202011349355A CN 112300898 A CN112300898 A CN 112300898A
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Abstract
The invention relates to biological medicine, in particular to a grid-divided culture medium plate and a method for detecting carbapenem-resistant enterobacter. A grid-divided culture medium flat plate comprises a bottom plate (1), a side wall (2) surrounding the bottom plate (1) and an upper cover (3), wherein a partition plate (4) for dividing the bottom plate (1) into more than 2 areas is arranged on the bottom plate (1); the inner wall of the upper cover (3) is provided with a slot (5) for inserting the clapboard (4), and the inner wall of the slot (5) is covered with a rubber layer. A method for detecting carbapenem-resistant enterobacteriaceae is characterized in that a carbapenemase activator is added into a first-region agar culture medium, EDTA is added into a second-region agar culture medium, tazobactam is added into a third-region agar culture medium, and sample inoculation cultivation is carried out in three regions. The method can effectively identify the carbapenem-resistant enterobacter.
Description
Technical Field
The invention relates to biological medicine, in particular to a grid-divided culture medium plate and a method for detecting carbapenem-resistant enterobacter.
Background
The culture medium plate is used for obtaining the most common solid culture medium form of pure culture of microorganisms, and is a culture medium solid plane formed by cooling solidified solid culture medium in a sterile culture dish, which is often referred to as a culture plane for short. Also called planar medium, plate medium. The plate culture medium is commonly used for single spore separation, the growth speed of hyphae is measured, the morphology of bacterial colonies is observed, antagonism experiment is carried out, and the number of mixed bacteria in an inoculation space is measured.
In order to screen, detect and/or identify the Enterobacteriaceae (CPE) producing carbapenemase, some commercialized culture mediums such as Brilliance CRE culture medium (Oxoid), CHROMagar KPC, etc. appear, carbapenem antibiotics are added into the culture medium to screen the Enterobacteriaceae bacteria which are resistant to carbapenem, and the specific enzyme and the substrate (such as indole-based substrate, umbelliferone substrate, etc.) of the bacteria react to present different colony colors to identify the genus species of the bacteria. The culture medium can directly screen penicillene-resistant Enterobacteriaceae bacteria and preliminarily identify the bacterial species, but the culture medium has the defects of low sensitivity and incapability of directly identifying the carbapenemase type.
Disclosure of Invention
The invention aims to provide a latticed culture medium plate and a detection method for carbapenem-resistant enterobacter, and provides a latticed culture medium plate forming a good sealed subarea and a detection method for identifying carbapenem-resistant enterobacter.
In order to achieve the purpose, the invention provides the following technical scheme:
a grid-divided culture medium flat plate comprises a bottom plate, a side wall surrounding the bottom plate and an upper cover, wherein the bottom plate is provided with a partition plate for dividing the bottom plate into more than one area; the inner wall of the upper cover is provided with a slot for inserting the clapboard, and the inner wall of the slot is covered with a rubber layer.
The partition plate divides the bottom plate into more than one area for culturing more than one strain in the same environment. The upper cover has the function of reducing the water evaporation rate, and when the upper cover is covered, the partition plate is inserted into the slot, and good sealing is formed between the partition plate and the slot, so that the microorganisms are prevented from being mixed.
Preferably, the bottom plate and the upper cover are circular, the number of the partition plates is three, the partition plates are arranged along the radial direction of the bottom plate and are connected with the side wall, the bottom plate is equally divided into three areas, and the environments of the three areas are equal.
Preferably, a downward protruding piece is arranged in the middle of the edge of the upper cover between the adjacent slots; the middle of the side wall edge between the adjacent partition plates is provided with a notch. The notches are for tab insertion.
Preferably, the tab has a length longer than the depth of the notch.
Preferably, the clapboard is a transparent body, and a color card is embedded in the clapboard.
A method for detecting carbapenem-resistant enterobacteriaceae comprises the steps of selecting three areas of a cellular culture medium plate, namely a first area, a second area and a third area, adding agar culture media into the three areas, adding a carbapenemase activator into the agar culture medium in the first area, adding EDTA into the agar culture medium in the second area, adding tazobactam into the agar culture medium in the third area, and inoculating and culturing samples in the three areas.
If there is growth of bacteria on the medium after inoculation of the sample, it is proved to be a carbapenem-resistant bacterium of the Enterobacteriaceae family; if one, two regions grow, it is indicated that the growing bacteria contain carbapenemase and are a class A enzymes in the Ambler classification; if one, three regions grow, it is indicated that the growing bacteria contain carbapenemase and are class B enzymes in the Ambler classification; if the first region, the second region and the third region all grow, the carbapenemase is D-type enzyme in the Ambler classification or is drug-resistant bacteria in the enterobacteriaceae family with a non-enzyme-containing mechanism.
Preferably, a chromogenic substrate and a carbapenem antibiotic are further added to the agar medium in the three regions.
Preferably, the following components: the agar culture medium of the three regions is also added with a third-number bile salt and amphotericin B.
Preferably, the chromogenic substrate is an indolyl substrate.
Preferably, the inoculation and cultivation temperature is 35-37 ℃, and the cultivation time is 18-36 h.
Compared with the prior art, the invention can at least produce one of the following beneficial effects:
the invention can reduce the water evaporation rate, and the separated areas form good isolation, thereby preventing the cultured microorganisms from mixing.
The method can effectively identify the carbapenem-resistant enterobacter.
Drawings
FIG. 1 is a schematic structural diagram of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The structure of such a compartmentalized culture medium plate is shown in FIG. 1, and several examples are listed below with reference to the figures.
Example 1:
a grid-divided culture medium flat plate comprises a bottom plate 1, a side wall 2 surrounding the bottom plate 1 and an upper cover 3, wherein a partition plate 4 is arranged in the middle of the bottom plate 1 to divide the bottom plate 1 into 2 areas; the inner wall of the upper cover 3 is provided with a slot 5 for inserting the clapboard 4, and the inner wall of the slot 5 is covered with a rubber layer.
The partition plates 4 divide the bottom plate 1 into 2 areas for simultaneously culturing 2 kinds of microorganisms in the same environment. The upper cover 3 is used for reducing the water evaporation rate, and when the upper cover 3 is covered, the partition plate 4 is inserted into the slot 5, and good sealing is formed between the partition plate 4 and the slot 5, so that the microorganisms are prevented from mixing.
Example 2:
a grid-divided culture medium flat plate comprises a bottom plate 1, a side wall 2 surrounding the bottom plate 1 and an upper cover 3, wherein the bottom plate 1 and the upper cover 3 are circular, partition plates 4 are arranged on the bottom plate 1 to divide the bottom plate 1 into 3 areas, the number of the partition plates 4 is three, the partition plates are arranged along the radial direction of the bottom plate 1 and connected with the side wall 2, and the bottom plate 1 is divided into three areas equally; the inner wall of the upper cover 3 is provided with a slot 5 for inserting the clapboard 4, and the inner wall of the slot 5 is covered with a rubber layer.
The partition plates 4 divide the bottom plate 1 into 3 areas for simultaneously culturing 3 kinds of microorganisms in the same environment. The upper cover 3 is used for reducing the water evaporation rate, and when the upper cover 3 is covered, the partition plate 4 is inserted into the slot 5, and good sealing is formed between the partition plate 4 and the slot 5, so that the microorganisms are prevented from mixing.
Example 3:
a grid-divided culture medium flat plate comprises a bottom plate 1, a side wall 2 surrounding the bottom plate 1 and an upper cover 3, wherein the bottom plate 1 and the upper cover 3 are circular, partition plates 4 are arranged on the bottom plate 1 to divide the bottom plate 1 into 3 areas, the number of the partition plates 4 is three, the partition plates are arranged along the radial direction of the bottom plate 1 and connected with the side wall 2, and the bottom plate 1 is divided into three areas equally; the inner wall of the upper cover 3 is provided with a slot 5 for inserting the clapboard 4, and the inner wall of the slot 5 is covered with a rubber layer. And a downward lug 6 is arranged in the middle of the edge of the upper cover 3 between the adjacent slots 5; a notch 7 is provided at the middle of the edge of the side wall 2 between the adjacent partitions 4.
The partition plates 4 divide the bottom plate 1 into 3 areas for simultaneously culturing 3 kinds of microorganisms in the same environment. The upper cover 3 is used for reducing the water evaporation rate, and when the upper cover 3 is covered, the partition plate 4 is inserted into the slot 5, and good sealing is formed between the partition plate 4 and the slot 5, so that the microorganisms are prevented from mixing. And because baffle 4 and slot 5 are all inside, difficult alignment when covering, consequently set up lug 6 at upper cover 3, set up notch 7 at lateral wall 2, notch 7 is used for lug 6 to insert, when covering upper cover 3, it can be to its baffle 4 and slot 5 to align lug 6 and notch 7, consequently more convenient.
Example 4:
in addition to embodiment 3, the length of the protruding piece 6 is further limited to be longer than the depth of the notch 7, so that the upper cover 3 is jacked up by the protruding piece 6, and a gap is formed between the upper cover 3 and the side wall 2, so that the upper cover 3 can still maintain the function of reducing water evaporation, and each area can be fully contacted with the set environment of the culture chamber.
Example 5:
a grid-divided culture medium flat plate comprises a bottom plate 1, a side wall 2 surrounding the bottom plate 1 and an upper cover 3, wherein the bottom plate 1 and the upper cover 3 are circular, partition plates 4 are arranged on the bottom plate 1 to divide the bottom plate 1 into 3 areas, the number of the partition plates 4 is three, the partition plates 4 are transparent bodies, and color cards are embedded in the partition plates 4. The bottom plate is arranged along the radial direction of the bottom plate 1 and is connected with the side wall 2, and the bottom plate 1 is divided into three areas; the inner wall of the upper cover 3 is provided with a slot 5 for inserting the clapboard 4, and the inner wall of the slot 5 is covered with a rubber layer. And a downward lug 6 is arranged in the middle of the edge of the upper cover 3 between the adjacent slots 5; a notch 7 is provided at the middle of the edge of the side wall 2 between the adjacent partitions 4.
The partition plates 4 divide the bottom plate 1 into 3 areas for simultaneously culturing 3 kinds of microorganisms in the same environment. The upper cover 3 is used for reducing the water evaporation rate, and when the upper cover 3 is covered, the partition plate 4 is inserted into the slot 5, and good sealing is formed between the partition plate 4 and the slot 5, so that the microorganisms are prevented from mixing. And because baffle 4 and slot 5 are all inside, difficult alignment when covering, consequently set up lug 6 at upper cover 3, set up notch 7 at lateral wall 2, notch 7 is used for lug 6 to insert, when covering upper cover 3, it can be to its baffle 4 and slot 5 to align lug 6 and notch 7, consequently more convenient. The color card embedded in the partition board can play a guiding role in a plurality of microorganism identification experiments, and the microorganism type identification is carried out through the microorganism color in the plurality of experiments.
Example 6:
a carbapenem-resistant enterobacter detection method comprises the steps of selecting three areas of a cellular culture medium plate in examples 1 to 5, namely a first area, a second area and a third area, adding agar culture medium into the three areas, adding a carbapenemase activator into the agar culture medium added into the first area, wherein the adding concentration is 0.08mM-0.12 mM; EDTA is added into the agar culture medium added in the second area to inhibit the activity of B-class enzyme in Ambler classification, and the addition concentration is 1.0-2.5 m mol/L; tazobactam is added into the agar culture medium added in the three zones, the adding concentration is 2mg/mL-3mg/mL, and sample inoculation and cultivation are carried out in all three zones.
If there is growth of bacteria on the medium after inoculation of the sample, it is proved to be a carbapenem-resistant bacterium of the Enterobacteriaceae family; if one, two regions grow, it is indicated that the growing bacteria contain carbapenemase and are a class A enzymes in the Ambler classification; if one, three regions grow, it is indicated that the growing bacteria contain carbapenemase and are class B enzymes in the Ambler classification; if the first region, the second region and the third region all grow, the carbapenemase is D-type enzyme in the Ambler classification or is drug-resistant bacteria in the enterobacteriaceae family with a non-enzyme-containing mechanism. Saves the time and the workload of identifying the strain type by using chromogenic plate culture, identifying whether the strain is a CRE strain by using a method such as Carba NP and the like, and separating and identifying the carbapenemase type.
Example 7:
in addition to the above example 6, a chromogenic substrate and carbapenem antibiotics were added to the agar medium added to the three regions. The chromogenic substrate is preferably an indolyl substrate, and the preferred chromogenic substrate is selected from 5-bromo-6-chloro-3-indole-beta-D-galactoside and 5-bromo-4-chloro-3-indole-beta-D-glucoside, the concentrations of which are 0.18g/L and 0.09g/L respectively, so that the chromogenic substrate has better and more obvious chromogenic effect and can develop color more quickly. Adding carbapenem antibiotics for inhibiting carbapenem antibiotic sensitive strain, preferably meropenem, with an addition concentration of 0.25 mg/L. The invention can carry out primary strain identification according to the growth color of the bacterial colony after the inoculated clinical strain is cultured. If the colony color is dark pink to red, the colony is carbapenem-resistant escherichia coli; the colony color is metal blue, and the colony is carbapenems-resistant Klebsiella and Citrobacter; the colony color is light green, and the colony is the carbapenem-resistant pseudomonas aeruginosa; the colony color is opaque cream, and the colony is carbapenem-resistant acinetobacter. Namely, the invention can effectively identify the carbapenem-resistant phenotype of the enterobacteriaceae bacteria.
Example 8:
on the basis of the above example 6, tripno salts and amphotericin B were added to the agar medium added to the three regions. The third-number bile salt is used for inhibiting gram-positive bacteria, and the addition concentration of the third-number bile salt is 1.8 g/L; the amphotericin B is added to inhibit the growth of bacteria such as fungi, the concentration of the amphotericin B is 1.0mg/L, and the result is more accurate.
The most preferred embodiment is as follows:
on the basis of the example 7, the inoculation and cultivation temperature is limited to be 35-37 ℃, and the cultivation time is 18-36 h. Under the cultivation environment, the identification result can be quickly displayed. According to experiments, compared with other temperatures, for example, under the condition that other conditions are inconvenient, the incubation temperature is set to be 30 ℃, the strain grows slowly, and the identification result can be displayed only when the incubation time is about 42 hours. When the temperature is higher than 37 ℃, the death rate of the strains is increased, and the color is obvious in about 40 hours.
Reference throughout this specification to multiple illustrative embodiments means that a particular structure described in connection with the embodiments is included in at least one embodiment described generally throughout this application. The appearances of the same phrase in various places in the specification are not necessarily all referring to the same embodiment. Further, while a structure has been described in connection with any one embodiment, it is to be understood that it is within the scope of the invention to implement such structure in connection with other embodiments.
Claims (10)
1. A grid-divided culture medium flat plate comprises a bottom plate (1), a side wall (2) surrounding the bottom plate (1) and an upper cover (3), wherein a partition plate (4) for dividing the bottom plate (1) into more than 2 areas is arranged on the bottom plate (1); the method is characterized in that: the inner wall of the upper cover (3) is provided with a slot (5) for inserting the clapboard (4), and the inner wall of the slot (5) is covered with a rubber layer.
2. A grid medium plate according to claim 1, wherein: the bottom plate (1) and the upper cover (3) are circular, the number of the partition plates (4) is three, the partition plates are arranged along the radial direction of the bottom plate (1) and are connected with the side wall (2), and the bottom plate (1) is equally divided into three areas.
3. A grid medium plate according to claim 2, wherein: a downward protruding piece (6) is arranged in the middle of the edge of the upper cover (3) between the adjacent slots (5); the middle of the edge of the side wall (2) between the adjacent partition boards (4) is provided with a notch (7).
4. A grid medium plate according to claim 3, wherein: the length of the lug (6) is longer than the depth of the notch (7).
5. A grid medium plate according to claim 1, wherein: the partition board (4) is a transparent body, and a color card is embedded in the partition board (4).
6. A method for detecting Enterobacter carbapenems-resistant on the basis of a divided cell culture medium plate according to any one of claims 1 to 5, wherein: selecting three areas in a grid culture medium plate, namely a first area, a second area and a third area, respectively, adding agar culture media into the three areas, adding a carbapenemase activator into the agar culture medium of the first area, adding EDTA into the agar culture medium of the second area, adding tazobactam into the agar culture medium of the third area, and inoculating and culturing samples in the three areas.
7. The method for detecting carbapenem-resistant Enterobacter sp. The agar culture medium of the three areas is also added with chromogenic substrate and carbapenem antibiotics.
8. The method for detecting carbapenem-resistant Enterobacter sp. The agar culture medium of the three regions is also added with a third-number bile salt and amphotericin B.
9. The method for detecting carbapenem-resistant Enterobacter sp. The chromogenic substrate is an indolyl substrate.
10. The method for detecting carbapenem-resistant Enterobacter sp. The inoculation cultivation temperature is 35-37 ℃, and the cultivation time is 18-36 h.
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| CN202011349355.3A CN112300898A (en) | 2020-11-26 | 2020-11-26 | Grid-divided culture medium plate and method for detecting carbapenem-resistant enterobacteriaceae |
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| CN202011349355.3A CN112300898A (en) | 2020-11-26 | 2020-11-26 | Grid-divided culture medium plate and method for detecting carbapenem-resistant enterobacteriaceae |
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| CN202011349355.3A Pending CN112300898A (en) | 2020-11-26 | 2020-11-26 | Grid-divided culture medium plate and method for detecting carbapenem-resistant enterobacteriaceae |
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