CN112779185A - Method for purifying staphylococcus aureus in proteus - Google Patents
Method for purifying staphylococcus aureus in proteus Download PDFInfo
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- CN112779185A CN112779185A CN202110059359.6A CN202110059359A CN112779185A CN 112779185 A CN112779185 A CN 112779185A CN 202110059359 A CN202110059359 A CN 202110059359A CN 112779185 A CN112779185 A CN 112779185A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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Abstract
The invention discloses a method for purifying staphylococcus aureus in proteus, belongs to a microorganism separation and purification technology, and particularly relates to a staphylococcus aureus separation and purification technology, aiming at solving the defect that the staphylococcus aureus can not be purified from the proteus, comprising the following steps: taking physiological saline; adding aztreonam drug sensitive paper sheets into the normal saline, and uniformly mixing to obtain aztreonam drug liquid; dipping aztreonam medicinal liquid with a cotton swab; uniformly smearing on a blood agar plate, and standing for 5-10 min to obtain the blood agar plate containing aztreonam medicine; selecting a mixture of proteus and staphylococcus aureus, and inoculating; and incubating for 16-24 hours in the incubator to separate staphylococcus aureus. As long as the mixture contains staphylococcus aureus, a large number of single pure bacterial colonies can be accurately separated, the bacterial colonies are not polluted by proteus, and the bacterial colonies can be directly used for drug sensitivity tests, so that the time period is greatly shortened.
Description
Technical Field
The invention discloses a method for purifying staphylococcus aureus in proteus, belongs to the technical field of microbial separation and purification, and particularly relates to the technical field of staphylococcus aureus separation and purification.
Background
Staphylococcus aureus (also called Staphylococcus aureus), belonging to Staphylococcus, is a common food-borne pathogenic microorganism, without spores and flagella, most of which are not capsule, gram-positive, the nutrition requirement of Staphylococcus aureus is not high, the Staphylococcus aureus grows well on a common culture medium, aerobically or facultatively anaerobically, the optimum growth temperature is 37 ℃, the optimum growth pH is 7.4, the bacterial colony on the flat plate is thick, glossy and round bulge, the diameter is 1-2mm, a transparent hemolytic ring is formed around the bacterial colony of the blood flat plate, the Staphylococcus aureus has high salt tolerance, can grow in 10-15% NaCl broth, can decompose glucose, maltose, lactose and cane sugar, produce acid and produce no gas, the methyl red reaction is positive, the VP reaction is weak positive, a plurality of bacterial strains can decompose arginine, hydrolyze urea, reduce nitrate and liquefy gelatin, staphylococcus aureus has a strong resistance, low sensitivity to sulfonamides, but high sensitivity to penicillin, erythromycin, and the like.
The proteus is a group of intestinal bacteria which can actively move and have extremely polymorphism, has spherical and filamentous shapes, has flagella, no capsule, no spore, gram negative and active movement, and is in diffusion growth on a solid culture medium to form a migration growth phenomenon, if 0.1 percent carbolic acid or 0.4 percent boric acid is added into the culture medium, the diffusion growth can be inhibited to form a common single colony, a round, flat and thin semitransparent colony can be formed on an SS plate, the colony is easy to be confused with other intestinal pathogenic bacteria, and the hemolysis phenomenon exists on a blood agar plate.
Because of the characteristic of the migration and growth of the proteus, the bacterial colony can cover the whole culture medium along with the growth of the proteus, so that the staphylococcus aureus growing in the culture medium can not be used for a medicament sensitivity test in a pure mode, the staphylococcus aureus is a pathogenic bacterium but can be polluted by the proteus in a clinical specimen provided by a patient, and the existing purification method is good.
Disclosure of Invention
The invention aims to: a method for purifying staphylococcus aureus in proteus, which aims to solve the defect that the existing staphylococcus aureus cannot be purified from the proteus due to the growth characteristics of the proteus in the early days.
The technical scheme adopted by the invention is as follows:
the method for purifying staphylococcus aureus in proteus comprises the following steps:
step 1, taking 2ml of 0.45-0.9% physiological saline;
step 2, putting 1 or more aztreonam drug sensitive paper sheets (with the size of 6mm and the aztreonam content of 30 microliters) into the physiological saline, clamping the aztreonam drug sensitive paper sheets by using forceps, and uniformly mixing to obtain aztreonam drug liquid;
step 3, dipping aztreonam medicinal liquid by using a cotton swab;
step 4, uniformly smearing the mixture on a blood agar plate, and standing for 5-10 minutes to obtain the blood agar plate containing the aztreonam drug;
step 5, selecting a mixture of proteus and staphylococcus aureus, inoculating the mixture to a blood agar plate containing aztreonam medicine, and marking lines in areas 3 or 4;
and 6, incubating in an incubator (a conventional incubator for bacterial culture) for 16-24 hours, and separating staphylococcus aureus.
In the technical scheme of the application: because aztreonam can inhibit the growth of proteus and has no effect on staphylococcus aureus, aztreonam drug sensitive paper is soaked in physiological saline to release aztreonam, then the whole plate is coated with liquid containing aztreonam, standing is carried out for 5-10 minutes, after the plate is dried, bacterial colony or bacterial liquid of a mixture of proteus and staphylococcus aureus is inoculated, and the plate is incubated in an incubator for 16-24 hours, only staphylococcus aureus grows and can be used for drug sensitive tests, and the proteus is completely inhibited. The method for purifying staphylococcus aureus in proteus reduces purification steps, greatly shortens purification time, achieves one step, and is good in repeatability, simple and easy to operate; as long as the staphylococcus aureus exists in the mixture, a large number of single pure bacterial colonies can be accurately separated, the bacterial colonies are not polluted by proteobacteria, the bacterial colonies can be directly used for drug sensitivity tests, the time period is greatly shortened, a drug sensitivity report can be issued within about 30 hours at the fastest speed, the working efficiency is high, and the bacterial colonies play a positive role in clinically and timely adjusting the drug administration.
Preferably, in step 1, saline is taken with a drug sensitive turbidimeter.
Preferably, the step 2 is carried out by a blending machine for 15 to 45 seconds.
Preferably, in step 2, the mixture is mixed for 30s by a mixer.
Preferably, any one of 0.5 to 6 aztreonam drug-sensitive paper sheets is put into the physiological saline in the step 2.
Preferably, in step 3, the aztreonam drug liquid is dipped by a cotton swab, and excess aztreonam drug liquid is wiped off.
More preferably, the aztreonam drug liquid is dipped by a cotton swab and evenly smeared on a blood agar plate, the smearing is repeated for 1 to 4 times, and the blood agar plate is turned at an angle of 60 degrees after each smearing is finished.
More preferably, the aztreonam drug liquid is dipped with a cotton swab and spread evenly on blood agar plates 3 times.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. according to the method for purifying staphylococcus aureus in proteus, the purification steps are reduced, the purification time is greatly shortened, the method is one-step, good in repeatability and simple and easy to operate;
2. in the invention, a large amount of single pure bacterial colonies can be accurately separated as long as staphylococcus aureus exists in the mixture, the single bacterial colonies are not polluted by proteus bacillus in the mixed bacteria, the pure bacteria are not required to be separated again, the pure bacteria can be directly used for a drug sensitivity test, the time period is greatly shortened, a drug sensitivity report can be provided within about 30 hours at the fastest speed, the working efficiency is greatly improved, and the method plays a positive role in clinically and timely adjusting the drug administration;
3. in the invention, 1/4 tablets/ml aztreonam solution can be used as the critical concentration, and 0.5 tablet/ml aztreonam solution can be used as the stable inhibitory concentration.
Drawings
FIG. 1 shows the growth of Proteus and Staphylococcus aureus at different aztreonam concentrations according to the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following 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.
Example 1
The method for purifying staphylococcus aureus in proteus comprises the following steps:
step 1, taking 2ml of 0.45% physiological saline by using a drug sensitive turbidimetric tube;
step 2, more than 2 pieces of aztreonam drug sensitive paper sheets are put into the physiological saline, and are mixed uniformly to obtain aztreonam drug liquid, and the mixture is mixed uniformly for 30s by a mixer;
step 3, dipping the aztreonam medicinal liquid by using a cotton swab, and wiping off redundant aztreonam medicinal liquid;
step 4, dipping the aztreonam medicine liquid by using a cotton swab, uniformly smearing the aztreonam medicine liquid on a blood agar plate, repeating the step for 3 times, turning the blood agar plate to an angle of 60 degrees after each smearing is finished, and standing for 8 minutes to obtain the blood agar plate containing the aztreonam medicine;
step 5, selecting a mixture of proteus and staphylococcus aureus, inoculating the mixture to a blood agar plate containing aztreonam medicine, and then drawing lines in a zone 4;
and 6, incubating for 18 hours in the incubator to separate staphylococcus aureus.
Example 2
The method for purifying staphylococcus aureus in proteus comprises the following steps:
step 1, taking 2ml of 0.45% physiological saline by using a drug sensitive turbidimetric tube;
step 2, more than 4 pieces of aztreonam drug sensitive paper sheets are put into the physiological saline, and are uniformly mixed to obtain aztreonam drug liquid, and the aztreonam drug liquid is uniformly mixed for 30s by a uniformly mixer;
step 3, dipping the aztreonam medicinal liquid by using a cotton swab, and wiping off redundant aztreonam medicinal liquid;
step 4, dipping the aztreonam medicine liquid by using a cotton swab, uniformly smearing the aztreonam medicine liquid on a blood agar plate, repeating for 2 times, turning the blood agar plate to an angle of 60 degrees after each smearing is finished, and standing for 10 minutes to obtain the blood agar plate containing the aztreonam medicine;
step 5, selecting a mixture of proteus and staphylococcus aureus, inoculating the mixture to a blood agar plate containing aztreonam medicine, and then drawing lines in a zone 3;
and 6, incubating for 20 hours in the incubator to separate staphylococcus aureus.
Example 3
The method for purifying staphylococcus aureus in proteus comprises the following steps:
step 1, taking 2ml of 0.9% physiological saline by using a drug sensitive turbidimetric tube;
step 2, more than 1 piece of aztreonam drug sensitive paper sheets are put into the physiological saline, and are mixed uniformly to obtain aztreonam drug liquid, and the mixture is mixed uniformly for 30s by a mixer;
step 3, dipping the aztreonam medicinal liquid by using a cotton swab, and wiping off redundant aztreonam medicinal liquid;
step 4, dipping the aztreonam medicine liquid by using a cotton swab, uniformly smearing the aztreonam medicine liquid on a blood agar plate, repeating the step for 3 times, turning the blood agar plate to an angle of 60 degrees after each smearing is finished, and standing for 6 minutes to obtain the blood agar plate containing the aztreonam medicine;
step 5, selecting a mixture of proteus and staphylococcus aureus, inoculating the mixture to a blood agar plate containing aztreonam medicine, and then drawing lines in a zone 4;
and 6, incubating for 16 hours in the incubator to separate staphylococcus aureus.
Experimental example 1
Control 1, 1/4 pieces of aztreonam drug sensitive paper were added to each ml of physiological saline, and only proteus was inoculated; control 2, add 3 aztreonam drug sensitive paper sheets per ml of normal saline, only inoculate staphylococcus aureus; 1/32, 1/20, 1/16, 1/8, 1/4 and 1/2 pieces of aztreonam drug sensitive paper (in the technical scheme of the invention, a series of aztreonam concentrations are obtained by a multiple dilution method) are respectively added into each milliliter of normal saline, and the rest is the same as the example 2, and the growth conditions of the bacteria are observed in 1 day, 2 days and 3 days after inoculation and are shown in a table 1 and a figure 1(35 ℃, 48 hours).
TABLE 1 growth conditions of the respective bacteria
From table 1, it is known that: the aztreonam drug sensitive paper sheet has no inhibiting effect on the growth of staphylococcus aureus; when the dosage of the aztreonam paper sheet is 1/4 sheets/ml, the inhibition effect on the proteus is not ideal when the inoculation amount of the proteus is large; 1/2 tablets/ml, the inhibition effect is obvious, so 1/4 tablets/ml aztreonam solution can be used as the critical concentration, and 0.5 tablet/ml can be used as the stable inhibition concentration.
Experimental example 2
The pure staphylococcus aureus on 0.5 piece/ml, 1 piece/ml, 2 pieces/ml and 3 pieces/ml aztreonam blood agar plates are used for enrichment culture for 5 times and a merriella VITEK 2compact instrument susceptibility test, and the experimental results are shown in tables 2 and 3.
TABLE 2 selection of growth of a single S.aureus bouillon enrichment culture after fractionation
As shown in Table 2, the concentration of 0.5 tablets/ml of aztreonam is effective in inhibiting the growth of Proteobacteria, but some of the Proteobacteria may still survive under the drug pressure, so that Staphylococcus aureus is isolated from Proteobacteria, and the concentration of aztreonam should be selected to be 0.5 tablets/ml or more.
TABLE 3 picking the results of the single staphylococcus aureus susceptibility test after fractionation and the single staphylococcus aureus susceptibility comparison
As shown in Table 3, the results of the single Staphylococcus aureus susceptibility test and the single Staphylococcus aureus susceptibility test after the sorting of the 0.5 pieces/ml aztreonam plate and the plates with the concentrations above are uniform. After 0.5 piece/ml aztreonam solution is purified by the method, staphylococcus aureus can not be interfered by proteus carried in the plate, so that a single staphylococcus aureus purified by the aztreonam plate can be directly used for a drug sensitivity test.
According to the data, after comprehensively considering a plurality of factors such as simple and easy operation, stable and reliable result, good repeatability, quick separation of drug sensitivity test and the like, the following test conclusion is obtained:
selecting 1 piece/ml or more of aztreonam solution to be smeared on a blood culture medium, diluting the mixed bacterial colony in the solution, inoculating 50-100 microliters (1-2 drops) of mixed solution on the plate, and incubating for 16-18 hours for carrying out isolated culture of staphylococcus aureus;
selecting 2 pieces/ml-3 pieces/ml aztreonam solution to smear a blood culture medium, diluting the mixed bacterial colony in the solution, inoculating 50-100 microliters (1-2 drops) of the mixed liquid on the plate, incubating for 20-24 hours, and performing direct single bacterial colony drug sensitivity test after the separation culture of staphylococcus aureus.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. The method for purifying staphylococcus aureus in proteus is characterized by comprising the following steps: the method comprises the following steps:
step 1, taking 2ml of 0.45-0.9% physiological saline;
step 2, 1 or more than 1 piece of aztreonam drug sensitive paper sheets are put into the physiological saline and evenly mixed to obtain aztreonam drug liquid;
step 3, dipping aztreonam medicinal liquid by using a cotton swab;
step 4, uniformly smearing the mixture on a blood agar plate, and standing for 5-10 minutes to obtain the blood agar plate containing the aztreonam drug;
step 5, selecting a mixture of proteus and staphylococcus aureus, inoculating the mixture to a blood agar plate containing aztreonam medicine, and marking lines in areas 3 or 4;
and 6, putting the blood agar plate obtained in the step 5 into a bacterial incubator with or without CO2 at 35 ℃ for incubation for 16-24 hours, and separating staphylococcus aureus.
2. The method for purifying staphylococcus aureus in proteus according to claim 1, wherein: in the step 1, saline is taken by a drug sensitive turbidimeter tube.
3. The method for purifying staphylococcus aureus in proteus according to claim 1, wherein: and (3) uniformly mixing for 15-45s by using a uniformly mixing device in the step 2.
4. The method for purifying staphylococcus aureus in proteus according to claim 1, wherein: and in the step 2, uniformly mixing for 30s by using a uniformly mixing device.
5. The method for purifying staphylococcus aureus in proteus according to claim 1, wherein: in the step 2, any one piece of aztreonam drug sensitive paper is put into 0.5 to 6 pieces of physiological saline.
6. The method for purifying staphylococcus aureus in proteus according to claim 1, wherein: in step 3, the aztreonam medicinal liquid is dipped by a cotton swab, and redundant aztreonam medicinal liquid is wiped off.
7. The method for purifying staphylococcus aureus in proteus as claimed in claim 6, wherein: dipping the aztreonam medicinal liquid with a cotton swab, uniformly smearing on a blood agar plate, repeating for 1-4 times, and turning the blood agar plate at an angle of 60 degrees after each smearing is finished.
8. The method for purifying staphylococcus aureus in proteus according to claim 7, wherein: the aztreonam drug liquid was dipped with a cotton swab and spread evenly on blood agar plates for 3 times.
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| W WOOD等: "Aztreonam selective agar for Gram positive bacteria", J CLIN PATHOL., vol. 46, no. 8, 31 August 1993 (1993-08-31), pages 769, XP009059061, DOI: 10.1136/jcp.46.8.769 * |
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