Method for preparing bacterial liquid for antimicrobial susceptibility testing

Abstract

Provided is a method for preparing a bacterial liquid for antimicrobial susceptibility testing. The method comprises: picking a bacterial colony from a sample for preparing the bacterial liquid; preparing the bacterial colony into a first bacterial liquid, and counting bacteria in the first bacterial liquid to obtain a first bacterial count of the first bacterial liquid; and deducing a first concentration of the first bacterial liquid on the basis of the first bacterial count, and diluting the first bacterial liquid on the basis of the first concentration to obtain a second bacterial liquid, wherein the second concentration of the second bacterial liquid is 105 to 106 / milliliters (cfu / mL); and the second bacterial liquid is the bacterial liquid for the antimicrobial susceptibility testing. By applying a counting method to the preparation of the bacterial liquid for the antimicrobial susceptibility testing, the time for preparing the bacterial liquid is greatly shortened, and the concentration of the sample used is almost lower than 100 times a bacterial liquid amount of 0.5MCF (1.5*108cfu / mL). That is to say, a bacterial liquid for antimicrobial susceptibility testing can be prepared at a low concentration, thus achieving an application innovation.

Description

A method for preparing bacterial solution for drug sensitivity test

[0001] This application is based on the Chinese application with CN application number 202410172103.X and application date February 6, 2024 and the Chinese application with CN application number 202410172166.5 and application date February 6, 2024, and claims the above-mentioned priority. The disclosed content of the above-mentioned CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of biomedicine, and more particularly to a method for preparing a bacterial solution for drug sensitivity testing. Background Art

[0003] The preparation of bacterial solution is an important part of routine operation of microbial susceptibility test (AST). The target concentration of bacterial solution inoculation is 10 5 -10 6 cfu / mL, the conventional operation is to use McFadden turbidimeter to measure the concentration of 0.5MCF (1.5*10 8 cfu / mL) of bacterial solution, which is then manually diluted to the target concentration. However, obtaining pure colonies of 0.5 McFarland's Ferguson in clinical practice requires a long wait. This is partly due to the slow growth of cultured bacteria, and partly because obtaining sufficient numbers of single pure colonies in actual clinical work is difficult. Due to the presence of colonizing bacteria, it is difficult to obtain a high concentration of pure colonies of pathogenic bacteria, so further transfection culture is required, which often takes 24 hours or even longer.

[0004] McFarland turbidimetry is one of the common methods to reflect the microbial content by detecting the scattered light of microorganisms in the suspension. The greater the content of microorganisms in the suspension, the less light passes through the suspension and the more light is scattered. That is, the concentration of the bacterial suspension is inversely proportional to the transmittance and directly proportional to the optical density within a certain range. It is mainly used in bacterial turbidity detection and drug sensitivity testing in the field of microbial detection.

[0005] Turbidity refers to the degree of obstruction to light transmission caused by suspended matter in water. Suspended matter in water generally consists of soil, sand, fine organic and inorganic matter, plankton, microorganisms, and colloids. The turbidity of water is not only related to the content of suspended matter in the water, but also to their size, shape, and refractive index. Turbidity can also be measured using a turbidimeter. A turbidimeter sends light through a sample and detects how much light is scattered by particles in the water at a 90-degree angle to the incident light. This method of measuring scattered light is called the scattering method. Any true turbidity must be measured in this way. Turbidimeters are suitable for both field and laboratory measurements and for continuous monitoring around the clock.

[0006] The turbidimetric method for testing bacterial solution concentration based on this principle uses a photoelectric turbidimetric detection method. By testing the transmittance and reflectance of the test solution, the turbidity value of the test solution is determined, and the result can be output in transmittance McFarland units. However, this method of using optical concentration to detect bacterial solution concentration is not accurate and the manual operation is cumbersome. If the measured McFarland result is too high, it is necessary to dilute and re-measure. If the measured McFarland result is too low, the bacteria must be picked and re-measured.

[0007] Therefore, a new method for preparing bacterial solution for drug sensitivity testing needs to be proposed to at least solve the technical problems in the prior art of long time for further culture of bacterial solution, inaccurate bacterial solution concentration and cumbersome manual operation.

[0008] Bloodstream infections (BSIs) are systemic infections caused by pathogens such as bacteria and fungi invading the bloodstream. Pathogens entering the bloodstream reproduce at an astonishing rate of one generation every 20 minutes, spreading rapidly and triggering a strong host response within hours. BSIs can rapidly progress to septic shock, disseminated intravascular coagulation (DIC), and multiple organ failure, with a mortality rate of 25-46%. For every hour of delay in treatment, the patient's mortality rate increases by 5%. Therefore, shortening the time required to identify the pathogenic bacteria and their antibiotic susceptibility will significantly improve the success rate of treatment.

[0009] Blood culture is the simplest, most accurate, and most commonly used method for detecting bloodstream infections. It provides the basis for confirming the etiology of bloodstream infections. Early blood culture testing and appropriate antimicrobial therapy are paramount measures for controlling bloodstream infections. Furthermore, as the gold standard for diagnosing bloodstream infections, blood culture accurately isolates the infecting pathogen and, when combined with antimicrobial susceptibility testing, allows for the development of accurate and precise treatment plans.

[0010] However, the traditional method requires that after a positive blood culture alarm is issued, the positive blood culture specimen be transferred to a blood plate and cultured for 18-24 hours, waiting for the formation of a single colony, and then the pure colony be subjected to a drug sensitivity test. The whole process takes 2-3 days, and the patient's condition often cannot wait for such a long time.

[0011] Therefore, there is an urgent need to develop a method for directly conducting drug sensitivity testing on blood culture-positive specimens to shorten the time. Summary of the Invention

[0012] The present invention provides a method for preparing a bacterial solution for drug sensitivity testing, which at least solves the technical problems in the prior art of long time required for further culture of the bacterial solution, inaccurate bacterial solution concentration and cumbersome manual operation.

[0013] The present invention provides a method for preparing a bacterial solution for drug sensitivity testing, the method comprising:

[0014] Picking the colony of the sample used to prepare the above bacterial solution;

[0015] preparing the colonies into a first bacterial liquid, and counting the number of bacteria in the first bacterial liquid to obtain a first bacterial count of the first bacterial liquid;

[0016] The first concentration of the first bacterial solution is inferred based on the first bacterial count, and the first bacterial solution is diluted based on the first concentration to obtain a second bacterial solution. The second concentration of the second bacterial solution is 10 5 to 10 6 cells / mL (cfu / mL);

[0017] The second bacterial solution is the bacterial solution used for the drug sensitivity test (as shown in Figure 2).

[0018] Optionally, the above-mentioned colonies are fastidious bacterial colonies, mixed bacterial colonies or pure colonies.

[0019] Optionally, the above-mentioned fastidious bacteria colony is a fastidious bacteria colony whose growth does not reach 0.5 McFarland, or the above-mentioned mixed bacteria colony includes a colony whose growth does not reach 0.5 McFarland, or the above-mentioned pure colony is a pure colony whose growth does not reach 0.5 McFarland.

[0020] Optionally, colonies of a sample for preparing the bacterial liquid are picked up and mixed into physiological saline to form the first bacterial liquid; and the first bacterial liquid is diluted with culture medium according to the first concentration to obtain the second bacterial liquid.

[0021] Optionally, a resistance counting method is used to count the number of bacteria in the first bacterial liquid to obtain a first bacterial count in the first bacterial liquid.

[0022] Optionally, an inoculation device is used to pick colonies from a sample of the bacterial solution.

[0023] Optionally, the above-mentioned inoculation device is an inoculation needle.

[0024] Optionally, the above method includes the following steps:

[0025] A. Pick up the above bacterial colonies and add 2 to 10 ml of normal saline solution, and mix thoroughly to form the above first bacterial solution;

[0026] B. counting the number of bacteria in the first bacterial solution to obtain a first bacterial count of the first bacterial solution;

[0027] C. Determine the first concentration of the first bacterial solution (C) based on the first bacterial count and the volume of the first bacterial solution. 初 ), according to the first concentration (C 初 ) dilute the first bacterial solution and mix it to obtain a second bacterial solution. The second concentration of the second bacterial solution is 10 5to 10 6 cells / ml (cfu / mL), and the second bacterial liquid is the bacterial liquid used for the drug sensitivity test.

[0028] Optionally, the above method includes the following steps:

[0029] A. Pick up the above bacterial colonies and add 3 to 5 ml of normal saline solution, and mix thoroughly to form the above first bacterial solution;

[0030] B. counting the number of bacteria in the first bacterial solution to obtain a first bacterial count of the first bacterial solution;

[0031] C. Determine the first concentration of the first bacterial solution (C) based on the first bacterial count and the volume of the first bacterial solution. 初 ), according to the first concentration (C 初 ) dilute the first bacterial solution and mix it to obtain a second bacterial solution. The second concentration of the second bacterial solution is 10 5 to 10 6 cells / ml (cfu / mL), and the second bacterial liquid is the bacterial liquid used for the drug sensitivity test.

[0032] Optionally, the above method includes the following steps:

[0033] A. Pick up the above bacterial colonies and add 6 to 8 ml of normal saline solution, and mix thoroughly to form the above first bacterial solution;

[0034] B. counting the number of bacteria in the first bacterial solution to obtain a first bacterial count of the first bacterial solution;

[0035] C. Determine the first concentration of the first bacterial solution (C) based on the first bacterial count and the volume of the first bacterial solution. 初 ), according to the first concentration (C 初 ) dilute the first bacterial solution and mix it to obtain a second bacterial solution. The second concentration of the second bacterial solution is 10 5 to 10 6 cells / ml (cfu / mL), and the second bacterial liquid is the bacterial liquid used for the drug sensitivity test.

[0036] Optionally, the second bacterial liquid obtained by the bacterial liquid preparation method for drug sensitivity test is used in the drug sensitivity test.

[0037] In addition, the present invention provides a method for directly preparing a bacterial solution for drug sensitivity testing from a blood culture-positive specimen, so as to at least solve the technical problem in the prior art that it takes too long to prepare a bacterial solution for drug sensitivity testing.

[0038] The present invention provides a method for directly preparing a bacterial liquid for a drug sensitivity test from a blood culture-positive specimen. The method comprises the following steps: aspirating the blood culture-positive specimen into a centrifuge tube containing a separation gel; mixing the blood culture-positive specimen with the separation gel to form a liquid to be centrifuged; centrifuging the liquid to be centrifuged of the blood culture-positive specimen so that target bacteria of the blood culture-positive specimen remain in a supernatant after centrifugation of the liquid to be centrifuged to form a first supernatant; counting the target bacteria in the first supernatant to obtain a first bacterial count in the first supernatant; inferring a first concentration of the first supernatant based on the first bacterial count in the first supernatant; diluting the first supernatant based on the first concentration of the first supernatant to obtain a bacterial liquid with a target concentration; and directly performing a drug sensitivity test on the bacterial liquid with the target concentration.

[0039] Optionally, counting the target bacteria in the first supernatant refers to detecting the number of the target bacteria.

[0040] Optionally, the separation gel retains the target bacteria in the first supernatant under centrifugation.

[0041] Optionally, the target bacteria are counted using the Coulter counting method.

[0042] Optionally, the second concentration of the target concentration bacterial solution is 10 4 to 10 6 The bacterial solution with the above target concentration was directly used for drug sensitivity test.

[0043] Optionally, 2 to 8 ml of the above-mentioned blood culture-positive specimen is aspirated into the above-mentioned centrifuge tube, and the above-mentioned liquid to be centrifuged containing the above-mentioned blood culture-positive specimen is centrifuged at a speed of 1000 to 6000 revolutions per minute (r / min) for 6 to 14 minutes, so that the above-mentioned target bacteria in the above-mentioned blood culture-positive specimen remain in the supernatant after centrifugation of the above-mentioned liquid to be centrifuged to form the above-mentioned first supernatant, and the above-mentioned first supernatant including the above-mentioned target bacteria is diluted 25-100 times to obtain the above-mentioned target concentration bacterial liquid.

[0044] Optionally, 3 to 6 ml of the above-mentioned blood culture-positive specimen is aspirated into the above-mentioned centrifuge tube, and the above-mentioned liquid to be centrifuged containing the above-mentioned blood culture-positive specimen is centrifuged at a speed of 2000 to 4000 rpm and a time of 8 to 12 minutes, so that the target bacteria of the above-mentioned blood culture-positive specimen remain in the supernatant after the centrifugation of the above-mentioned liquid to be centrifuged to form the above-mentioned first supernatant. The above-mentioned first supernatant including the above-mentioned target bacteria is diluted 40 to 60 times to obtain the above-mentioned bacterial liquid with the target concentration.

[0045] Optionally, the target bacteria of the above-mentioned blood culture-positive specimen remain in the supernatant after the above-mentioned liquid to be centrifuged is centrifuged to form the above-mentioned first supernatant, and other impurities of the above-mentioned blood culture-positive specimen sink to the lower end of the above-mentioned centrifuge tube.

[0046] Optionally, the bacterial solution of the target concentration is transferred into a test kit of a drug sensitivity analyzer to perform a drug sensitivity test.

[0047] The present invention provides a method for directly preparing a bacterial solution for drug sensitivity testing from a blood culture-positive specimen. Target bacteria are directly obtained from the blood culture-positive specimen by centrifugation, thus saving the time for plate culture.

[0048] The present invention provides a method for preparing bacterial liquid for direct drug sensitivity testing of blood culture-positive specimens, which provides a reference for the formulation of clinical treatment plans.

[0049] The main steps of this method are as follows:

[0050] 5 ml of blood was drawn into a blood culture bottle and inoculated with 50 cfu / mL of ATCC25922 Escherichia coli and cultured in a commercially available blood culture device; after the blood culture sample was positive, a volume a of the positive specimen was aspirated and added to a centrifuge tube containing separation gel, and centrifuged so that the target bacteria in the positive blood culture specimen remained in the supernatant and other impurities settled to the bottom of the centrifuge tube; the supernatant was diluted b times and transferred to a plate equipped with a fully automatic rapid drug sensitivity analyzer, and the target bacteria were counted on the analyzer; the target bacteria were then diluted and subjected to a subsequent rapid drug sensitivity test. The process flow diagram of the drug sensitivity test was shown in Figure 4.

[0051] Therefore, the method of the present invention can be applied to almost all drug sensitivity tests, because other impurities sink to the lower end of the centrifuge tube, and the target concentration bacterial solution does not contain other impurities, which is more conducive to the counting of target bacteria and the determination of turbidity.

[0052] In a preferred embodiment, the volume a of the blood culture-positive sample drawn is 2-8 ml, the suboptimal volume is 3 ml, and the optimal volume is 4 ml. In another preferred embodiment, the centrifugation speed is 1000-6000 rpm, the suboptimal volume is 4000 rpm, and the optimal volume is 3000 rpm. In another preferred embodiment, the centrifugation time is 6-14 minutes, the suboptimal volume is 12 minutes, and the optimal volume is 10 minutes. In another preferred embodiment, the dilution factor b of the supernatant after centrifugation is 25-100 times, the suboptimal volume is 60 times, and the optimal volume is 50 times.

[0053] Separation gel is a reagent used for serum separation, separating blood into serum and blood cells. Separation gel uses a special colloid to separate serum and blood cells. This not only reduces cross-contamination between serum and blood cells, improving experimental accuracy, but also allows for rapid bacterial count and drug susceptibility testing from serum. Separation gel is characterized by its ability to separate blood into serum and blood cells without mixing pathogens with blood cells, retaining them in the serum for subsequent direct count and drug susceptibility testing. The main components of this separation gel are polyolefin, polyester, and propylene.

[0054] The technical solution of the present invention can obtain the clinically required bacterial liquid concentration, measure the bacterial content, and dilute to the target bacterial liquid concentration. Its advantages are:

[0055] 1. Compared with the turbidimetric method, the method of the present invention has the disadvantage that the bacterial solution needs to be diluted with physiological saline and frequently operated manually, and the bacterial solution can only be put into the machine for experiment after finally reaching a concentration of 0.5. The method of the present invention can directly put the bacterial solution into the machine after being diluted with physiological saline, because the instrument first measures the initial bacterial value, and then automatically analyzes the dilution factor according to the software. The instrument dilutes the bacterial solution according to the dilution factor before culturing and testing on the machine. Compared with the turbidimetric method, it avoids frequent manual operations, saves a lot of time and manual operations, and is more automated.

[0056] 2. When the bacterial growth is small and insufficient to meet the 0.5 McFarland concentration, it is necessary to wait for a considerable time. The technical solution of the present invention can completely avoid this situation, that is, greatly shorten the time.

[0057] 3. During clinical operation, the specimen does not exist as a single colony, but there are many colonizing bacteria. It is difficult for a single colony of pathogenic bacteria to reach a concentration of 0.5 McFarland. Generally, it is necessary to culture the specimen for 24 hours to reach a pure colony before conducting the test. If the technical solution of the present invention is adopted, time can be saved and the test can be carried out directly without culture.

[0058] Specifically, bacterial identification and drug sensitivity require an initial concentration standard. The initial concentration of the prepared specimen must be around this standard, neither too high nor too low. The method commonly used in the prior art is turbidimetry, as shown in Figure 1. Pick up the colony and add it to 5 ml of normal saline, mix it, put it into the turbidimeter, and observe the McFarland result. If the McFarland result is too high, the turbidimeter will continue to measure after the normal saline is diluted until the result is normal. If the McFarland result is too low, continue to pick up the colony and add it to the normal saline, mix it, and continue to measure the turbidimeter until the bacterial solution finally reaches 0.5 McFarland concentration before being put on the machine. The whole process requires frequent manual operation. The technical solution of the present invention is fully automatic. First, the concentration in the sample is measured by impedance method. Then, according to the result of the measurement, it is determined whether dilution is needed and how many times the dilution should be made. Then, the bacterial solution sample is automatically diluted. After the dilution is completed, its concentration should be very close to the standard concentration because the instrument automatically calculates the difference from the standard concentration based on the number of bacteria measured by the impedance method.

[0059] It is very difficult to obtain pure colonies using the turbidimetric method in the existing technology because during clinical operations, there are many colonizing bacteria in the specimen, making it difficult for a single colony of pathogenic bacteria, that is, a pure colony, to reach a McFarland concentration of 0.5. Therefore, it is necessary to culture the specimen, which takes a considerable amount of time. The method of the present invention does not require culture, and pure colonies can be directly picked from the specimen plate and diluted by counting method to achieve the bacterial solution concentration used for drug sensitivity testing. This is an innovative application of the counting method in the preparation of bacterial solution for drug sensitivity testing.

[0060] When the sample isolated from the clinic is infected with mixed bacteria and the concentration of single colonies cannot reach 0.5 McFarland, the turbidimetric method of the prior art requires a lot of time to culture and dilute. In the technical solution of the present invention, the inoculation needle does not need to pick up pure colonies with a concentration of 0.5 McFarland, but only needs to reach 10 5 -10 6 cfu / mL or above, you can start preparing the bacterial solution before the drug sensitivity test, because the final bacterial solution concentration requirement for the drug sensitivity test is 10 5 -10 6 cfu / mL is sufficient, and the reason why the existing technology must reach a concentration of 0.5 McFarland is because of the requirements of the turbidimetric method itself. Otherwise, it is impossible to use the turbidimetric method for dilution. Therefore, the counting method is cleverly applied to the preparation of bacterial solution for drug sensitivity test, which greatly shortens the time for preparing bacterial solution. The concentration of the sample used in the scheme of the present invention is almost lower than 0.5MCF (1.5*10 8 cfu / mL), which means that the bacterial solution preparation for drug sensitivity testing can be started at a low concentration, achieving application innovation.

[0061] There are many methods for bacterial identification or agar dilution drug sensitivity testing in the existing technology, but they have not been widely used in clinical laboratories. The main reason is that the most common method used in existing clinical laboratories is the broth dilution method to obtain the MIC drug sensitivity test; and the gray-white precipitate at the edge of the separation gel after centrifugation needs to be washed and centrifuged again. This repeated centrifugation and washing is unacceptable in the busy clinical work. The key point solved by the present invention is to meet the requirements of the broth dilution drug sensitivity test with minimal manual operation: after the blood culture is positive, the concentration of the bacterial solution is 10 8 CFU / mL, while the concentration required for broth dilution drug sensitivity test is 5*10 5 CFU / mL. Although existing techniques typically use the off-white precipitate at the edge of the separation gel after centrifugation to collect the majority of bacteria, and in routine experiments, most bacteria are indeed concentrated there, approximately 10% to 20% of bacteria in the supernatant remain unspun down. This fraction of bacteria fully meets the requirements for broth dilution susceptibility testing. However, existing techniques employ identification and agar dilution methods after bacterial selection, which require a high initial bacterial concentration and therefore dispense with the supernatant. Furthermore, the key to the success of the present invention lies in the use of the Coulter counting technique to count and dilute bacteria to the target concentration. The microbroth dilution method for susceptibility testing has clear requirements for the initial bacterial concentration; a higher concentration is not necessarily better. High concentrations can easily lead to high MICs, which can result in drug resistance in drug susceptibility tests, while too low a concentration can easily lead to low MICs and sensitivity in drug susceptibility tests. Therefore, the present invention addresses this problem through the Coulter counting method. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The above and other objects, features and advantages of the exemplary embodiments of the present invention will become readily apparent by reading the following detailed description with reference to the accompanying drawings, in which several embodiments of the present invention are shown by way of example and not limitation, in which:

[0063] FIG1 is a schematic diagram of a flow chart of an optional turbidimetric measurement provided by the prior art;

[0064] FIG2 is a schematic diagram of a process for preparing an optional bacterial solution for drug sensitivity testing according to an embodiment of the present invention;

[0065] FIG3-1 is a schematic diagram of an optional bacterial solution for drug sensitivity testing according to an embodiment of the present invention after culturing multiple bacteria for 16-24 hours;

[0066] Figure 3-2 is a schematic diagram of the target bacteria circled in Figure 3-1 after 24 hours of inoculation;

[0067] FIG4 is a schematic diagram of a process for directly preparing a bacterial solution of target concentration from a blood culture-positive specimen and then conducting a drug sensitivity test, as provided by the technical solution of the present invention.

[0068] The following specific embodiments are used to further illustrate but not limit the present invention. The following examples are only a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0069] The principles and spirit of the present invention will be described below with reference to several exemplary embodiments. It should be understood that these embodiments are provided solely to enable those skilled in the art to better understand and implement the present invention, and are not intended to limit the scope of the present invention in any way. Rather, these embodiments are provided to make the disclosure of this application more thorough and complete, and to fully convey the scope of the disclosure of this application to those skilled in the art.

[0070] Common clinical contaminants are divided into pure bacterial infections and polymicrobial infections, which can manifest as follows during sample processing and transfection: For polymicrobial infections (Figure 3-1 shows the results of polymicrobial infections with Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae), the target pathogen is Streptococcus pneumoniae, as shown in the circle in Figure 3-1. A single colony of the target pathogen is difficult to reach a McFarland concentration of 0.5. After 24 hours of transfection, the resulting pure colony is shown in Figure 3-2. For polymicrobial infections, see Examples 1 and 2, and for pure bacterial infections, see Examples 3 and 4.

[0071] Example 1

[0072] For mixed bacterial infections, since the pathogenic bacteria cannot meet the 0.5 McFarland requirement, drug sensitivity testing cannot be performed. It is necessary to continue culturing for 4-8 hours until the bacterial concentration meets the 0.5 McFarland requirement. After culturing under the same conditions for 16-24 hours, the bacterial concentration is measured using a bacteria counter (4*10 6 cfu / mL, which failed to meet the requirement of 0.5 McFarland. Put it into the device and set the target concentration (1.5*10 5 cfu / mL, the device will automatically determine the dilution multiple based on the initial concentration, automatically aspirate the stock solution, and finally obtain the target concentration (1.5*10 5 cfu / mL).

[0073] Therefore, for mixed bacterial infections, after 16-24 hours of separation and culture, the culture may present the following form as shown in Figure 3-1: The circle marks the target pathogens. If a drug sensitivity test is to be performed, the concentration of the target pathogens must be prepared to 10 5 -10 6 cfu / mL;

[0074] 1.1 Conventional operation mode:

[0075] As shown in Figure 3-1, the target pathogenic bacteria colonies are too few to meet the 0.5 McFarland concentration. The target colonies shown in the circle are further cultured for 16-24 hours to obtain the colony morphology shown in Figure 3-2. Then, the colonies are picked and thoroughly mixed in 5 mL of normal saline. The suspension is placed in a turbidimeter to prepare a uniform bacterial suspension with a concentration of approximately 0.5 McFarland units. 100 μL of the bacterial suspension is then transferred to 10 mL of diluent and labeled as tube 1 for later use.

[0076] 1.2 The operation mode of the method of the present invention is:

[0077] Use the inoculation needle of the inoculation device to directly pick up the target colony shown in Figure 3-1 and add it to 5 ml of normal saline to mix thoroughly. Count the bacteria and calculate the bacterial solution concentration C 初 , then according to the target concentration C 终 Calculate the dilution multiple to obtain the target concentration tube, and mark it as tube 2 for future use;

[0078] Calculate the bacterial concentration C 初 3*10 7 cfu / mL, and then according to the target concentration C 目 (1.5*10 5 cfu / mL), the device automatically calculates the dilution multiple and automatically draws the stock solution (V 移 ), inject it into the target tube (the target tube solution has been set in the device, V 设 The specific dilution basis is: according to the formula:

[0079] C 目 To set the target concentration, C 初 is the concentration of the original solution, V 初 To set the volume of the stock solution, V 设 To set the target volume, V 移 Transfer the stock solution into the target tube volume.

[0080] Substitute the data into the formula

[0081] Get V 移 The final target concentration is 1.5*10 6 cfu / mL, and the solution volume was 5.263 mL.

[0082] 1.3 Count the colonies in tubes 1 and 2 according to the colony counting method. The colony counting method is as follows:

[0083] (1) Take the bacterial solution in tube 1 and tube 2 and perform 2*10 5Dilution was made into 12 mL of the total volume, 50 μL of which was taken out for agar plate coating. The coated plate was placed upside down in a 37°C constant temperature incubator and cultured for 16-24 h.

[0084] (2) Manually count the cultured plates and calculate the actual number of colonies based on the dilution factor.

[0085] The results of the colony counts obtained by the two methods are shown in (Table 1):

[0086] Table 1: Note: For the results shown in Table 1, repeated counting experiments were performed for each method.

[0087] Experimental conclusion: By comparing the conventional operation mode and the method of the embodiment of the present invention for Streptococcus pneumoniae, the colony counting results obtained are relatively consistent, and it is concluded that the turbidimeter counting and the dilution method counting of the embodiment of the present invention can both meet the requirements.

[0088] Here, the colony counting is a comparison of the two methods. The purpose is to observe whether the method of the embodiment of the present invention is feasible by counting the colonies of the diluted bacterial solution obtained by the two methods. The conclusion is that the technical solution of the present invention is feasible.

[0089] Example 2 Comparison of drug sensitivity test accuracy

[0090] Experimental purpose: The bacterial solution preparation in Example 1 is the basis for drug sensitivity testing. Through drug sensitivity testing, the bacterial solution preparation method is verified.

[0091] Experimental methods:

[0092] 2.1 Conventional operation: Since the pathogenic bacteria colonies are too few to meet the 0.5 McFarland concentration, continue to culture the target colony shown in the circle for 18-24 hours to the colony morphology shown in Figure 3-2. Then, pick the colony, add it to 5 mL of normal saline and mix it thoroughly. Place it in a turbidimeter to make a uniform bacterial suspension of about 0.5 McFarland units. Pipette 260 μL of the bacterial suspension into 26 mL of dilution culture medium and mark it as tube 3 for later use.

[0093] 2.2 The operation mode of the method of the present invention is: use an inoculation needle to directly pick up the target colony shown in Figure 3-1 and add it to 5 ml of normal saline to mix thoroughly, count the bacteria, and calculate the bacterial solution concentration C 初 , then according to the target concentration C 目 Calculate the dilution multiple and dilute with culture medium to obtain the target concentration tube, which is labeled as tube 4 for future use. This means that the 24 hours required to form Figure 3-2 are omitted.

[0094] 2.3 Prepare the drug sensitivity test plate: Place the test plate at room temperature to recover for 15-30 minutes;

[0095] 2.4 Open the packaging bag and take out the test kit; use a micropipette or automatic sampler to take the drug sensitivity enrichment solution in tubes 3 and 4 respectively and add it to the drug sensitivity test plate, adding 500μL to each well, stick the sealing film, and the inoculation is complete;

[0096] 2.5 Load the inoculated test kit into the constant temperature incubation module for timed constant temperature incubation;

[0097] 2.6 After the constant temperature incubation is completed, the fully automatic rapid drug sensitivity analyzer begins to detect the number of bacteria in each well of the drug sensitivity plate. The software summarizes and analyzes the test data and determines the sensitivity of each antibiotic.

[0098] The experimental results are shown in Table 2:

[0099] Table 2:

[0100] Comparing the results analyzed in Table 2 with the quality control results in Table 2, it can be seen that the drug sensitivity test results in Table 2 are consistent with the quality control results in Table 2, thereby proving that the results obtained by turbidimeter measurement and the results obtained by colony counting in the embodiment of the present invention are feasible, that is, the drug sensitivity test results obtained based on the colony counting in the embodiment of the present invention meet the quality control standards.

[0101] Example 3 Comparative experiment on the accuracy of drug sensitivity test of pure strains cultured in the laboratory

[0102] For the pure colonies that have been prepared by the laboratory, the colony count and drug sensitivity test of the above two methods are performed simultaneously to observe whether the method of the embodiment of the present invention is feasible and can be used for routine laboratory operations.

[0103] 3.1 Prepare standard strains: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Perform colony counts using both methods simultaneously.

[0104] 3.1.1 Use a disposable sterile inoculating loop to pick three F1 generations of the pure strains to be tested, streak three lines on an agar plate, and place it upside down in a 37°C constant temperature incubator for 16-24 hours;

[0105] 3.1.2 Conventional Procedure: Use a disposable sterile inoculating loop to pick a portion of individual F2 colonies, add them to 5 mL of normal saline, mix thoroughly, and place in a turbidimeter to create a uniform bacterial suspension of approximately 0.5 McFarland units. Pipette 100 μL of the bacterial suspension into 10 mL of diluent and label tubes A to A for later use.

[0106] The method is as follows: pick up a single colony of the F2 generation with an inoculating needle, add it to 5 ml of normal saline and mix it thoroughly, count the bacteria, and calculate the bacterial solution concentration C 初 , then according to the target concentration C目 Calculate the dilution multiple to obtain the target concentration tube, and mark it as tube DF for future use;

[0107] 3.1.3 Count the colonies on tubes AF according to the colony counting method. The colony counting method is as follows:

[0108] 【1】 Take the bacterial solution in AF tube and use drug sensitive inoculation culture medium (common type) for 2*10 5 Dilution was made into 12 mL of the total volume, 50 μL of which was taken out for agar plate coating. The coated plate was placed upside down in a 37°C constant temperature incubator and cultured for 16-24 h.

[0109] 【2】Manually count the cultured plates and calculate the actual number of colonies based on the dilution factor. The results of the two methods of colony counting are shown in the following table (Table 3):

[0110] Table 3:

[0111] Experimental conclusion: By comparing the conventional operation mode and the method of the embodiment of the present invention on three standard strains - Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, the colony counting results obtained are relatively consistent. It is concluded that the turbidimeter counting and the dilution method counting of the embodiment of the present invention can both meet the requirements, and the technical solution of the present invention is feasible.

[0112] 3.2 Prepare standard strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Perform drug susceptibility testing using both methods described above.

[0113] 3.2.1 Use a disposable sterile inoculating loop to pick three pure F1 generations of the strains to be tested, streak three lines on an agar plate, and place it upside down in a 37°C constant temperature incubator for 16-24 hours;

[0114] 3.2.2 Conventional Procedure: Use a disposable sterile inoculating loop to pick a portion of individual F2 colonies, add them to 5 mL of normal saline, mix thoroughly, and place in a turbidimeter to create a uniform bacterial suspension of approximately 0.5 McFarland units. Pipette 260 μL of the bacterial suspension into 26 mL of dilution medium and label the tubes A'-C' for later use.

[0115] The method is as follows: use an inoculation needle to pick up single colonies of the F2 generation and add them to 5 ml of normal saline, mix them thoroughly, count the bacteria, and calculate the bacterial solution concentration C 初 , then according to the target concentration C 目 Calculate the dilution multiple and dilute with culture medium to obtain the target concentration tubes, which are labeled as D'-F' tubes for future use;

[0116] 3.2.3 Prepare the drug sensitivity test plate: Allow the test plate to recover at room temperature for 15-30 minutes;

[0117] 3.2.4 Open the packaging bag and take out the test kit; use a micropipette or automatic sampler to add 500 μL of the drug sensitivity enrichment solution from tubes A'-F' to the drug sensitivity test plate, respectively, to each well. Attach the sealing film, and the inoculation is complete;

[0118] 3.2.5 Load the inoculated test kit into the constant temperature incubation module for timed constant temperature incubation;

[0119] 3.2.6 After the constant temperature incubation is completed, the fully automatic rapid drug sensitivity analyzer begins to detect the number of bacteria in each well of the drug sensitivity plate. The software summarizes and analyzes the test data and determines the sensitivity of each antibiotic.

[0120] The experimental results are shown in Table 4:

[0121] Table 4:

[0122] Table 5 is attached for the quality control standards of each strain:

[0123] Table 5:

[0124] Comparing the results analyzed in Table 4 with the quality control results in Table 5, it can be seen that the drug sensitivity test results in Table 4 are consistent with the quality control results in Table 5, thereby proving that the results obtained by turbidimeter measurement and the results obtained by colony counting in the embodiment of the present invention are feasible, that is, the drug sensitivity test results obtained based on the colony counting in the embodiment of the present invention meet the quality control standards.

[0125] Example 4: Streptococcus pneumoniae (fastidious bacteria)

[0126] After inoculating Streptococcus pneumoniae into the culture medium and culturing for 16-24 hours, if Streptococcus pneumoniae cannot meet the requirement of 0.5 McFarland, the drug sensitivity test cannot be performed. It is necessary to continue culturing for 4-8 hours until the bacterial concentration meets the requirement of 0.5 McFarland. The 0.5 McFarland preparation solution is manually prepared. Under the same conditions, a strain of Streptococcus pneumoniae is also inoculated. After culturing for 16-24 hours, the bacterial concentration is measured using a bacteria counter (3*10 6 cfu / mL, which failed to meet the requirement of 0.5 McFarland. Put it into the device and set the target concentration (1.5*10 5 cfu / mL, the device will automatically determine the dilution multiple based on the initial concentration, automatically aspirate the stock solution, and finally obtain the target concentration (1.5*10 5 cfu / mL).

[0127] In summary, the method of the embodiment of the present invention provides a method for preparing bacterial solution for drug sensitivity testing, which solves the defects caused by conventional laboratory operation methods: first, it is difficult to obtain 0.5 McFarland pure bacterial solution for fastidious bacteria infection or mixed bacteria specimens, which saves a lot of culture time and prepares for quickly entering the drug sensitivity stage; secondly, in the laboratory pure strain experiment that has been cultured, it also avoids the defect of finally reaching a 0.5 McFarland concentration of the bacterial solution before the experiment can be carried out on the machine, avoids complicated manual operations, and solves the problem of inaccurate concentration. This shows that the method of the embodiment of the present invention is feasible.

[0128] Example 5

[0129] Comparison of the time required to obtain the target concentration of bacterial solution between the traditional blood culture method and the method of the present invention

[0130] The experimental method is as follows:

[0131] 5.1 Experimental purpose: To compare the time to obtain target bacteria using traditional blood culture method and the method of the present invention.

[0132] 5.2 Preparation of experimental drugs

[0133] Syringe, incubator, inoculating loop, alcohol lamp, turbidimeter, pipette, blood plate

[0134] 5.3 Experimental steps

[0135] 5.3.1 Draw 5 ml of blood into a blood culture bottle and inoculate it with 50 cfu / mL of ATCC25922 Escherichia coli. Culture the blood in a commercially available blood culture apparatus. If the blood culture sample is positive, divide the sample into two equal portions to prepare for the two subsequent methods.

[0136] 5.3.2 One portion is processed using traditional methods:

[0137] (1) After the blood culture is positive, 200 μL of the sample is pipetted onto a blood plate in a clean bench and evenly spread. The plate is then placed in an incubator and cultured overnight at 37°C. After 24 hours, the colonies are observed to obtain pure bacterial colonies.

[0138] (2) Use an inoculating loop to pick up pure colonies and place them in a bacterial dilution solution (5 ml). Use a McFarland turbidimeter to determine the bacterial concentration, adjusting it to 0.5 McFarland. Set aside for later use.

[0139] (3) Use a micropipette to draw 260 microliters of the 0.5 ml of the prepared broth (1) into 26 ml of the enrichment culture medium and mix thoroughly. At the same time, draw 50 microliters of the 0.5 ml of the broth (1) into 5 ml of the diluent and mix thoroughly as a negative control. This will give the bacterial solution for the fully automatic rapid drug sensitivity analyzer.

[0140] 5.3.3 One portion is processed according to the method of the present invention:

[0141] (1) Pipette 4 ml of positive specimen into a serum separation gel tube (a disposable vacuum blood collection tube containing separation gel) and mix thoroughly;

[0142] (2) After mixing, place the mixture in a centrifuge and centrifuge at 3000 rpm for 10 minutes;

[0143] (3) Dilute the supernatant 50 times with sample diluent and transfer it to the plate of the automatic rapid antimicrobial susceptibility analyzer to count the target bacteria;

[0144] (4) Determine the target bacterial count value for the machine, and then dilute it with the enrichment culture medium to the amount required for the fully automatic rapid drug sensitivity analyzer.

[0145] 5.4 Experimental Results

[0146] 5.5 Experimental Conclusion

[0147] The experimental results show that there is no significant difference between the number of bacteria obtained by plate counting using the traditional method and the number of bacteria obtained by this method, and this method takes 24 hours less than the traditional method.

[0148] Example 6 Comparison of rapid drug sensitivity results between conventional blood culture method and the method of the present invention

[0149] The experimental method is as follows:

[0150] 6.1 Experimental purpose: To compare the rapid drug sensitivity results obtained by traditional blood culture method and the method of the present invention.

[0151] 6.2 Experimental supplies: syringe, incubator, inoculating loop, alcohol lamp, turbidimeter, pipette, blood plate, rapid drug sensitivity analyzer.

[0152] 6.3 Experimental steps

[0153] 6.3.1 A clinically positive blood culture bottle, identified as a single bacterium, Escherichia coli, sourced from the Clinical Microbiology Laboratory of Union Hospital, was cultured in a commercially available blood culture apparatus. After the blood culture sample was reported positive, the specimen was divided equally into two aliquots in preparation for the two subsequent methods.

[0154] 6.3.2 One portion is processed using traditional methods:

[0155] (1) After the blood culture is positive, 200 μL of the sample is pipetted onto a blood plate in a clean bench and evenly spread. The plate is then placed in an incubator and cultured overnight at 37°C. After 24 hours, the colonies are observed to obtain pure bacterial colonies.

[0156] (2) Use an inoculating loop to pick up pure colonies and place them in a bacterial dilution solution (5 ml). Use a McFarland turbidimeter to determine the bacterial concentration, adjusting it to 0.5 McFarland. Set aside for later use.

[0157] (3) Use a micropipette to draw 260 μl of the 0.5% Melvin solution prepared in (1) and inject it into 26 ml of the enrichment culture medium, and mix thoroughly. At the same time, draw 50 μl of the 0.5% Melvin solution in (1) and inject it into 5 ml of the diluent, and mix thoroughly as a negative control. The bacterial solution for the drug sensitivity analyzer can be obtained.

[0158] 6.3.3 One portion is processed according to the method of the present invention:

[0159] (1) Pipette 4 ml of positive specimen into a centrifuge tube containing separation gel (disposable vacuum blood collection tube containing separation gel) and mix thoroughly;

[0160] (2) After mixing, place the mixture in a centrifuge and centrifuge at 3000 rpm for 10 minutes;

[0161] (3) Dilute the supernatant 50-fold with sample diluent and transfer it to the plate of the antimicrobial susceptibility analyzer to count the target bacteria.

[0162] (4) Determine the target bacterial count value on the machine, and then dilute it with the enrichment culture medium to the amount required for the drug sensitivity analyzer.

[0163] 6.4 Experimental Results

[0164] The results are shown in Table 6. The MIC results obtained by the rapid drug sensitivity test of the two bacterial suspensions were consistent.

[0165] Table 6 Rapid drug sensitivity results of traditional method and method of the present invention

[0166] 6.5 Experimental Conclusion

[0167] The MIC results obtained from the rapid drug sensitivity test of the two bacterial suspensions were consistent, demonstrating that the method of the present invention is less time-consuming (about 24 h).

[0168] Example 7

[0169] Comparative study on the effect of obtaining target bacterial solution from supernatant and precipitate after centrifugation of serum separation tube

[0170] 7.1 Experimental Objective: To explore the accuracy of the supernatant and precipitated bacterial fluid after centrifugation and to explore effective ways to reduce the difficulty of experimental operation by comparing the content and drug sensitivity test data of the bacterial fluid in the positive blood culture bottle after clinical identification.

[0171] 7.2 Preparation of experimental drugs

[0172] Syringe, incubator, inoculation loop, alcohol lamp, turbidimeter, pipette, blood plate, rapid drug sensitivity analyzer

[0173] 7.3 Experimental steps

[0174] 7.3.1 The positive blood culture bottles that have been clinically identified were identified as a single bacterium, Escherichia coli, which was sourced from the Clinical Microbiology Laboratory of Union Hospital.

[0175] 7.3.2 Processing according to the method of the present invention:

[0176] (1) Control group:

[0177] 18-24 hours after the positive blood culture bottle was transferred by plate streak method, the bacteria were picked and prepared into 0.5 McFarland suspension for use. 4 —10 5 cfu / mL was added to the Gram-negative bacillus drug sensitivity plate according to the drug sensitivity test procedures, and then placed in the drug sensitivity analyzer for detection and recorded the MIC respectively.

[0178] (2) Experimental group:

[0179] 1) Pipette 4 ml of the positive sample into two tubes containing serum separation gel, mix thoroughly, and mark them as tube 1 and tube 2. After mixing, place them in a centrifuge and centrifuge at 3000 rpm for 10 minutes.

[0180] 2) Aspirate the supernatant from tube 1, measure the turbidity with a turbidimeter, and dilute the sample to the required target bacterial concentration with sample diluent. Transfer the sample to the plate of the fully automatic rapid antimicrobial susceptibility analyzer, count the target bacteria and antimicrobial susceptibility results, and perform a blood plate count at the same time.

[0181] 3) Remove the supernatant from tube 2, dilute the precipitate with 4 ml of sample diluent, pipette repeatedly, measure the turbidity with a turbidimeter, and dilute with sample diluent to the required target bacterial concentration. Transfer the sample to the plate of the fully automatic rapid antimicrobial susceptibility analyzer, count the target bacteria and antimicrobial susceptibility results, and perform a blood plate count at the same time.

[0182] 4) Determine the target bacterial count value, and then dilute it with enrichment culture medium to the amount that can be used on the fully automatic rapid drug sensitivity analyzer.

[0183] 7.4 Experimental Results

[0184] Table 7 Comparison of target bacteria count values

[0185] Table 8 Comparison of rapid drug sensitivity results using supernatant and precipitate

[0186] 7.5 Experimental Conclusion

[0187] The experimental results showed that the number of bacteria obtained from the supernatant was of the same order of magnitude as the clinical results and met the requirements; the number of bacteria obtained from the precipitate was lower than the clinical standard. The reason was that different types of resins were added to the blood culture bottles to reduce the risk of false negatives caused by the patient's early use of antibiotics during use. Some resins were distributed on the upper layer of the separation gel together with the bacteria, which had no effect on bacterial identification and traditional drug sensitivity results, but would cause the Coulter counting method to mistake the resin for bacteria. In the rapid drug sensitivity analysis, the supernatant drug sensitivity results were consistent with the clinical results and met the requirements, while the precipitate showed abnormal values ​​(imipenem, tigecycline, ertapenem resistance, high count values), which did not meet the requirements. This is because the precipitate contains resin, which affects the bacterial count and causes the analyzer to clog. In addition, the use of supernatant operations can meet the clinical bacterial count requirements, and there is no need to discard the supernatant and repeatedly blow and aspirate. The simpler operation provides a better basis for the later automation of the entire method.

Claims

1. A method for preparing a bacterial solution for drug sensitivity testing, characterized in that: The method comprises: Picking a colony of a sample for preparing the bacterial solution; preparing the colonies into a first bacterial liquid, and counting the number of bacteria in the first bacterial liquid to obtain a first bacterial count of the first bacterial liquid; The first concentration of the first bacterial solution is inferred based on the first bacterial count, and the first bacterial solution is diluted based on the first concentration to obtain a second bacterial solution. The second concentration of the second bacterial solution is 10 5 to 10 6 cells / mL (cfu / mL); The second bacterial liquid is the bacterial liquid used for drug sensitivity test.

2. The method according to claim 1, characterized in that The colony is a fastidious bacterial colony, a mixed bacterial colony or a pure bacterial colony.

3. The method according to claim 2, characterized in that The fastidious bacterial colony is a fastidious bacterial colony whose growth does not reach 0.5 McFarland, or the mixed bacterial colony includes a colony whose growth does not reach 0.5 McFarland, or the pure colony is a pure colony whose growth does not reach 0.5 McFarland.

4. The method according to claim 1, wherein The colonies of the sample for preparing the bacterial liquid are picked up and mixed into physiological saline to form the first bacterial liquid; and the first bacterial liquid is diluted with culture medium according to the first concentration to obtain a second bacterial liquid.

5. The method according to claim 1, wherein The number of bacteria in the first bacterial liquid is counted using a resistance counting method to obtain a first bacterial count of the first bacterial liquid.

6. The method according to claim 1, characterized in that The colonies of the sample for preparing the bacterial solution are picked up using an inoculation device.

7. The method according to claim 1, wherein: A. Pick up the bacterial colony and add it to 2 to 10 ml of normal saline, and mix thoroughly to form the first bacterial solution; B. counting the number of bacteria in the first bacterial liquid to obtain a first bacterial count of the first bacterial liquid; C. Determine the first concentration of the first bacterial solution based on the number of the first bacteria and the volume of the first bacterial solution (C 初 ), according to the first concentration (C 初 ) dilute and mix the first bacterial solution to obtain a second bacterial solution, wherein the second concentration of the second bacterial solution is 10 5 to 10 6 cells / ml (cfu / mL), and the second bacterial liquid is the bacterial liquid used for drug sensitivity test.

8. The method according to claim 7, wherein: A. Pick up the bacterial colony and add it to 3 to 5 ml of normal saline, and mix thoroughly to form the first bacterial solution; B. counting the number of bacteria in the first bacterial liquid to obtain a first bacterial count of the first bacterial liquid; C. Determine the first concentration of the first bacterial solution based on the number of the first bacteria and the volume of the first bacterial solution (C 初 ), according to the first concentration (C 初 ) dilute and mix the first bacterial solution to obtain a second bacterial solution, wherein the second concentration of the second bacterial solution is 10 5 to 10 6 cells / ml (cfu / mL), and the second bacterial liquid is the bacterial liquid used for drug sensitivity test.

9. The method according to claim 7, wherein: A. Pick up the bacterial colony and add it to 6 to 8 ml of normal saline, and mix thoroughly to form the first bacterial solution; B. counting the number of bacteria in the first bacterial liquid to obtain a first bacterial count of the first bacterial liquid; C. Determine the first concentration of the first bacterial solution based on the number of the first bacteria and the volume of the first bacterial solution (C 初 ), according to the first concentration (C 初 ) dilute and mix the first bacterial solution to obtain a second bacterial solution, wherein the second concentration of the second bacterial solution is 10 5 to 10 6 cells / ml (cfu / mL), and the second bacterial liquid is the bacterial liquid used for drug sensitivity test.

10. Use of the second bacterial liquid obtained by the method for preparing a bacterial liquid for drug sensitivity test according to any one of claims 1 to 9 in the drug sensitivity test.

11. A method for directly preparing bacterial liquid for drug sensitivity testing from blood culture-positive specimens, characterized in that: The blood culture-positive specimen is drawn into a centrifuge tube containing a separation gel. The blood culture-positive specimen and the separation gel are mixed to form a liquid to be centrifuged. The liquid to be centrifuged of the blood culture-positive specimen is centrifuged so that the target bacteria of the blood culture-positive specimen remain in the supernatant after centrifugation of the liquid to be centrifuged to form a first supernatant. The target bacteria in the first supernatant are counted to obtain a first bacterial count in the first supernatant. A first concentration of the first supernatant is inferred based on the first bacterial count in the first supernatant. The first supernatant is diluted based on the first concentration of the first supernatant to obtain a bacterial solution of a target concentration. The bacterial solution of the target concentration is directly subjected to a drug sensitivity test.

12. The method according to claim 11, characterized in that Counting the target bacteria in the first supernatant refers to detecting the number of the target bacteria.

13. The method according to claim 11, characterized in that The separation gel retains the target bacteria in the first supernatant under the action of centrifugation.

14. The method according to claim 12, characterized in that The target bacteria were counted using the Coulter counting method.

15. The method according to claim 11, characterized in that The second concentration of the target concentration bacterial solution is 10 4 to 10 6 The target concentration bacterial solution was directly used for drug sensitivity test.

16. The method according to claim 11, characterized in that 2 to 8 milliliters of the blood culture-positive specimen is drawn into the centrifuge tube, and the liquid to be centrifuged containing the blood culture-positive specimen is centrifuged at a speed of 1000 to 6000 revolutions per minute (r / min) for 6 to 14 minutes, so that the target bacteria in the blood culture-positive specimen remain in the supernatant after centrifugation of the liquid to be centrifuged to form the first supernatant. The first supernatant containing the target bacteria is diluted 25-100 times to obtain the bacterial solution of the target concentration.

17. The method according to claim 16, characterized in that 3 to 6 milliliters of the blood culture-positive specimen is drawn into the centrifuge tube, and the liquid to be centrifuged containing the blood culture-positive specimen is centrifuged at a speed of 2000 to 4000 rpm for 8 to 12 minutes, so that the target bacteria of the blood culture-positive specimen remain in the supernatant after centrifugation of the liquid to be centrifuged to form the first supernatant. The first supernatant containing the target bacteria is diluted 40 to 60 times to obtain the bacterial solution of the target concentration.

18. The method according to claim 11, characterized in that The target bacteria of the blood culture positive specimen remain in the supernatant after the liquid to be centrifuged is centrifuged to form the first supernatant, and other impurities of the blood culture positive specimen sink to the lower end of the centrifuge tube.

19. The method according to claim 11, wherein The bacterial solution of the target concentration is transferred into the reagent kit of the drug sensitivity analyzer to perform a drug sensitivity test.

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