Bacterial culture test method

Abstract

Provided is a method for shortening the induction phase in the proliferation of bacteria. A growth stimulation solution obtained by (1) a step that inoculates a culture solution with Staphylococcus to obtain a cultured culture solution and (2) a step that removes the Staphylococcus from the cultured culture solution, or a growth stimulation powder obtained by drying the growth stimulation solution is used.

Description

Bacterial culture test method

[0001] The present invention relates to a method for shortening the lag phase in bacterial culture tests, and to a growth stimulating solution and a growth stimulating powder used to shorten the lag phase in culture tests, as well as methods for producing the same.

[0002] Culture testing is the global standard for bacterial detection and sterility testing, both in clinical and non-clinical settings. The presence or absence of bacteria (viable bacteria) in a sample is determined by checking whether the microorganisms grow in culture medium.

[0003] The present inventors have proposed a method for quantifying bacteria using a real-time PCR method with specific primers (Patent Document 1), and a method for quantifying bacteria accurately and with high sensitivity using a PCR method with a specific DNA polymerase (Patent Document 2).

[0004] Conventional microbial testing methods prescribed in the Pharmacopoeia require more than a week (7 to 14 days) to determine "sterility." For example, in the conventional method (the most standard method), a sample is mixed into a liquid culture medium, and then the presence or absence of turbidity in the culture medium is visually confirmed. However, since a large number of bacteria is required to cause turbidity in the culture medium, a culture period of 14 days is required to confirm sterility. In addition, the BACT / ALERT method, a rapid sterility testing method prescribed in the Pharmacopoeia, uses CO2 emitted by bacteria. ２ It detects the presence or absence of CO ２ Because a certain number of bacteria is required to detect bacteria, a seven-day incubation period is required to confirm sterility. In other words, both the conventional method and the BacteAlert method have low detection sensitivity, so a long incubation period is required to determine sterility.

[0005] International Publication No. WO 2019 / 123692 International Publication No. WO 2010 / 082640

[0006] The reason it takes so long for the culture to become positive is because of the long "lag phase," which is the stage preceding the growth phase (logarithmic phase) (see Figure 1). If this lag phase could be shortened, culture results could be obtained more quickly. However, there is currently no method to further shorten the lag phase under optimal culture conditions (culture temperature, etc.).

[0007] Obtaining culture results more quickly could save patients' lives in clinical testing, and in sterility testing, it could improve the safety of platelet preparations, regenerative medicine products, etc. The present invention aims to develop a method to "shorten the lag phase" of bacterial growth, i.e., a method to turn on the growth start switch, and to introduce this method in practice, thereby enabling culture results to be obtained more quickly than currently possible.

[0008] The present inventors have found that the lag period can be shortened by inoculating a culture solution with Staphylococcus species, culturing the culture solution, and then using a medium from which Staphylococcus species have been removed.

[0009] That is, the present invention provides the following: [1] A method for producing a growth stimulator solution, comprising: (1) a step of inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after culture; and (2) a step of removing Staphylococcus sp. from the culture medium after culture. [2] The method according to [1], wherein the step (2) comprises a step of removing the supernatant of the culture medium after culture by centrifugation and filtering the supernatant. [3] The method according to [1] or [2], wherein the Staphylococcus sp. is Staphylococcus aureus. [4] A method for producing a growth stimulator powder, comprising: (1) a step of inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after culture; (2) a step of removing Staphylococcus sp. from the culture medium after culture to obtain a growth stimulator solution; and (3) a step of drying the growth stimulator solution. [5] The method according to [4], wherein the step (2) comprises removing the supernatant of the culture solution after the culture by centrifugation and filtering the supernatant. [6] The method according to [4] or [5], wherein the Staphylococcus genus is Staphylococcus aureus. [7] A method for shortening the lag period of a culture test, comprising mixing a specimen with a growth stimulator solution obtained by the method according to any one of [1] to [3] and culturing the specimen. [8] A method for shortening the lag period of a culture test, comprising mixing a specimen with a growth stimulator powder obtained by the method according to any one of [4] to [6] and culturing the specimen. [9] A method for culturing a specimen, comprising: (1) a step of inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after culturing; (2) a step of removing Staphylococcus sp. from the culture medium after culturing to obtain a growth stimulator solution; (3) a step of mixing a specimen with the growth stimulator solution to obtain a mixture; and (4) a step of culturing the mixture.

[10] A method for culturing a specimen, comprising: (1) inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after cultivation; (2) removing Staphylococcus sp. from the culture medium after cultivation to obtain a growth stimulating solution (Growth Stimulator solution); (3) drying the growth stimulating solution (Growth Stimulator solution) to obtain a growth stimulating powder (Growth Stimulator powder); (4) mixing a specimen with the growth stimulating powder (Growth Stimulator powder) to obtain a mixture; and (5) culturing the mixture.

[11] The method according to [9] or

[10] , wherein the Staphylococcus sp. is Staphylococcus aureus.

[12] A growth stimulating solution (Growth Stimulator solution) obtained by removing Staphylococcus sp. from a Staphylococcus sp. culture medium.

[13] A growth stimulator powder, which is a dried product of the growth stimulator solution according to

[12] .

[14] A culture solution containing the growth stimulator solution according to

[12] or the growth stimulator powder according to

[13] .

[15] A culture bottle containing the culture solution according to

[14] .

[0010] According to the present invention, the growth of a specimen can be confirmed early by shortening the "lag phase" which is the stage preceding the logarithmic phase in bacterial growth.

[0011] FIG. 1 is a diagram of a typical growth curve showing the change in cell number over time.

[0012] <Growth Stimulator Solution and Manufacturing Method Thereof> The growth stimulator solution according to the present invention is a Staphylococcus genus culture solution from which the genus Staphylococcus has been removed.

[0013] [Staphylococcus culture medium] The Staphylococcus culture medium is a culture medium (after culture) obtained by inoculating a Staphylococcus species into a culture medium (before culture) and culturing the Staphylococcus species.

[0014] That is, the growth stimulator solution according to the present invention can be produced by a method comprising at least the following steps: (1) inoculating a culture solution with Staphylococcus bacteria to obtain a culture solution (Staphylococcus culture solution) after culture; and (2) removing Staphylococcus bacteria from the culture solution after culture. Each step will be described in detail below.

[0015] [Step (1)] The Staphylococcus genus used in the present invention is not particularly limited, and examples thereof include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus caprae, Staphylococcus hominis, etc. Among these, Staphylococcus aureus (hereinafter also referred to as "S. aureus") is preferred.

[0016] In the present invention, a method for culturing Staphylococcus in a culture medium and obtaining a culture medium (Staphylococcus culture medium) after culture includes, for example, inoculating Staphylococcus into a culture medium placed in any container and culturing the Staphylococcus. The container for the culture medium is not limited as long as it is capable of culturing bacteria. Specific examples of the container include, but are not limited to, flasks, beakers, culture bottles, and mass culture devices. In the present invention, the term "culture bottle" refers to a culture vessel that already contains a culture medium. In other words, when a culture bottle is used as the container, it is not necessary to add a culture medium separately. Furthermore, the culture medium is not limited, and any known bacterial culture medium can be used.

[0017] The culture temperature is not particularly limited, but can be, for example, 30 to 37° C. The number of days for culture is not particularly limited as long as it is a number of days that allows the Staphylococcus genus to be cultured. The number of days for culture can be, for example, 1 to 14 days, preferably 3 to 10 days.

[0018] The step of obtaining a Staphylococcus culture solution is preferably carried out in a controlled environment to prevent external contamination. When a culture bottle is used as the container, the type of culture bottle is not particularly limited, and examples include aerobic culture bottles such as iAST culture bottles and anaerobic culture bottles such as iNST culture bottles. From the viewpoint of industrial production of a Staphylococcus culture solution, it is preferable to use aerobic culture bottles, which allow bacteria to grow more rapidly.

[0019] [Step (2)] The method for removing Staphylococcus from a Staphylococcus culture is not particularly limited, but examples thereof include centrifuging the Staphylococcus culture and separating the supernatant after culture. The conditions for centrifugation are not particularly limited, but examples thereof include centrifuging the culture at 500 to 4,000 × g for 1 to 20 minutes.

[0020] Furthermore, Staphylococcus can be removed more reliably by filtering the separated supernatant. There are no particular limitations on the filtration conditions, but for example, a 0.1 to 1 μm filter that does not allow Staphylococcus to pass through can be used.

[0021] The supernatant of the Staphylococcus culture thus obtained serves as the growth stimulator solution of the present invention. As described above, the growth stimulator solution is a solution obtained by removing Staphylococcus from a Staphylococcus culture. Therefore, the growth stimulator solution does not contain (living) Staphylococcus. The growth stimulator solution of the present invention can be directly added to a culture solution together with a specimen (sample) at the time of use. The growth stimulator solution of the present invention can also be mixed in advance with the culture solution in a culture bottle and stored until use.

[0022] <Growth Stimulator Powder and Method for Producing the Same> The growth stimulator powder of the present invention is a dried product of the growth stimulator solution obtained by drying the growth stimulator solution of the present invention. That is, the growth stimulator powder of the present invention can be produced by a method including at least the following steps: (1) a step of inoculating a culture solution (before culture) with Staphylococcus to obtain a culture solution (Staphylococcus culture solution) after culture; (2) a step of removing Staphylococcus from the culture solution after culture to obtain a growth stimulator solution; and (3) a step of drying the growth stimulator solution. Steps (1) and (2) correspond to steps (1) and (2) in the method for producing a growth stimulator solution of the present invention.

[0023] [Step (3)] The conditions for drying the growth stimulating solution are not particularly limited, but for example, the solution can be pre-frozen at -80 to -30°C and then vacuum freeze-dried for 5 to 48 hours using a vacuum freeze dryer. The resulting growth stimulating powder can be stored and transported at room temperature while maintaining its activity. Furthermore, the growth stimulating powder can be directly added to the culture solution together with the specimen (sample) at the time of use. Furthermore, the growth stimulating powder can be mixed in advance with the culture solution in a culture bottle and stored until use.

[0024] <Method for shortening the lag period in a culture test> The method for shortening the lag period in a culture test according to the present invention includes at least culturing a mixture obtained by mixing the growth stimulating solution or growth stimulating powder with a specimen. Specifically, the method for shortening the lag period of bacterial culture growth using the growth stimulating solution or growth stimulating powder is as follows: First, the growth stimulating solution or growth stimulating powder is placed in a sterilized container such as a vial, and the specimen (sample) is added to this container using a syringe or the like, and the growth stimulating solution or growth stimulating powder and the specimen (sample) are mixed. At this time, the growth stimulating solution or growth stimulating powder and the specimen (sample) can be easily mixed by adding and removing the growth stimulating solution or growth stimulating powder from the syringe several times.

[0025] When the sample is blood, 5 to 50 mL of blood can be added with a syringe to a vial or the like containing the growth stimulating solution or growth stimulating powder, mixed with the syringe still inserted, and the blood sample mixed with the growth stimulating solution or growth stimulating powder can be removed with the syringe.

[0026] Next, the mixture of the growth stimulating solution or growth stimulating powder and the specimen (sample) is placed in a culture bottle to begin culturing. The culturing can be carried out by a known method. The culture bottle is not particularly limited, and aerobic culture bottles such as iAST culture bottles or anaerobic culture bottles such as iNST culture bottles can be used. In this way, the lag phase of bacterial culture growth can be shortened. That is, the lag phase of the culture test can be shortened.

[0027] Although the specimen is not limited thereto, any of blood, cerebrospinal fluid, synovial fluid, pericardial fluid, amniotic fluid, ascites, drainage fluid, alveolar lavage fluid, and abscess can be used as a clinical specimen. Other specimens that can be used are platelet preparations, regenerative medicine products, infusion preparations, eye drops, water for daily use, and cosmetics for sterility testing (culture testing). Among these, specimens that require rapidity in particular are preferred, including clinical specimens such as blood and cerebrospinal fluid, platelet preparations, and regenerative medicine products.

[0028] <Method for culturing a specimen> The method for culturing a specimen according to the present invention may include at least the following steps: (1) a step of inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after culture (Staphylococcus sp. culture medium); (2) a step of removing Staphylococcus sp. from the culture medium after culture to obtain a growth stimulator solution (Growth Stimulator solution); (3) a step of mixing a specimen with the growth stimulator solution (Growth Stimulator solution) to obtain a mixture; and (4) a step of culturing the mixture.

[0029] Another method for culturing a specimen according to the present invention may include at least the following steps: (1) inoculating a culture medium with Staphylococcus sp. to obtain a culture medium (Staphylococcus sp. culture medium) after the culture; (2) removing Staphylococcus sp. from the culture medium after the culture to obtain a growth stimulator solution (Growth Stimulator solution); (3) drying the growth stimulator solution (Growth Stimulator solution) to obtain a growth stimulator powder (Growth Stimulator powder); (4) mixing a specimen with the growth stimulator powder to obtain a mixture; and (5) culturing the mixture.

[0030] The steps in the method for culturing a specimen according to the present invention correspond to the steps in the method for producing the growth stimulating solution and growth stimulating powder according to the present invention and the method for shortening the lag period in a culture test, and detailed explanations will be omitted. According to the method for culturing a specimen according to the present invention, the use of the growth stimulating solution or growth stimulating powder shortens the lag period in bacterial growth, thereby shortening the time required for a positive determination.

[0031] In the present invention, the method for confirming the growth of a specimen is not particularly limited. For example, a method for confirming the turbidity (cloudiness) of the culture medium, a method for confirming the CO released by the bacteria in the culture medium, and the like are available. ２ Examples of the method include a method for confirming an increase in the number of bacteria and a method for confirming the formation of colonies.

[0032] <Culture Solution and Culture Bottle> As shown in the examples, the growth stimulating solution and growth stimulating powder of the present invention remain active even when stored in an appropriate container such as a sterile tube, sealed with a rubber stopper, or the like, at room temperature for an extended period of time. This demonstrates that the activity of the growth stimulating solution and growth stimulating powder of the present invention remains unchanged, i.e., the effect of shortening the induction period in culture tests is maintained. Therefore, by premixing the growth stimulating solution or growth stimulating powder of the present invention with the culture solution in a culture bottle, when a test such as a sterility test is required, the test results can be obtained quickly by using the culture bottle. In other words, the culture solution of the present invention contains at least the growth stimulating solution or growth stimulating powder of the present invention. Furthermore, the culture bottle of the present invention is a culture bottle containing the culture solution of the present invention, and is a culture bottle containing a culture solution containing at least the growth stimulating solution or growth stimulating powder of the present invention.

[0033] The culture medium is not limited, and any known bacterial culture medium can be used. The culture bottle is also not limited, and for example, aerobic culture bottles such as iAST culture bottles or anaerobic culture bottles such as iNST culture bottles can be used.

[0034] EXAMPLES The present invention will be described below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0035] Example 1 Preparation of Growth Stimulator Solution 50 CFU of Staphylococcus aureus were inoculated into an aerobic culture bottle (containing 40 mL of culture solution) and cultured at 32.5°C for 7 days to obtain a Staphylococcus aureus culture solution. The resulting culture solution was centrifuged at 2,280 x g for 10 minutes, and the supernatant was removed. The removed supernatant was filtered through a 0.22 µm filter to obtain 35 mL of growth stimulator solution.

[0036] Example 2 Preparation of Growth Stimulator Powder A portion (12 mL) of the growth stimulator solution obtained in Example 1 was pre-frozen at −80° C. and then vacuum freeze-dried for 24 hours to obtain 490 mg of growth stimulator powder.

[0037] 1. Measurement of the time (days) required for a positive determination using a growth stimulator solution [Example 3] The time (days) required for a positive determination of S. aureus in a specimen was measured under the following conditions: Specimen: Staphylococcus aureus (ATCC6538 strain) Quantity: 100 μL (50 CFU) Equipment used: Automatic culture device (product name: BACT / ALERT) Culture bottle: iAST (aerobic bottle) Culture temperature: 32.5°C Total specimen inoculation volume (growth stimulator solution + S. aureus bacterial solution): 10 mL

[0038] Comparative Example 1 Culture was carried out under the same conditions as in Example 3, except that the growth stimulating solution was changed to iAST medium.

[0039] Comparative Example 2 The S. aureus bacterial solution was cultured under the same conditions as in Example 3, except that the iAST medium was used.

[0040] The culture conditions and the time required for a positive determination in the Examples and Comparative Examples are shown in Table 1.

[0041] 2. Measurement of the time (days) required for a positive determination using growth stimulator powder [Example 4] Culture was carried out under the same conditions as in Example 3, except that a mixture of growth stimulator powder and distilled water (410 mg of growth stimulator powder + 9.5 mL of distilled water) was used instead of the growth stimulator solution.

[0042] Comparative Example 3 The S. aureus bacterial solution was cultured under the same conditions as in Example 4, except that the iAST medium was used.

[0043] The culture conditions and the time required for a positive determination in the Examples and Comparative Examples are shown in Table 1.

[0044]

[0045] As shown in Table 1, it was found that culturing using the growth stimulating solution of the present invention enabled a positive result in 0.80 days (Example 3). On the other hand, with the conventional method (Comparative Example 1) using iAST medium instead of the growth stimulating solution, it took 3.14 days to reach a positive result. Therefore, by using the growth stimulating solution of the present invention, the time to reach a positive result could be shortened by an average of 2.34 days = 56.16 hours (74.5%) compared to the conventional method. This is because the use of the growth stimulating solution shortened the lag period of bacterial growth.

[0046] In Comparative Example 2, in which the S. aureus bacterial solution in Example 3 was replaced with iAST medium, and in Comparative Example 3, in which the S. aureus bacterial solution in Example 4 was replaced with iAST medium, negative results were obtained even after 7 days had passed. This confirmed that the growth stimulating solution and growth stimulating powder did not contain any bacteria.

[0047] Furthermore, it was found that when growth stimulation powder was prepared from the growth stimulation solution and the growth stimulation powder was returned to the growth stimulation solution with distilled water at the time of use (Example 4), the same effect as when the growth stimulation solution was used directly (Example 3) was obtained.

[0048] 3. Measurement of bacterial count after 24 hours of culture Next, in Example 3 and Comparative Examples 1 and 2, 1 mL of sample was taken from the culture bottle after 24 hours of culture, and DNA was extracted using the magnetic bead method. The number of bacteria contained in a PCR tube per 40 μL of culture solution was then measured using real-time PCR (a calibration curve was drawn using the bacterial solution in which the number of bacteria was measured using flow cytometry). The results are also shown in Table 1. In Table 1, "ND" means not detected, and "-" means not measured.

[0049] As shown in Table 1, in Example 3, the number of bacteria, which was 426 before culture, increased to 156,930,000 after 24 hours of culture. On the other hand, in the conventional method (Comparative Example 1), only 28 bacteria were detected even after 24 hours of culture. In Comparative Example 2, 391 bacteria were detected before culture, but this was because the growth stimulation solution originally contained DNA derived from damaged (dead) S. aureus.

[0050] 4. Verification of the effect of growth stimulating solutions on bacterial species other than S. aureus Cultivation was carried out under the same conditions as in Example 3, except that bacterial solutions of the bacterial species shown in Table 2 were used instead of the S. aureus bacterial solution, and the time required for a positive determination was measured. The results are shown in Table 2. Note that "normal cultivation" means that the cultivation was carried out without adding a growth stimulating solution.

[0051]

[0052] As shown in Table 2, the effect of quickly obtaining positive culture results was observed not only for bacterial species with a long lag period (species that start growing slowly) but also for bacterial species with a short lag period.

[0053] 5. Long-Term Storage Stability Test of Growth Stimulatory Solution and Growth Stimulatory Powder After preparation, the growth stimulatory solution and growth stimulatory powder according to the present invention were placed in sterilized Falcon tubes, sealed, and stored at room temperature (25°C) and 4°C for a set period (1 month, 3 months, and 8 months). After each storage period, the growth stimulatory solution and growth stimulatory powder were cultured under the same conditions as in Example 3, and the time (days) required for a positive test was measured. The results are shown in Table 3. In the table, the bacterial load refers to the result of counting the number of colonies (CFU) after plating the same amount of S. aureus liquid as the iAST culture bottle used in each test on a medium. While significant differences in bacterial load values ​​may affect the time required for a positive test, we confirmed that the variation shown in Table 3 is not a problem. "NC" denotes a negative control, in which S. aureus was cultured in an iAST culture bottle (containing only culture medium) without the growth stimulatory solution or growth stimulatory powder. The time required for a positive determination is the average of three samples, and the figure in parentheses indicates the difference from the time required for a positive determination when no growth stimulating solution or growth stimulating powder was added (NC condition).

[0054]

[0055] As shown in Table 3, it was revealed that the growth stimulator of the present invention is stable over long periods at room temperature, not only in powder form (growth stimulating powder) but also in liquid form (growth stimulating solution), and its activity is not lost. In other words, it is possible to prepare culture bottles containing the growth stimulating powder or growth stimulating solution of the present invention in advance in a culture medium, and store the culture bottles at room temperature for long periods.

[0056] 6. Verification of the effect of growth stimulating solution using Cutibacterium acnes When growth stimulating solution and growth stimulating powder are applied to sterility testing of platelet preparations, the most frequently detected contaminant is Cutibacterium acnes (hereinafter referred to as "C. acnes"). Therefore, an experiment was conducted to verify the effect of the growth stimulating solution using C. acnes.

[0057] Reference Example 1 Preparation of C. acnes Post-Culture Supernatant 194 CFU (0.1 mL) of C. acnes was inoculated into an iNST culture bottle (containing 40 mL of culture medium) and cultured at 32.5°C for 7 days to obtain a cultured C. acnes culture medium. The obtained culture medium was centrifuged at 2,280 × g for 10 minutes, and the supernatant was removed. The removed supernatant was filtered through a 0.22 μm filter to obtain 38 mL of C. acnes post-culture supernatant.

[0058] Example 5 Preparation of S. aureus Post-Culture Supernatant 50 CFU (0.1 mL) of S. aureus was inoculated into an iNST culture bottle (containing 40 mL of culture medium) and cultured at 32.5°C for 3 days to obtain a cultured S. aureus culture medium. The resulting culture medium was centrifuged at 2,280 × g for 10 minutes, and the supernatant was removed. The removed supernatant was filtered through a 0.22 μm filter to obtain 38 mL of S. aureus post-culture supernatant (growth stimulation solution).

[0059] Reference Example 2 The time (days) required for a sample to be determined to be positive for C. acnes was measured under the following conditions: Sample: Cutibacterium acnes (NBRC 107605 strain) Quantity: 100 μL (71 CFU) Equipment used: Automatic culture device (product name: BACT / ALERT) Culture bottle: iNST (BacteAlert anaerobic bottle) Culture temperature: 32.5°C Total sample volume inoculated (C. acnes culture supernatant + C. acnes bacterial solution): 10 mL

[0060] [Example 6 and Comparative Examples 4 to 6] The inoculated specimens were cultured under the same conditions as in Reference Example 2, except that they were changed as shown in Table 4. Table 4 shows the time required for a positive determination for each test example.

[0061]

[0062] As shown in Table 4, when the C. acnes culture supernatant was used (Reference Example 2), the positive result was 0.53 days (6.2%) earlier than when the culture supernatant was not used (Comparative Example 4), and when the S. aureus culture supernatant (the growth stimulating solution according to the present invention) was used (Example 6), the positive result was 4.03 days (46.9%) earlier. In other words, it was demonstrated that the growth stimulating solution according to the present invention can also shorten the time required for a positive determination of C. acnes.

[0063] While certain preferred embodiments of the present invention have been shown and described in detail above, it should be understood that the present invention is not limited to the above-described embodiments, but that various changes and modifications are possible without departing from the spirit or scope of the appended claims.

[0064] This application claims priority based on Japanese Patent Application No. 2024-082538, filed May 21, 2024, the entire contents of which are incorporated herein by reference.

[0065] The inventors have discovered that Staphylococcus cultures have the effect of shortening the lag phase of bacterial growth and accelerating the development of positive culture results. This effect was observed not only for bacterial species with a long lag phase (i.e., species that start growth slowly) but also for bacterial species with a short lag phase. To make this culture practical, the Staphylococcus cells were completely removed from the culture, and the supernatant was then vacuum freeze-dried to form a powder, allowing for stable storage and transportation without losing its activity even at room temperature.

[0066] The growth stimulator solution and growth stimulator powder according to the present invention can be easily prepared, and their use enables rapid production of culture results. Rapid production of culture results will contribute to a wide range of benefits to society, including saving patients' lives in clinical testing and improving the safety of platelet preparations and regenerative medicine products in sterility testing.

Claims

1. A method for producing a growth stimulator solution, comprising: (1) a step of inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after culture; and (2) a step of removing Staphylococcus sp. from the culture medium after culture.

2. The method according to claim 1, wherein step (2) comprises the steps of removing the supernatant of the culture solution after the culture by centrifugation and filtering the supernatant.

3. The method according to claim 1 or 2, wherein the Staphylococcus genus is Staphylococcus aureus.

4. A method for producing a growth stimulator powder, comprising: (1) inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after cultivation; (2) removing Staphylococcus sp. from the culture medium after cultivation to obtain a growth stimulator solution; and (3) drying the growth stimulator solution.

5. The method according to claim 4, wherein step (2) comprises the steps of removing the supernatant of the culture solution after the culture by centrifugation and filtering the supernatant.

6. The method according to claim 4 or 5, wherein the Staphylococcus genus is Staphylococcus aureus.

7. A method for shortening the induction period of a culture test, comprising mixing a specimen with a growth stimulator solution obtained by the method of claim 1 or 2 and culturing the mixture.

8. A method for shortening the induction period of a culture test, comprising mixing a specimen with the growth stimulator powder obtained by the method of claim 4 or 5 and culturing the mixture.

9. A method for culturing a specimen, comprising: (1) inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after cultivation; (2) removing Staphylococcus sp. from the culture medium after cultivation to obtain a growth stimulator solution; (3) mixing a specimen with the growth stimulator solution to obtain a mixture; and (4) culturing the mixture.

10. A method for culturing a specimen, comprising: (1) inoculating a culture medium with Staphylococcus sp. to obtain a culture medium after cultivation; (2) removing Staphylococcus sp. from the culture medium after cultivation to obtain a growth stimulator solution; (3) drying the growth stimulator solution to obtain a growth stimulator powder; (4) mixing a specimen with the growth stimulator powder to obtain a mixture; and (5) culturing the mixture.

11. The method according to claim 9 or 10, wherein the Staphylococcus is Staphylococcus aureus.

12. Growth stimulator solution in which Staphylococcus genus has been removed from Staphylococcus culture medium.

13. A growth stimulator powder, which is a dried product of the growth stimulator solution of claim 12.

14. A culture solution comprising the growth stimulator solution according to claim 12 or the growth stimulator powder according to claim 13.

15. A culture bottle containing the culture medium according to claim 14.

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