CN112924633A - Sterility detection method for ceftiofur oil suspension injection - Google Patents
Sterility detection method for ceftiofur oil suspension injection Download PDFInfo
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Abstract
The invention discloses a sterile detection method of ceftiofur oil suspension injection, which comprises the following steps: shaking ceftiofur uniformly, adding penicillinase, and mixing uniformly to obtain a mixed solution; adding the mixed solution into a fluid culture medium containing tween-80 and a modified martin culture medium containing tween-80, shaking up again, and performing constant temperature treatment to obtain a test solution. After constant temperature treatment, the sample solution is directly inoculated for aseptic detection. The sterility test method has the best effect on ceftiofur oil suspension injection with the content of 5% and 10%. The method can effectively improve the accuracy and the reproducibility of the detection data, has strong operability and good specificity, and the oily auxiliary materials have no interference on the sterility test.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, particularly relates to a sterile detection method of a veterinary pharmaceutical preparation, and particularly relates to a sterile detection method of ceftiofur oil suspension injection.
Background
Ceftiofur (Ceftiofur), also known as cefafur, is a veterinary-specific third-generation cephalosporin that was successfully developed in the 80 th century in the united states, and the drug was first marketed in the united states in 1988, and is officially approved for the treatment of respiratory diseases in beef cattle, cows, horses, pigs and sheep in some countries in the united states, canada, japan and europe due to its excellent antibacterial activity and pharmacokinetic characteristics. The chemical name of the compound is (6R,7R) -7- [2- (2-aminothiazole-4-yl) (methoxyimino) acetamido ] -3- [ (2-furyl carbonyl) thiomethyl ] -8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, the molecular formula is C19H17N5O7S3, the molecular weight is 523.56, and the chemical structural formula is shown in the following figure. Ceftiofur has an amino group and a carboxyl group, and is an amphoteric compound which is easily decomposed under strong acid, strong base and high temperature conditions, but the sodium salt and the hydrochloride salt of ceftiofur are stable.
Ceftiofur is a third-generation cephalosporin antibiotic special for animals, has wide antibacterial spectrum and strong antibacterial activity, and has strong antibacterial activity on gram-positive bacteria, gram-negative bacteria and anaerobic bacteria, wherein the gram-positive bacteria are close to or weaker than the first-generation cephalosporin, and the gram-negative bacteria such as escherichia coli, salmonella typhi, pasteurella multocida and streptococcus have strong antibacterial activity. The compound bactericide is suitable for respiratory tract and urinary tract infection caused by various sensitive bacteria, and is particularly suitable for preventing and treating early death of chicks caused by escherichia coli, salmonella, pseudomonas aeruginosa, staphylococcus and the like, wound infection caused by umbilical cord cutting, ear number punching, tooth cutting, tail cutting and the like of newborn piglets, infectious pleuropneumonia of pigs, streptococcus suis, swine plague, yellow and white scour of piglets and the like caused by haemophilus actinobacillus.
The ceftiofur and the oil for injection are prepared into suspension type injection, which has good antibacterial action and can be widely used in clinical application, and the currently nationally approved ceftiofur oil suspension preparations mainly comprise three types, namely ceftiofur hydrochloride oil suspension injection, ceftiofur oil suspension injection and ceftiofur crystal injection. As an injection, the sterility test is required according to the related requirements of the pharmacopoeia of the people's republic of China 2015 edition. The aseptic inspection method comprises a filter membrane filtration method and a direct inoculation method, the ceftiofur oil suspension injection belongs to beta-lactam suspension, if a filter membrane method is adopted, ceftiofur can not pass through a filter membrane, so the aseptic inspection method of the ceftiofur oil suspension injection adopts the direct inoculation method, penicillin is used for enzymolysis of ceftiofur to inactivate the ceftiofur, and then the direct inoculation method is adopted for aseptic inspection. At present, a document (CN 201310665166.0) describes that a ceftiofur oil-mixed suspension type injection sterile detection method adopts 0.1% tween-80% peptone solution for pretreatment, and in this treatment mode, when the drug is subjected to sterile detection and culture for 14 days, an observation phenomenon is not obvious, a negative or positive result cannot be accurately judged, and a one-step inoculation treatment needs to be performed, that is, a culture solution is inoculated on a slant culture medium, bacteria are cultured for 2 days, fungi are cultured for 3 days, and the detection is performed according to the method. Not only consumes manpower and increases the risk of pollution, but also prolongs the detection period. Aiming at ceftiofur oil suspension injection, a sterile detection method with high accuracy and convenient operation needs to be established.
Disclosure of Invention
Based on the above problems, the present invention aims to provide a method for detecting the sterility of ceftiofur oil suspension injection. The detection method is a direct inoculation method, is convenient to operate and high in sensitivity, and the test result is easy to observe and judge and high in practicability.
The invention provides a method for asepsis detection of ceftiofur oil suspension injection, which comprises the following steps: shaking ceftiofur uniformly, adding penicillinase, and mixing uniformly to obtain a mixed solution; adding the mixed solution into a fluid culture medium containing tween-80 and a modified martin culture medium containing tween-80, shaking up again, and performing constant temperature treatment to obtain a test solution. And (3) carrying out constant temperature treatment on the pre-treated test solution, then directly inoculating the test solution into a culture medium suitable for the growth of corresponding bacteria, and detecting according to a method after culturing, wherein the specific detection method is carried out according to relevant regulations of Chinese veterinary pharmacopoeia. The sterility test method has the best effect on ceftiofur oil suspension injection with the content of 5% and 10%.
The pretreatment steps of the sterile detection method of the ceftiofur oil suspension injection provided by the invention comprise:
(1) shaking the ceftiofur oil suspension injection evenly, and then fully and evenly mixing the ceftiofur oil suspension injection with penicillinase to prepare a mixed solution of which each 1mg of ceftiofur penicillinase is more than or equal to 20000 units.
(2) Adding the mixed solution into a thioglycollate fluid culture medium containing 2-8% of tween-80, and uniformly mixing to prepare a thioglycollate test sample solution, wherein the volume ratio of the mixed solution to the thioglycollate fluid culture medium is 1: 5-10; and adding the mixed solution into an improved martin culture medium containing 2-8% of tween-80, and uniformly mixing to prepare an improved martin test solution, wherein the volume ratio of the mixed solution to the improved martin culture medium is 1: 5-10.
(3) Keeping the temperature of the thioglycolate test solution and the improved martin test solution constant at 37 ℃ for 1-4 hours.
Preferably, when the mixed solution is prepared by pretreatment, 6% of tween-80 is adopted in a thioglycolate fluid culture medium, and 6% of tween-80 is also adopted in an improved martin culture medium.
Preferably, in the step (2), when the sample solution is prepared, the volume ratio of the mixed solution to the thioglycolate fluid medium is 1:9, and the volume ratio of the mixed solution to the modified martin medium is also 1: 9.
Further, 30ml of the mixed solution is added into 270ml of thioglycolate fluid culture medium, the thioglycolate test solution is prepared after uniform mixing, and the improved martin test solution is prepared by adding 30ml of the mixed solution into 270ml of improved martin culture medium.
Preferably, in the step (3), the thioglycolate test solution and the improved martin test solution are kept at the constant temperature of 37 ℃ for 1 hour.
The sterility test pretreatment method is suitable for ceftiofur oil-soluble suspension injection with ceftiofur content of 5 percent and 10 percent. Based on the pretreatment of the ceftiofur oil suspension injection sterility test method provided by the invention, the specific ceftiofur oil suspension injection sterility test method comprises the following steps:
(1) shaking the ceftiofur oil suspension injection evenly, and then fully and evenly mixing the ceftiofur oil suspension injection with penicillinase to prepare a mixed solution of which each 1mg of ceftiofur penicillinase is more than or equal to 20000 units.
(2) Adding the mixed solution into a thioglycollate fluid culture medium containing 2-8% of tween-80, and uniformly mixing to prepare a thioglycollate test sample solution, wherein the volume ratio of the mixed solution to the thioglycollate fluid culture medium is 1: 5-10; and adding the mixed solution into an improved martin culture medium containing 2-8% of tween-80, and uniformly mixing to prepare an improved martin test solution, wherein the volume ratio of the mixed solution to the improved martin culture medium is 1: 5-10.
(3) Keeping the temperature of the thioglycolate test solution and the improved martin test solution constant at 37 ℃ for 1-4 hours.
(4) Respectively and directly inoculating the constant-temperature thioglycollate test sample solution and the improved martin test sample solution into a culture medium suitable for growth of corresponding bacteria, or a suitable culture medium indicated in pharmacopoeia, and culturing for 14 days; and directly observing to judge whether the bacteria is sterile or not.
The specific steps of the more preferable sterile detection of the ceftiofur oil suspension injection comprise:
(1) shaking the ceftiofur oil suspension injection evenly, and then fully and evenly mixing the ceftiofur oil suspension injection with penicillinase to prepare a mixed solution of which each 1mg of ceftiofur penicillinase is more than or equal to 20000 units.
(2) Adding the mixed solution into a thioglycollate fluid culture medium containing 6% of tween-80, and uniformly mixing to prepare a thioglycollate test sample solution, wherein the volume ratio of the mixed solution to the thioglycollate fluid culture medium is 1: 9; and adding the mixed solution into a modified martin culture medium containing 6% of tween-80, and uniformly mixing to obtain a modified martin test solution, wherein the volume ratio of the mixed solution to the modified martin culture medium is 1: 9.
(3) Keeping the temperature of the thioglycolate test solution and the improved martin test solution constant at 37 ℃ for 1 hour.
(4) Respectively and directly inoculating the constant-temperature thioglycollate test sample solution and the improved martin test sample solution into a culture medium suitable for growth of corresponding bacteria, or a suitable culture medium indicated in pharmacopoeia, and culturing for 14 days; and directly observing to judge whether the bacteria is sterile or not.
The method comprises the following more specific steps:
(1) shaking the ceftiofur oil suspension injection evenly, and then fully and evenly mixing the ceftiofur oil suspension injection with penicillinase to prepare a mixed solution of which each 1mg of ceftiofur penicillinase is more than or equal to 20000 units.
(2) Adding 30ml of the mixed solution into 270ml of a thioglycolate fluid culture medium containing 6% Tween-80, and uniformly mixing to prepare a thioglycolate test sample solution; adding 30ml of the mixed solution into 270ml of improved martin culture medium containing 6% Tween-80, and mixing to obtain improved martin test solution.
(3) Keeping the temperature of the thioglycolate test solution and the improved martin test solution constant at 37 ℃ for 1 hour.
(4) Respectively and directly inoculating the constant-temperature thioglycollate test sample solution and the improved martin test sample solution into a culture medium suitable for growth of corresponding bacteria, or a suitable culture medium indicated in pharmacopoeia, and culturing for 14 days; and directly observing to judge whether the bacteria is sterile or not.
Has the advantages that:
the aseptic detection method of the ceftiofur oil suspension injection provided by the invention has the following advantages:
1. the processing method is convenient and quick to operate, and the experimental result can be judged by visual inspection.
2. The culture medium selected in the test sample pretreatment method has good pertinence and full dissolution, and the use of the penicillinase enables the inactivation effect to be better, thereby being beneficial to avoiding the interference in the subsequent direct inoculation culture process.
3. According to the aseptic detection method based on the pretreatment method, the test result can be directly judged by eye observation without carrying out inoculation culture again after the culture medium is cultured for 14 days, the detection period is shortened, and the problem of secondary pollution is avoided.
4. The detection method of the invention has good specificity, and the oily auxiliary materials have no interference to the sterility test.
Detailed Description
The invention will now be further described by way of the following examples, which are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that equivalent substitutions for the technical features of the present invention, or corresponding modifications, can be made within the scope of the present invention.
Example 1 method for the sterility testing of ceftiofur oil suspension injection
(1) The culture medium for test includes thioglycollate fluid culture medium, trypticase soytone liquid culture medium, Sabouraud's glucose liquid culture medium, and Sabouraud's glucose agar. Various culture mediums used in the examples of the present invention were prepared and sterilized according to the Chinese veterinary drug classical method. The bacteria and strains for the test are all regulated and controlled by the inspection of Chinese medicinal biological products.
(2) Results of Medium sterility test
The culture medium was randomly taken out of 5 flasks, and each culture medium was incubated at a predetermined temperature for 14 days, the results of which are shown in Table 1 below. As can be seen from the table, the blank medium grows aseptically for 14 days, and meets the requirements of the sterility test.
TABLE 1 examination of the sterility of the culture Medium
(3) Preparation of test bacterial solution
Inoculating a fresh culture of staphylococcus aureus, pseudomonas aeruginosa and bacillus subtilis to a trypticase soytone liquid culture medium or a trypticase soytone agar culture slant, inoculating a fresh culture of clostridium sporogenes to a thioglycolate fluid culture medium, and culturing for 18-24 hours at the temperature of 30-35 ℃; inoculating a fresh culture of Candida albicans to a Sabouraud's dextrose liquid culture medium or a Sabouraud's dextrose agar culture medium, and culturing for 24-48 hours at 20-25 ℃. The above culture was made into a bacterial suspension containing less than 100cfu (colony forming units) per 1ml of bacteria using a sterile sodium chloride-peptone buffer solution of pH7.0 or a 0.9% sterile sodium chloride solution.
Inoculating a fresh culture of Aspergillus niger to a Sha's glucose agar culture slant culture medium, culturing at 20-25 ℃ for 5-7 days, adding 3-5 ml of pH7.0 sterile sodium chloride-peptone buffer solution containing 0.05% (ml/ml) polysorbate 80 or 0.9% sterile sodium chloride solution, eluting spores, sucking out a spore suspension by adopting a proper method into a sterile test tube, and preparing the spore suspension containing less than 100cfu spores per 1ml by using the pH7.0 sterile sodium chloride-peptone buffer solution containing 0.05% (ml/ml) polysorbate 80 or the 0.9% sterile sodium chloride solution.
(4) Medium sensitivity test
Taking 7 thioglycollate fluid culture media with the volume of 12ml per tube, respectively inoculating 2 of staphylococcus aureus, pseudomonas aeruginosa and clostridium sporogenes, and culturing for 3 days without inoculating 1 of the culture media as a blank control; taking 7 trypticase soy peptone liquid culture media with the loading of 9ml per tube, respectively inoculating 2 bacillus subtilis, candida albicans and aspergillus niger with the loading of less than 100cfu, and taking the other 1 non-inoculated bacillus subtilis, candida albicans and aspergillus niger as blank controls, culturing for 5 days, observing the results day by day, and obtaining the results shown in the table below.
TABLE 2 examination results of sensitivity of culture Medium
Example 2 sterility test method and verification of ceftiofur oil suspension injection
When a thioglycolate fluid culture medium containing 6% of Tween-80 and a modified martin culture medium containing 6% of Tween-80 are selected in the sterile detection treatment process, the reaction temperature is controlled within the range of the optimal reaction activity condition of penicillinase at 37 ℃, the purpose of adding the Tween-80 into the culture medium is to uniformly disperse an oil phase in a buffer solution, and the dosage of the penicillinase is deduced through the related calculation of enzyme activity, the conversion number of penicillinase and the like, and theoretically, the dosage is enough to completely inactivate the full amount of ceftiofur within 4 hours at 37 ℃. Meanwhile, an aseptic detection verification scheme is designed in a laboratory, and the feasibility of the operation of the aseptic detection method is demonstrated. The specific verification method comprises the following steps:
the verification test is divided into four groups:
a first group: and (5) testing the test article group. According to the sterility test method of the invention, a proper amount of ceftiofur oil suspension injection is taken and shaken up, mixed with penicillinase solution (each 1mg of ceftiofur is added with penicillinase not less than 2 ten thousand units), 30ml of mixed solution is respectively taken and added into 270ml of thioglycolate fluid culture medium containing 6 percent of Tween-80 and 270ml of improved martin culture medium containing 6 percent of Tween-80, and a sample is placed in a thermostatic water bath at 37 ℃ for 1 hour; taking a proper amount of test solution, respectively inoculating the test solution into 20ml of thioglycolate fluid medium and 20ml of trypticase soytone liquid medium by the method, culturing the inoculated thioglycolate fluid medium at 30-35 ℃ for 14 days, culturing the inoculated trypticase soytone liquid medium at 20-25 ℃ for 14 days, observing, and filling the results into the following table.
Second group: the method verifies the group. According to the sterility test method of the invention, 30ml of ceftiofur oil suspension injection is shaken up and added with a proper amount of penicillinase solution, every 1mg of ceftiofur penicillinase solution is not less than 2 ten thousand units, 270ml of thioglycolate fluid culture medium containing 6 percent of Tween-80 and 270ml of improved martin culture medium containing 6 percent of Tween-80 are added, and a test sample is placed in a thermostatic water bath at 37 ℃ for 1 hour; taking a proper amount of a test solution for sterile detection, dividing the test solution into six groups I, II, III, IV, V and VI, wherein each group of parallel samples comprises 2 samples; the I, II and III groups are fluid culture medium groups containing 20ml of thioglycollate, the IV, V and VI groups are liquid culture medium groups containing 20ml of trypticase soytone, different standard bacterial liquids less than 100cfu are added after the groups are uniformly mixed, and staphylococcus aureus is added into the group I; adding pseudomonas aeruginosa in group II; adding clostridium sporogenes into group III; adding bacillus subtilis into the group IV; adding Candida albicans into group V; aspergillus niger is added into group VI, after shaking up, group I-III is cultured for 14 days at 30-35 deg.C, group IV-VI is cultured for 14 days at 20-25 deg.C, and the results are filled in the following table.
TABLE 3 sterile test method verification results for ceftiofur oil suspension injection
The results of the verification tests showed that the test bacteria in each tube of the second group (test article group) grew well compared to the control tubes. The test quantity of the test sample is proved to have no bacteriostasis under the test condition, the dosage of the penicillinase is enough to inactivate the test sample completely, in addition, the dosage of the tween-80 can lead the oil phase to be dispersed evenly, and has no bacteriostasis, and the dosages of the penicillinase and the tween-80 are suitable for the test of the test sample. The sterility test method can fully verify that the sterility test of the test article meets the regulations, and can carry out the sterility test of the test article according to the test method and the test conditions.
EXAMPLE 35% ceftiofur oil suspension injection sterility test method comparative experiment
Test samples: the ceftiofur oil suspension injection is selected, and the content specification is 5%.
Test protocol: selecting two detection modes for aseptic comparison detection, which are respectively as follows:
firstly, shaking a proper amount of ceftiofur oil suspension injection uniformly, mixing the mixture with a proper amount of penicillinase solution uniformly to prepare a mixed solution of which each 1mg of ceftiofur and penicillinase is not less than 2 ten thousand units, adding 30ml of the mixed solution into 270ml of thioglycolate fluid culture medium containing 6% of Tween-80 and 270ml of improved martin culture medium containing 6% of Tween-80, and placing a sample in a thermostatic water bath at 37 ℃ for 1 hour; taking a proper amount of sample solution, respectively inoculating the sample solution into 20ml of thioglycolate fluid medium and 20ml of trypticase sose liquid medium correspondingly according to the method, culturing the inoculated thioglycolate fluid medium at 30-35 ℃ for 14 days, and culturing the inoculated trypticase soytose liquid medium at 20-25 ℃ for 14 days, observing, and filling the results into the following table.
The method comprises the steps of taking 25ml of ceftiofur oil suspension injection, adding sterile 1% Tween-80 and 250ml of 0.1% peptone solution, fully shaking to disperse, adding penicillinase solution (each 1mg of ceftiofur is added with penicillinase not less than 20000 units), fully shaking for 30 minutes, uniformly mixing, taking 10ml of mixed solution, respectively adding 10ml of thioglycolate fluid medium and trypticase soy peptone fluid medium, shaking for 14 days under corresponding conditions, taking the culture solution to inoculate on a slant culture medium, culturing for 3 days, checking according to law, and determining that the result meets the specification.
TABLE 4 test results of two test methods
In the method, after the scheme is cultured for 14 days, obvious negative reaction is presented in a thioglycollate fluid culture medium and a tryptone and soytone liquid culture medium solution when the observation and judgment are carried out, and the judgment result is not interfered by turbidity or other phenomena. Secondly, when the culture is observed and judged after 14 days, the thioglycollate fluid culture medium and the trypticase soytone liquid culture medium solution are in a turbid state, accurate judgment cannot be carried out, the culture solution is continuously taken and inoculated on the same fresh culture medium, the culture is carried out for 3 days, the result judgment is negative by examination according to the law, and the explanation that the treatment process has interference on the result judgment is provided.
Example 410% ceftiofur oil suspension injection sterility test method comparative experiment
Test samples: the ceftiofur oil suspension injection is selected, and the content specification is 10%.
Test protocol: selecting two detection modes for aseptic comparison detection, which are respectively as follows:
firstly, shaking a proper amount of ceftiofur oil suspension injection uniformly, mixing the mixture with a proper amount of penicillinase solution uniformly to prepare a mixed solution of which each 1mg of ceftiofur and penicillinase is not less than 2 ten thousand units, adding 30ml of the mixed solution into 270ml of thioglycolate fluid culture medium containing 6% of Tween-80 and 270ml of improved martin culture medium containing 6% of Tween-80, and placing a sample in a thermostatic water bath at 37 ℃ for 1 hour; taking a proper amount of the test solution, checking according to the law, and observing.
The third method is that a proper amount of ceftiofur oil suspension injection is taken and shaken evenly and mixed with a proper amount of penicillinase solution to prepare mixed solution of which each 1mg of ceftiofur and penicillinase are not less than 2 ten thousand units, 30ml of the mixed solution is respectively taken and added into 270ml of thioglycolate fluid culture medium containing 1 percent of Tween-80 and 270ml of improved martin culture medium containing 1 percent of Tween-80, and a sample is placed in a thermostatic water bath at 37 ℃ for 1 hour; checking according to law and observing.
Shaking a proper amount of ceftiofur oil suspension injection uniformly, mixing the mixture with a proper amount of penicillinase solution uniformly to prepare a mixed solution of which each 1mg of ceftiofur and penicillinase is not less than 2 ten thousand units, adding 270ml of 0.1% peptone solution containing 6% Tween-80 into 30ml of the mixed solution respectively, and placing a test sample in a 37 ℃ constant-temperature water bath for 1 hour; checking according to law and observing.
TABLE 5 test results of the three test methods
| Detection method | Method 1 | Method 3 | Method iv |
| Thioglycolate stream Body culture medium | Negative of | When the solution is observed for 14 days, the solution is turbid and cannot be judged And (4) cutting off, continuing inoculating and culturing, and judging as negative. | When the solution is observed for 14 days, the solution is turbid and can not be judged, and judging the result as negative after continuing the inoculation culture. |
| Trypticase soybean peptone liquid Body culture medium | Negative of | When observed for 14 days, the solution is in a turbid state,can not make a judgment And (4) cutting off, continuing inoculating and culturing, and judging as negative. | When the solution is observed for 14 days, the solution is turbid and can not be judged, and judging the result as negative after continuing the inoculation culture. |
In the method, when observation and judgment are carried out after 14 days of culture, a thioglycollate fluid culture medium and a tryptone and soybean peptone liquid culture medium solution present obvious negative reaction, and no turbidity or other phenomena interfere with the judgment result. When the culture is observed and judged after 14 days of culture, the thioglycolate fluid culture medium and trypticase soy peptone liquid culture medium solution are in a turbid state, accurate judgment cannot be carried out, 1% tween-80 cannot better emulsify oily injection, meanwhile, the culture medium of 6% tween-80 and 0.1% peptone solution is not suitable for sterile detection of ceftiofur oil suspension injection, the culture solution needs to be continuously inoculated on a slant culture medium after 14 days of culture for 3 days, and the result judgment is negative through examination by an law, which indicates that the treatment process of the method (III) and the method (IV) has interference on the result judgment.
Claims (9)
1. A pretreatment method for sterile detection of ceftiofur oil suspension injection is characterized by comprising the following steps:
(1) shaking the ceftiofur oil suspension injection evenly, and then fully and evenly mixing the ceftiofur oil suspension injection with penicillinase to prepare a mixed solution of which each 1mg of ceftiofur penicillinase is more than or equal to 20000 units;
(2) adding the mixed solution into a thioglycollate fluid culture medium containing 2-8% of tween-80, and uniformly mixing to prepare a thioglycollate test sample solution, wherein the volume ratio of the mixed solution to the thioglycollate fluid culture medium is 1: 5-10; adding the mixed solution into an improved martin culture medium containing 2-8% of tween-80, and uniformly mixing to prepare an improved martin test solution, wherein the volume ratio of the mixed solution to the improved martin culture medium is 1: 5-10;
(3) keeping the temperature of the thioglycolate test solution and the improved martin test solution constant at 37 ℃ for 1-4 hours.
2. The method for pretreating a ceftiofur oil suspension injection for sterility test according to claim 1, wherein in step (2), tween-80 is used in an amount of 6% when preparing the thioglycolate test solution and the modified martin test solution.
3. The method for pretreating a ceftiofur oil suspension injection for sterility test according to claim 1, wherein in step (2), the volume ratio of the mixed solution to the thioglycolate fluid medium and the modified martin medium is 1:9 when the thioglycolate test solution and the modified martin test solution are prepared.
4. The pretreatment method for sterile detection of ceftiofur oil suspension injection according to claim 3, wherein 30ml of the mixed solution is added to 270ml of thioglycolate fluid medium, and the mixture is uniformly mixed to obtain a thioglycolate test solution; and adding 30ml of the mixed solution into 270ml of improved martin culture medium, and uniformly mixing to obtain an improved martin test solution.
5. The pretreatment method for sterile detection of ceftiofur oil suspension injection according to claim 1, wherein in the step (3), the thioglycolate test solution and the modified martin test solution are incubated at a constant temperature of 37 ℃ for 1 hour.
6. The method for pretreating a ceftiofur oil suspension injection according to any one of claims 1-5, wherein the method is suitable for ceftiofur oil suspension injections with ceftiofur content of 5% and 10%.
7. A sterile detection method of ceftiofur oil suspension injection is characterized by comprising the following steps:
(1) shaking the ceftiofur oil suspension injection evenly, and then fully and evenly mixing the ceftiofur oil suspension injection with penicillinase to prepare a mixed solution of which each 1mg of ceftiofur penicillinase is more than or equal to 20000 units;
(2) adding the mixed solution into a thioglycollate fluid culture medium containing 2-8% of tween-80, and uniformly mixing to prepare a thioglycollate test sample solution, wherein the volume ratio of the mixed solution to the thioglycollate fluid culture medium is 1: 5-10; adding the mixed solution into an improved martin culture medium containing 2-8% of tween-80, and uniformly mixing to prepare an improved martin test solution, wherein the volume ratio of the mixed solution to the improved martin culture medium is 1: 5-10;
(3) keeping the temperature of the thioglycolate test solution and the improved martin test solution constant at 37 ℃ for 1-4 hours;
(4) respectively and directly inoculating the constant-temperature thioglycollate sample solution and the improved martin sample solution into a culture medium, and culturing for 14 days; and directly observing to judge whether the bacteria is sterile or not.
8. The method for testing the sterility of ceftiofur oil suspension injection according to any one of claim 7, comprising the following steps:
(1) shaking the ceftiofur oil suspension injection evenly, and then fully and evenly mixing the ceftiofur oil suspension injection with penicillinase to prepare a mixed solution of which each 1mg of ceftiofur penicillinase is more than or equal to 20000 units;
(2) adding the mixed solution into a thioglycollate fluid culture medium containing 6% of tween-80, and uniformly mixing to prepare a thioglycollate test sample solution, wherein the volume ratio of the mixed solution to the thioglycollate fluid culture medium is 1: 9; adding the mixed solution into an improved martin culture medium containing 6% of tween-80, and uniformly mixing to obtain an improved martin test solution, wherein the volume ratio of the mixed solution to the improved martin culture medium is 1: 9;
(3) keeping the temperature of the thioglycolate test solution and the improved martin test solution constant at 37 ℃ for 1 hour;
(4) respectively and directly inoculating the constant-temperature thioglycollate sample solution and the improved martin sample solution into a culture medium, and culturing for 14 days; and directly observing to judge whether the bacteria is sterile or not.
9. The method for testing the sterility of ceftiofur oil suspension injection according to claim 7, comprising the following steps:
(1) shaking the ceftiofur oil suspension injection evenly, and then fully and evenly mixing the ceftiofur oil suspension injection with penicillinase to prepare a mixed solution of which each 1mg of ceftiofur penicillinase is more than or equal to 20000 units;
(2) adding 30ml of the mixed solution into 270ml of a thioglycolate fluid culture medium containing 6% Tween-80, and uniformly mixing to prepare a thioglycolate test sample solution; adding 30ml of the mixed solution into 270ml of improved martin culture medium containing 6% Tween-80, and mixing uniformly to obtain an improved martin test solution;
(3) keeping the temperature of the thioglycolate test solution and the improved martin test solution constant at 37 ℃ for 1 hour;
(4) respectively and directly inoculating the constant-temperature thioglycollate sample solution and the improved martin sample solution into a culture medium, and culturing for 14 days; and directly observing to judge whether the bacteria is sterile or not.
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