Establishment of microbial limit test method for cefradine capsule
Cefradine capsule is a cephalosporin antibiotic. It is commonly used in acute pharyngitis, acute tonsillitis, otitis media, bronchitis, pneumonia and other respiratory infections, genitourinary tract infections and skin and soft tissue infections caused by cefradine-sensitive bacteria. In the case of microbial limit testing, such antibacterial drugs need to adopt appropriate methods to eliminate the interference of the antibacterial activity of the drug, so as to ensure the scientificity of the test method and the accuracy of the test results.
In this experiment, three strains of bacteria and two strains of fungi prescribed in the 2005 Chinese Pharmacopoeia were used to verify the microbial limit test methods of three batches of cefradine capsules to establish a microbial limit test method for cefradine capsules.
1, material
1.1 Instrument: electronic balance; desktop low speed centrifuge; collection instrument.
1.2, medium nutrient agar medium, rose red sodium agar medium, modified Martin medium, nutrient broth medium BL medium, MUG medium, pH 7.0 sodium chloride-peptone buffer.
1.3 Test sample: cefradine capsule batch number is 200609020060902, 20060903).
1.4 strains: Escherichia coli [CMCC (B) 44102], Bacillus subtilis [CMCC (B) 63501], Staphylococcus aureus LCMCC (B) 26003], Aspergillus niger [CMCC (F) 98003], white rosary Bacteria [CMCC (F) 98001].
2, methods and results
2.1 Preparation of bacterial solution The fresh cultures of the above five strains were inoculated separately into the prescribed medium, and the bacteria were cultured for 16 hours; the yeast was cultured for 24 hours; and the mold was cultured for 5 days, and the suspension of the bacteria was prepared and used.
2.2 Preparation of test solution According to the provisions of the test sample 10g, add pH7.0 sterile sodium chloride - egg packet monitoring buffer to 100ml, in a 45 ° C water bath to keep the capsule dissolved, mix, that is 1:10 Test solution; according to the provisions of the test sample 10g aseptic operation, the capsule shell and the contents are separated, weighed in the same container, add pH7.0 sterile sodium chloride-peptone buffer to 100ml, mix evenly, Immediately pipette the suspension into a centrifuge tube, centrifuge at 500 r•rmin-1 for 4 min, and take a supernatant of 1:10 for the test solution.
2.3 Verification of bacterial, mold and yeast counting methods
2.3.1 Take appropriate amount of test solution and 50-100 CFU test bacteria, respectively, into the same dish, pour the corresponding agar medium, culture count, for the test group; take the same amount of test bacteria, determine the number of test bacteria added , for the bacterial liquid group; take the test solution to determine the number of background bacteria for the test sample, for the test product control group; according to the following formula: recovery rate% = [(test group average number of colonies - test sample control group average The number of colonies was determined by the average number of colonies in the sputum solution group × 100%.
2.3.2 Pre-test of mold and yeast Take test solution 11, 0.5mL, respectively, and place 2 dishes as a group, and add 1 kind of fungal test bacteria liquid to each group, a total of 2 groups. The recovery rate of 1,0.50 mL/dish 2 strains was 70% or more.
2.3.3 Bacterial pretest
2.3.3.1 Take test solution 11; 0.5; 0.33; 0.25; 0.2; 0.1mL respectively, 2 dishes as a group, each group added 1 kind of bacterial test bacteria liquid, a total of 3 groups. The recovery rate of the three strains was 0%.
2.3.3.2 Take the test solution 21; 0.50; 0.33; 0, 25; 0.2; 0, lmL respectively, 2 dishes as a group, each group added 1 kind of bacterial test bacteria liquid, a total of 3 groups. The recovery rate of the three strains was 0%.
2.3.3.3 Take the test solution 2 The supernatant after centrifugation per 10 ml is added to the appropriate volume of pH 7.0 sterile sodium chloride-peptone buffer container, and the membrane is sterilized by filtration at pH 7.0. The sodium-peptone buffer was washed in portions, and 50-100 CFU test bacteria were added to the rinsing solution after the Zui, and the membranes were respectively attached to the nutrient agar medium, and the culture count was used as the test group, and the three test bacteria were washed twice per membrane. 300mL, of which 300mL flushing rate of bacteria recovery rate of up to 70%; bacterial liquid group, test product control group by the same method.
2.3.3.4 Diluent control group Take pH710 sterile sodium chloride-peptone buffer solution, a total of 3 parts, respectively, add 3 test bacterial bacteria solution, so that the concentration of Zui terminal bacteria is 50 ~ 100 CFU / mL, the following operation is the same as 2113113.
2.3.3.5 According to the above test results, Escherichia coli, Bacillus subtilis and Staphylococcus aureus were subjected to low-speed centrifugation plus membrane filtration, and 2 strains of fungi were tested by conventional method 1 mL•dish.
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