Particles on the microbial limit ammonia Jinhuang Min method validation

[Abstract] Objective To verify the ammonia Jinhuang Min microbial limit test particles are applied to the inspection of the test. Methods using low-speed centrifugation of bacteria count - medium dilution method, mold and yeast count of the conventional test method, the conventional control bacteria Check the law. Results thinner than the control group 70% of bacterial recovery, the experimental group of bacteria is greater than 70% recovery of the negative control group were not detected in the negative control bacteria, the experimental group showed positive test bacteria. conclusions can use the microbial limit test for ammonia Jinhuang Min particle inspection.

[Keywords:] ammonia Jinhuang Min verify microbial limit test particles
Ammonia Jinhuang Min particles microbial limit test of the company before the conventional method, in order to ensure the scientific testing methods, is the company's ammonia Jinhuang Min microbial limit test particle method validation methods to verify whether this method is applicable to the examination of the test.

1 Materials and equipment
1.1 The name of the drug test sample: Ammonia Jinhuang Min particles, batch number: 090901,090902,090903, Source: Company production.

1.2 validation instruments
1.2.1 electric oven, refrigerator, electric incubator, constant temperature water bath, centrifuge, steam sterilization pot, set the instrument, such as bacteria.

1.2.2 glass flask, Petri dish, graduated cylinder, test tubes, straws and so on.

1.3 Authentication of strains of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Candida albicans; Aspergillus niger.

1.4 Authentication and thinner with nutrient agar medium, agar rose sodium, lactose bile medium, nutrient broth, modified Martin medium, eosin methylene blue agar.

2 Verify that the conditions
Sterile room cleanliness class 10,000 environment, the cleanliness of 100 local level, more than 30min purification and disinfection, and sterilization of the test apparatus such as ultraviolet light disinfection by the transfer window set to sterile indoor exposure to 30min, after the wear testing personnel hand disinfection sterile services into the operating room.

3 Methods and Results
3.1 Preparation of inoculum of Staphylococcus aureus bacterium, Escherichia coli, Bacillus subtilis fresh slant culture were inoculated into nutrient broth medium at 35 ℃ for 18 ~ 24 hours, the bacteria were collected by adding 1ml culture medium 9ml sterile 0.9% sodium chloride solution, 10 times followed by dilution, S. aureus was diluted to 10-5, to 10-7 dilution of E. coli, Bacillus subtilis was diluted to 10-5, which is about 50 ~ 100cfu / ml, set aside.

Candida albicans inoculated to fresh culture medium modified Martin, at 23 ~ 28 ℃ 23 ~ 48 hours of training, take 1ml culture medium in 0.9% sterile sodium chloride solution, 9ml, 10-fold diluted to 10-5 in turn , about 50 ~ 100cfu/ml, set aside.

Take a fresh slant of Aspergillus niger cultures, with 0.9% sterile sodium chloride solution 5ml wash the spores, the lessons of this solution, the setting of a sterile 1ml cuvette, add 0.9% sterile sodium chloride solution and the standard amount turbidimetry turbidity tube, taking turbidity 1ml of bacterial suspension after adding 9ml of 0.9% sterile sodium chloride solution, 10-fold diluted to 10-4 in turn, is about 50 ~ 100cfu/ml, set aside. reposted elsewhere Free Download Center http://www.hi138.com 3.2 paper prepared for the test solution taken for the test materials 10g, plus sterile sodium chloride pH7.0 - peptone buffer to 100ml, and mix with a decentralized instrument homogenate as a : 10 for the test solution.

3.3 bacteria, mold and yeast count verification tests
3.3.1 test group with low-speed centrifugation bacterial count - medium dilution method, taking 1:10 for the test solution 10ml, 500 r / separation heart 5 minutes, take all the supernatant with sterile sodium chloride pH7.0 - peptone complement to the original volume of buffer, mixing, and then take 1ml from which 5 were added in the petri dish (each dish 0.2ml, adding the bacteria-positive bacteria test 1ml, immediately pour the corresponding agar medium, and placing 32 ℃ constant temperature incubator 48h, determine the number of bacteria. mold and yeast count of the conventional method, taking 1:10 for the test solution 1ml, add the mold and yeast test bacteria 1ml, sodium rose immediately poured agar medium, set the 25 ℃ incubator cultured 72h, determination of the number of mold and yeast.

3.3.2 bacilli each group were diluted broth 1ml, were added to petri dish, add the appropriate medium, provided the conditions set training, measured by the number of test bacteria added.

3.3.3 the test method for the control group according to the experimental group, the test measured the number of background bacteria.

3.3.4 diluent for the control group were thinner alternative test solution, the method with the test group.

Test group test group, the average strain rate (%={( colonies - the test group the average number of colonies / bacilli group, the average number of colonies} × 100%
Diluent control bacteria recovery (% = (thinner control group, the average number of colonies / bacilli group, the average number of colonies × 100%
3.4 The inspection method validation control bacteria
3.4.1 experimental group were 1:10 for the test solution 10ml, set centrifugation under sterile conditions (500 rev / min for 5 minutes, the supernatant sterile test tube home, pH7.0 sterile sodium chloride - peptone buffer liquid supplement to the original volume, mix, add to 100ml of bile salt lactose medium, coupled with positive control strains of E. coli bacterium 1ml, shake, set 35 ~ 37 ℃ incubator for 18 to 24 hours. the same concentration of E. coli bacteria, mold and yeast count verification.

3.4.2 Method for the negative control group with experimental group, add 1ml negative bacteria (ie Staphylococcus aureus, the same concentration of bacteria, mold and yeast count verification), set 35 ~ 37 ℃ incubator for 18 to 24 hours.

3.5 Analysis
3.5.1 of bacteria, mold and yeast counts in the three tests verify the conclusion, the thinner the control group of bacteria was more than 70% recovery rate, recovery rate of the test group were more than 70% of the bacteria, it can be prepared according to the sample solution methods and particle counting method for the determination of ammonia Jinhuang Min bacteria, mold and yeast count.

3.5.2 The conclusion of the control bacteria in the three tests, the negative control group, negative control bacteria were not detected in the experimental group showed positive test bacteria, so use this method for the preparation for the test solution and control the bacteria test for the control of ammonia Jinhuang Min particles bacteria checks.

4 Discussion
4.1 features many drugs themselves (such as antibacterial activity will affect the test results, the need for the test proper pretreatment to eliminate interference caused by its own.

Microbial drugs 4.2 Drugs used to ensure the safety check is an important inspection items, each species must be validated test methods.

4.3 During the experiment, norms should be strictly observed to ensure the reliability of the results.

References
[1] Chinese Pharmacopoeia (2010 edition) two: Appendix 107.

[2] China Drug Control Standard Operating Procedures (2010 edition: 351.

[3] Dong Zhang, Wang Lanqing. Chemicals, inspection and Health Quality of microbial limit test [J]. Modern Applied Pharmacy, 1997,14 (1): 44. Links to Research Papers Download http://www.hi138 . com