Baoyuan detoxification pill microbial limit test for the validation study
【Abstract】 Objective To confirm the method used for the test chemicals for microbial limit. That is tested to confirm the amount of goods on the test, the test conditions, no
antibacterial activity or its antibacterial activity to be fully eliminate enough to be negligible. Methods The 2005 edition of "Chinese Pharmacopoeia" provisions of the authentication method. Results count results are accurate,
meet the requirement. Conclusion The experiment is suitable Yasumoto detox pill microbial limit method validation.
Key words Yasumoto detox pill microbial limit method validation
Theoretically speaking, each sample of the micro-
organisms will affect the extent of the impact, testing methods can ensure that microbial contamination can be tested out, it is necessary to verify through testing in order to ensure the accuracy of microbial limit test results, while drug passing rate of testing the existence of Quality
Supervision, not the clear, comprehensive, real outstanding issues . In accordance with "Chinese Pharmacopoeia" 2005 edition of the former provisions of microbial limits testing methods, there are many bacteria undetected, which is difficult to ensure that drug microbial limit test results are accurate and reliable, but also to bring hidden dangers clinical use. Bao-yuan Department of our hospital
detoxification pill famous old Chinese doctor Zhang Zhijian, director of long-term clinical experience side. Clinical use
of chronic nephritis, chronic renal failure, see there is dizziness fatigue, Shenpi limb soft, oliguria foot swelling, waist and knee pain, torpid intake pan vomiting, sleepless nights, looking dull, tongue pale and tender, thin moss Wong,
greasy and other diseases, a wider clinical use, it is necessary to ensure Yasumoto detox pill microbial limit test results accurate and reliable.
1.1 Equipment Clean Bench, SW CJ ICU (Su-Net Group
Aetna); biological safety cabinets, BHC 1300ILA/B3 (Su-Net
Group Aetna); electronic balance, AV412 Mettler Toledo, Inc. (Shanghai Limited company); watertight constant temperature incubator PYX DHS, (Shanghai an Infineon Technologies Co., Ltd.); mold incubator, MJ 180S ? (Shanghai Xinmiao
Medical Equipment Manufacturing Co., Ltd.); vertical sterilizer, LMQ. R 3260B (Shandong Xinhua Medical
Instrument Manufacturing Co., Ltd.).
1.2 medium nutrient agar medium, Qingdao Hi-tech Park
Haibo Bio-Technology Co., Ltd. (20,060,309); Rose Red sodium agar, Yixing Yongxin Bio Co., Ltd. (050915); pH 7.0 sterile sodium chloride - peptone buffer , Yixing Yongxin Bio Co., Ltd. (051027); bile acid medium, Yixing Yongxin Bio Co., Ltd. (050,509); nutrient broth, Yixing Yongxin Bio Co., Ltd.
(051,206); mannitol, high-salt agar, Qingdao Hi-tech Park
Haibo Biotechnology Co., Ltd. (20060112);
cetyltrimethylammonium bromide agar, Yixing Yongxin Bio Co., Ltd. (050,921); modified Martin medium, Yixing Yongxin Bio Co., Ltd. (050,829).
1.3 Authentication with strains of Escherichia coli (Escherichia coli) [CMCC (B) 44 102], 4th generation; Staphylococcus aureus (Staphylococcus aureus) [CMCC (B) 26 003], 4th generation; Bacillus subtilis ( Bacillus subtilis) [CMCC (B) 63 501], 4th generation; Pseudomonas aeruginosa (Pseudomonas aeruginosa) [CMCC (B) 10 104], the 3rd
generation; Candida albicans (Candida albicans) [CMCC (F) 98001] 4th generation; Aspergillus (Aspergillus niger) [CMCC (F) 98 003] 3rd Generation.
1.4 for the test chemicals and reagents Yasumoto Detox Pills (060.81 thousand), Changzhou City, Jiangsu Province Chinese Medicine Hospital preparation; sodium chloride solution, AR.
2.1 bacteria, mold and yeast count method validation 
2.1.1 Preparation for the test solution was obtained for the test quality assurance yuan detoxification pill 10 g, plus pH sterile sodium chloride - peptone buffer to 100 ml, at
45 ? water bath in the heat, soak, oscillation dissolution, made a: 10 for the test solution.
2.1.2 Preparation of the Bacterium Escherichia coli, Staphylococcus aureus, Bacillus subtilis were inoculated with fresh culture medium to the nutrient broth, 30 ~ 35 ? culture
24 ~ 48 h, were collected from these cultures with 0.9% sterile sodium chloride solution made of 50 ml per 100 cfu (colony number) in bacilli.
To Candida albicans were inoculated into fresh culture medium to the modified Martin, Chi 23 ~ 28 ? incubator 24 ~
48 h, to take the culture with 0.9% sterile sodium chloride solution per ml made 50 ~ 100 cfu of bacilli.
Will be a fresh culture of Aspergillus niger were inoculated to the culture medium improved slope Ma Dingqiong
fat, 23 ~ 28 ? cultured 7 d, so that a large number of spores produced. Then add 5 ml0.9% sterile sodium chloride solution, using sterile glass rod gently spores eluted, and then use a nozzle packed with sterile cotton, and can filter hyphae 10 ml
straw to suck out the liquid spore-free bacteria test tube,
with 0.9% sterile sodium chloride solution will bacilli per ml diluted to 50 ~ 100 cfu of bacilli.
2.1.3 Authentication Methods Plate method (conventional method).
Experimental group: take 10 Plate, 2 a group divided into 5 groups, do a good job of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger tags. 1:10 from each of the tested quality assurance yuan detoxification pill 1 ml, were added to 10 petri dish in, and then turn to join the above-mentioned five kinds of bacteria
of the bacterial suspension 1 ml (50 ~ 100 Ge cfu), the parallel preparation of 2 bacteria per plant a petri dish and immediately pour into nutrient agar medium and Rose Red sodium
agar, respectively, 30 ~ 35 ? train home 48 h, and 23 ~ 28 ?
cultured 72 h, the bacteria determine the number of experimental groups.
Bacilli: the dilution of the bacilli were collected from 1 ml, into the petri dish, each strain in parallel Plate
Preparation of 2, immediately poured into nutrient agar and Rose Red sodium agar, respectively, 30 ~ 35 ? train home 48 h
and 23 ~ 28 ? cultured 72 h, measured in the increase of the number of test bacteria.
Test products for the control group: 1:10 for the test solution from each of detox pill Baoyuan 1 ml were injected into 4 petri dish, he will immediately pour nutrient agar medium and Rose Red sodium agar, respectively, set 30 ~ 35 ?
for 48 h, and 23 ~ 28 ? cultured 72 h, to determine the base for the test chemicals.
Diluent control group: This product did not use the special handling and preparation, it is not done. Parallel experiments three times the above method.
Strain recovery calculated as follows: the experimental group of bacteria in experimental group, the average recovery rate of (%)=( colonies - the control group for test items, the average number of colonies) / bacilli the average number of colonies × 100% reposted elsewhere in the paper for free
Experimental Results: Table 1.
2.2 The control of bacteria inspection methods validation 
2.2.1 Escherichia coli inspection methods validation
Experimental group: 1:10 to take for the test solution was 10 ml, inoculated to 100 ml of bile salt lactose enrichment medium incorporating 47 cfu / ml E. coli 1 ml, 35 ~ 37 ? culture 18 ~ 24 h, take the above-mentioned culture
medium 0.2 ml to 5 ml MUG medium inoculated in vitro, the
culture 5,24 h, observed under ultraviolet light at 345 nm, which are the fluorescence response, dropping indole test solution, liquid was red roses.
-Negative bacteria control group: 1:10 to take for the test solution was 10 ml, inoculated to 100 ml of bile salt lactose enrichment medium incorporating 66 cfu / ml Staphylococcus aureus 1 ml, 35 ~ 37 ? for 18 ~ 24 h, taking
the above-mentioned culture medium 0.2 ml to 5 mlMUG medium inoculated in vitro, the culture 5,24 h, observed under
ultraviolet light at 365 nm, no fluorescence reaction, dropping indole test solution, liquid reagent was true colors.
2.2.2 Coliform inspection methods validation
Experimental group: 1:10 to take for the test solution 1
ml, inoculated 10 ml bile salt lactose fermentation medium tube, add 47 cfu / ml of Escherichia coli 1 ml, set 35 ~ 37 ?
culture 18 ~ 24 h. Medium appears turbid, acid gas production.
Table 1 Baoyuan detoxification pills bacteria, mold and
yeast recovery of the assay (Plate method) (omitted)
-Negative bacteria control group: 1:10 to take for the
test solution 1 ml, inoculated 10 ml bile salt lactose fermentation medium tube, add 66 cfu / ml of Staphylococcus aureus 1 ml, set 35 ~ 37 ? culture 18 ~ 24 h . No acid gas
2.2.3 Salmonella verification inspection methods
Experimental group: take 10 g's for the direct inoculation of test solution to 200 ml of nutrient broth medium, adding with 66 cfu / ml Staphylococcus aureus 1 ml,
set 35 ~ 37 ? culture 18 ~ 24 h, take the train solution 1 ml, inoculated to 10 ml four sulfur sodium light green medium, cultured 18 ~ 24 h, crossed inoculated Salmonella, Shigella agar of the plate, cultured 18 ~ 24 h, the growth plate a
colorless, translucent, dark colony colony center.
-Negative bacteria control group: 10 g taken for the direct inoculation of test items to 200 ml of nutrient broth medium, mixing, adding 66cfu/ml Staphylococcus aureus containing 1 ml, set 35 ~ 37 ? culture 18 ~ 24 h, take the
above-mentioned culture medium 1 ml, inoculated to 10 ml four sulfur sodium light green medium, cultured 18 ~ 24 h, crossed inoculated Salmonella, Shigella agar of the plate, cultured 18 ~ 24 h, No colony growth plate.
Table 1 shows that by Paul yuan detox pill to use Plate colony count, 3 times in parallel experiments, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida
albicans, Aspergillus niger colony recovery rate has reached
more than 70% shows that the method for bacteria, mold and yeast count of the results are accurate.
Control bacteria inspection methods validation experiment Note: the experimental group detected E. coli, Salmonella
paratyphi;-negative bacteria were not detected in control group, Staphylococcus aureus; Salmonella paratyphi. Note Baoyuan detox pill no inhibitory effect on E. coli, no inhibitory effect on the coliform bacteria, no inhibitory effect on Salmonella.
This product is thinner control group did not make any special handling and preparation of bacteria without damage, so not done validation.
Colonies in the preparation required to determine the number of bacilli in the process, through the experiments in
our laboratory to explore the dilution level for each type of bacteria: Staphylococcus aureus 10-6, 10-5, Candida albicans,
Escherichia coli 10-7, Bacillus Bacillus 10-5, 10-5,
Aspergillus niger, exactly in line with experimental colonies
50 ~ 100 cfu / ml or 10 ~ 100 cfu / ml requirements.
 JIANG. Drug testing pass rate and the Quality of Medicines (?) [J]. China Pharmaceutical, 2005,19 (1): 17.
 National Pharmacopoeia Committee. Chinese
Pharmacopoeia, ? Department of [S]. Beijing: Chemical Industry Press, 2005: Appendix 71, Appendix 73. Reposted elsewhere in the paper for free download http://