CN108315384A - The microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule - Google Patents
The microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule Download PDFInfo
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Abstract
The invention belongs to drug measurement techniques fields, and in particular to a kind of microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule.Prepared by bacterium solution, prepared by test liquid, aerobic bacteria, yeast and mold sum measurement, it in 0.5~2 range is qualified that test group clump count, which subtracts the value of test sample group clump count with the ratio of bacterium solution group clump count, controls the inspection of bacterium.Inventive samples processing can preferably eliminate Nifuratel, reduce biocidal property, operating process is simple, and the probability of pollution is small, prevents the appearance of sample false negative in checkout procedure, patient medication safe.
Description
Technical field
The invention belongs to drug measurement techniques fields, and in particular to a kind of micro- life of Nifuratel nystatin expandable vaginal soft capsule
Object limit test method.
Background technology
Bacterial vaginitis illness is leucorrhea increasing, has bad smell, sexual intercourse postemphasis with pruritus vulvue or can burn
Sense.But 10%~40% patient has no any symptom, is only just found in physical examination.
Monilial vaginitis illness is pruritus vulvue, vulva and vagina cusalgia, and leucorrhea increasing is in bean curd slag specimen, sometimes companion
There are frequent micturition, an odynuria, intercourse pain, the white membranoid substance of attachment on the inside of when gynecologial examination visible nymphae and on vagina mucosa, after erasing
Expose red and swollen mucosal surface, acute stage visible impaired rotten to the corn face.
Nifuratel nystatin expandable vaginal soft capsule, indication are bacterial vaginosis BV, outside trichomonas vaginitis, monilial
Cloudy vaginitis, vagina mixed infection.Nifuratel be nitrofuran analog derivative, have broad spectrum antimicrobial effect, to trichomonad,
Bacterium, Candida albicans etc. all have activity.
Microbial limit tests about Nifuratel nystatin expandable vaginal soft capsule not yet have been reported that so far.
Invention content
The object of the present invention is to provide a kind of microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule, solutions
Certainly the false negative phenomenon in Nifuratel nystatin expandable vaginal soft capsule Micro biological Tests, patient medication are safe.
The microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule of the present invention, steps are as follows：
(1) prepared by bacterium solution
Staphylococcus aureus bacteria suspension, pseudomonas aeruginosa bacteria suspension, bacillus subtilis bacteria suspension, white is prepared respectively
Color candida albicans bacteria suspension and aspergillus niger spore suspension；
(2) prepared by test liquid
Nifuratel nystatin expandable vaginal soft capsule test sample 10g is weighed, is shredded and is placed in sterile homogenizing bag with sterile scissors,
43 DEG C of sterile isopropyl myristates for adding 80ml are patted 1min with homogenizer, are poured into sterile triangular flask, capsule shells stay in nothing
43 DEG C of pH7.0 sterile NaCls-peptone buffer agents of addition 50ml are rubbed broken with hand handle capsule shells in bacterium homogenizing bag, then pour into three
After being shaked in the bottle of angle, 43 DEG C of pH7.0 sterile NaCls-peptone buffer agent 350ml are added and shake standing 5 minutes, with sterile note
Emitter draws upper layer isopropyl myristate and the solution of test sample mixing discards, then adds sterile digestion instrument needle with asepsis injector
The layer 240-260ml that fetch water is moved into sterile triangular flask, as 1：40 test liquid；
(3) aerobic bacteria, yeast and mold sum measurement
Aerobic bacteria be staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, Candida albicans and aspergillus niger,
Mould is aspergillus niger, and saccharomycete is Candida albicans.
1. test group：Take 1:40 test liquid 1ml set 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
It in pH7.0 sterile NaCls-peptone buffer agent 500ml, handles through membrane-filter procedure, is separately added into when last time filters
Staphylococcus aureus bacteria suspension, pseudomonas aeruginosa bacteria suspension, bacillus subtilis bacteria suspension, Candida albicans bacteria suspension and
Aspergillus niger spore suspension removes filter membrane and is affixed on to set in 30-35 DEG C of incubator on pancreas junket soya peptone agar medium and cultivates 3 days；Separately
The filter membrane for being separately added into Candida albicans bacteria suspension and aspergillus niger spore suspension is affixed on Sabouraud glucose agar again outside
On set in 20-25 DEG C of incubator and cultivate 5 days；
2. bacterium solution group：It takes pH7.0 sterile NaCls-peptone buffer agent to replace test liquid, is operated by test group and bacterium is added
Liquid measures the bacterium number of added bacterium solution；
3. test sample group：The test liquid prepared is taken, bacterium solution is replaced with pH7.0 sterile NaCls-peptone buffer agent,
He operates with test group；It sets in 30-35 DEG C of incubator and cultivates 5 days on pancreas junket soya peptone agar medium；Sabouraud's dextrose agar
It sets in 20-25 DEG C of incubator and cultivates 7 days on culture medium；
4. culture after carry out aerobic bacteria, yeast and mold sum measurement,
Test group clump count subtracts the value of test sample group clump count and the ratio of bacterium solution group clump count in 0.5~2 range
It is as qualified；
(4) inspection of bacterium is controlled
1. the inspection of staphylococcus aureus
Test sample group：Take 1：40 test liquid 40ml are added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 100mlTSB culture mediums is added,
Mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation in mannitol sodium chloride agar
On culture medium flat plate, 30~35 DEG C are cultivated 18~72 hours；If it is feminine gender not have bacterium colony or qualification result on tablet, judgement is for examination
Staphylococcus aureus is not detected in product；
Positive controls：Take 1：40 test liquid 40ml are added to 43 DEG C and contain 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
PH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter all filtering be added 100mlTSB culture mediums,
Staphylococcus aureus mixing is added, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation
In on mannitol sodium chloride agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If there is no bacterium colony or identification on tablet
As a result it is feminine gender, staphylococcus aureus is not detected in judgement positive controls；
Negative control group：It takes pH7.0 sterile NaCls-peptone buffer agent 40ml to be added to 43 DEG C and contains 3% polysorbate
80 and 0.3% soybean lecithin pH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter whole mistake
100mlTSB culture mediums are added in filter, and mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Above-mentioned culture is taken to cross
It is inoculated on mannitol sodium chloride agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If do not have on tablet bacterium colony or
Qualification result is feminine gender, and staphylococcus aureus is not detected in judgement negative control group；
2. the inspection of pseudomonas aeruginosa
Test sample group：Take 1：40 test liquid 40ml are added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 100mlTSB culture mediums is added,
Mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation in cetyltrimethylammonium
On ammonium agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If it is feminine gender not have bacterium colony or qualification result on tablet, sentence
Determine test sample and pseudomonas aeruginosa is not detected；
Positive controls：Take 1：40 test liquid 40ml are added to 43 DEG C and contain 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
PH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter all filter, be added 100mlTSB culture mediums
In, pseudomonas aeruginosa mixing is added, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Above-mentioned culture scribing line is taken to connect
In on cetab agar medium tablet, 30~35 DEG C are cultivated 18~72 hours kind；If there is no bacterium on tablet
It falls or qualification result is feminine gender, pseudomonas aeruginosa is not detected in judgement positive controls；
Negative control group：It takes pH7.0 sterile NaCls-peptone buffer agent 40ml to be added to 43 DEG C and contains 3% polysorbate
80 and 0.3% soybean lecithin pH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter whole mistake
Filter is added in 100mlTSB culture mediums, mixing, and 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Above-mentioned culture is taken to draw
Line is inoculated on cetab agar medium tablet, and 30~35 DEG C are cultivated 18~72 hours；If not having on tablet
It is feminine gender to have bacterium colony or qualification result, and pseudomonas aeruginosa is not detected in judgement negative control group；
3. the inspection of Candida albicans
Test sample group：Test liquid 40ml is taken to be added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 500ml Sabouraud dextroses are added
In fluid nutrient medium, mixing, 30~35 DEG C are cultivated 3~5 days, and culture is obtained；Take above-mentioned culture streak inoculation in Sharpe Portugal
On grape sugar agar medium tablet, 30~35 DEG C are cultivated 24~48 hours；If it is feminine gender not have bacterium colony or qualification result on tablet,
Candida albicans is not detected in judgement test sample；
Positive controls：Test liquid 40ml is taken to be added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 500ml Sabouraud dextroses are added
In fluid nutrient medium, Candida albicans mixing is added, 30~35 DEG C are cultivated 3~5 days, and culture is obtained；Above-mentioned culture is taken to draw
Line is inoculated on Sabouraud glucose agar tablet, and 30~35 DEG C are cultivated 24~48 hours；If do not have on tablet bacterium colony or
Qualification result is feminine gender, and Candida albicans is not detected in judgement positive controls；
Negative control group：It takes pH7.0 sterile NaCls-peptone buffer agent 40ml to be added to 43 DEG C and contains 3% polysorbate
80 and 0.3% soybean lecithin pH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter whole mistake
Filter is added in 500ml Sabouraud dextrose fluid nutrient mediums, mixing, and 30~35 DEG C are cultivated 3~5 days, and culture is obtained；It takes above-mentioned
In on Sabouraud glucose agar tablet, 30~35 DEG C are cultivated 24~48 hours for culture streak inoculation；If not having on tablet
It is feminine gender to have bacterium colony or qualification result, and Candida albicans is not detected in judgement negative control group.
The preparation method of staphylococcus aureus bacteria suspension is learnt from else's experience 30~35 DEG C and is cultivated 18~24 hours in step (1)
Every 1ml is made containing the bacterium that bacterium number is 50~100cfu with 0.9% aseptic sodium chloride solution in staphylococcus aureus fresh cultured object
Suspension.
The preparation method of pseudomonas aeruginosa bacteria suspension is 30~35 DEG C of copper for cultivating 18~24 hours of learning from else's experience in step (1)
It is outstanding containing the bacterium that bacterium number is 50~100cfu that every 1ml is made with 0.9% aseptic sodium chloride solution in green pseudomonad fresh cultured object
Liquid.
The preparation method of bacillus subtilis bacteria suspension is 30~35 DEG C of cultures, 18~24 hours withered of learning from else's experience in step (1)
It is outstanding containing the bacterium that bacterium number is 50~100cfu that every 1ml is made with 0.9% aseptic sodium chloride solution in careless bacillus fresh cultured object
Liquid.
The preparation method of Candida albicans bacteria suspension is 20~25 DEG C of Candida albicans for cultivating 2~3 days of learning from else's experience in step (1)
Every 1ml is made containing the bacteria suspension that bacterium number is 50~100cfu with 0.9% aseptic sodium chloride solution in bacterium fresh cultured object.
In step (1) preparation method of aspergillus niger spore suspension be learn from else's experience 20~25 DEG C culture 5~7 days aspergillus niger it is new
Fresh culture adds 3~5ml to contain 0.9% aseptic sodium chloride solution of 0.05% polyoxyethylene sorbitan monoleate, spore is eluted, and spore is sucked out
In suspension to sterile test tube, it is outstanding containing the spore that spore count is 50~100cfu that every 1ml is made with 0.9% aseptic sodium chloride solution
Liquid.
Compared with prior art, the present invention having the advantages that：
Inventive samples processing can preferably eliminate Nifuratel, reduce biocidal property, and operating process is simple, pollution it is several
Rate is small, prevents the appearance of sample false negative in checkout procedure, patient medication safe.
Specific implementation mode
The present invention is described further with reference to embodiments.
Embodiment 1
(1) prepared by bacterium solution
1. staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis
Learn from else's experience 30~35 DEG C culture 18~24 hours pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis
Every 1ml is made containing the bacteria suspension that bacterium number is 50~100cfu with 0.9% aseptic sodium chloride solution in fresh cultured object.
2. Candida albicans
It learns from else's experience 20~25 DEG C of cultures, 2~3 days Candida albicans fresh cultured objects, with 0.9% aseptic sodium chloride solution system
At every 1ml containing the bacteria suspension that bacterium number is 50~100cfu.
3. aspergillus niger
Learn from else's experience 20~25 DEG C culture 5~7 days aspergillus niger fresh cultured object, add 3~5ml containing 0.05% (ml/ml) gather
0.9% aseptic sodium chloride solution of sorb ester 80, spore is eluted, and is sucked out in spore suspension to sterile test tube, sterile with 0.9%
Every 1ml is made containing the spore suspension that spore count is 50~100cfu in sodium chloride solution.
(2) prepared by test liquid
Nifuratel nystatin expandable vaginal soft capsule test sample 10g is weighed, is shredded and is placed in sterile homogenizing bag with sterile scissors,
43 DEG C of sterile isopropyl myristates for adding 80ml are patted 1min with homogenizer, are poured into sterile triangular flask, capsule shells stay in nothing
43 DEG C of pH7.0 sterile NaCls-peptone buffer agents of addition 50ml are rubbed broken with hand handle capsule shells in bacterium homogenizing bag, then pour into three
After being shaked in the bottle of angle, 43 DEG C of pH7.0 sterile NaCls-peptone buffer agent 350ml are added and shake standing 5 minutes, with sterile note
Emitter draws upper layer isopropyl myristate and the solution of test sample mixing discards, then adds sterile digestion instrument needle with asepsis injector
The layer 240-260ml that fetch water is moved into sterile triangular flask, as 1：40 test liquid；
(3) aerobic bacteria, yeast and mold sum measurement
1. test group：Take 1:40 test liquid 1ml set 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
It in pH7.0 sterile NaCls-peptone buffer agent 500ml, handles through membrane-filter procedure, is separately added into when last time filters
Staphylococcus aureus bacteria suspension, pseudomonas aeruginosa bacteria suspension, bacillus subtilis bacteria suspension, Candida albicans bacteria suspension and
Aspergillus niger spore suspension removes filter membrane and is affixed on to set in 30-35 DEG C of incubator on pancreas junket soya peptone agar medium and cultivates 3 days, counts
Number the results are shown in Table 1；In addition the filter membrane for being separately added into Candida albicans bacteria suspension and aspergillus niger spore suspension is affixed on Sharpe Portugal again
It sets in 20-25 DEG C of incubator and cultivates 5 days on grape sugar agar medium, count results are shown in Table 5；
2. bacterium solution group：It takes pH7.0 sterile NaCls-peptone buffer agent to replace test liquid, is operated by test group and bacterium is added
Liquid measures the bacterium number of added bacterium solution；Bacterium number on pancreas junket soy agar culture medium is shown in Table 3, Sabouraud glucose agar
On bacterium number be shown in Table 6.
3. test sample group：The test liquid prepared is taken, bacterium solution is replaced with pH7.0 sterile NaCls-peptone buffer agent,
He operates with test group；It sets in 30-35 DEG C of incubator and cultivates 5 days on pancreas junket soya peptone agar medium, count results are shown in Table 2；
It sets in 20-25 DEG C of incubator and cultivates 7 days on Sabouraud glucose agar, count results are shown in Table 2；
4. culture after carry out aerobic bacteria, yeast and mold sum measurement,
Test group clump count subtracts the value of test sample group clump count and the ratio of bacterium solution group clump count in 0.5~2 range
It is as qualified；Measurement result is shown in Table 4, table 7.Aerobic bacteria, yeast and mold ratio in 0.5~2 range, detection for examination
Product are qualified.
1 aerobic bacteria test group result (unit of table：cfu)
2 test sample group result (unit of table：cfu)
| Aerobic bacteria (1:40) | Yeast and mold (1:40) |
| 0 | 0 |
3 aerobic bacteria bacterium solution group result (unit of table：cfu)
4 aerobic bacteria ratio of table
5 yeast and mold test group result (unit of table：cfu)
| Candida albicans | Aspergillus niger |
| 71 | 51 |
6 yeast and mold bacterium solution group result (unit of table：cfu)
| Candida albicans | Aspergillus niger |
| 70 | 49 |
7 yeast and mold ratio of table
| Candida albicans | Aspergillus niger |
| 1.01 | 1.04 |
(4) inspection of bacterium is controlled
1. the inspection of staphylococcus aureus
Test sample group：Take 1：40 test liquid 40ml are added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 100mlTSB culture mediums is added,
Mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation in mannitol sodium chloride agar
On culture medium flat plate, 30~35 DEG C are cultivated 18~72 hours；If it is feminine gender not have bacterium colony or qualification result on tablet, judgement is for examination
Staphylococcus aureus is not detected in product；
Positive controls：Take 1：40 test liquid 40ml are added to 43 DEG C and contain 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
PH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter all filtering be added 100mlTSB culture mediums,
Staphylococcus aureus mixing is added, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation
In on mannitol sodium chloride agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If there is no bacterium colony or identification on tablet
As a result it is feminine gender, staphylococcus aureus is not detected in judgement positive controls；
Negative control group：It takes pH7.0 sterile NaCls-peptone buffer agent 40ml to be added to 43 DEG C and contains 3% polysorbate
80 and 0.3% soybean lecithin pH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter whole mistake
100mlTSB culture mediums are added in filter, and mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Above-mentioned culture is taken to cross
It is inoculated on mannitol sodium chloride agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If do not have on tablet bacterium colony or
Qualification result is feminine gender, and staphylococcus aureus is not detected in judgement negative control group；
Staphylococcus aureus inspection result is shown in Table 8.
2. the inspection of pseudomonas aeruginosa
Test sample group：Take 1：40 test liquid 40ml are added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 100mlTSB culture mediums is added,
Mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation in cetyltrimethylammonium
On ammonium agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If it is feminine gender not have bacterium colony or qualification result on tablet, sentence
Determine test sample and pseudomonas aeruginosa is not detected；
Positive controls：Take 1：40 test liquid 40ml are added to 43 DEG C and contain 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
PH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter all filter, be added 100mlTSB culture mediums
In, pseudomonas aeruginosa mixing is added, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Above-mentioned culture scribing line is taken to connect
In on cetab agar medium tablet, 30~35 DEG C are cultivated 18~72 hours kind；If there is no bacterium on tablet
It falls or qualification result is feminine gender, pseudomonas aeruginosa is not detected in judgement positive controls；
Negative control group：It takes pH7.0 sterile NaCls-peptone buffer agent 40ml to be added to 43 DEG C and contains 3% polysorbate
80 and 0.3% soybean lecithin pH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter whole mistake
Filter is added in 100mlTSB culture mediums, mixing, and 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Above-mentioned culture is taken to draw
Line is inoculated on cetab agar medium tablet, and 30~35 DEG C are cultivated 18~72 hours；If not having on tablet
It is feminine gender to have bacterium colony or qualification result, and pseudomonas aeruginosa is not detected in judgement negative control group；
Pseudomonas aeruginosa inspection result is shown in Table 9.
3. the inspection of Candida albicans
Test sample group：Test liquid 40ml is taken to be added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 500ml Sabouraud dextroses are added
In fluid nutrient medium, mixing, 30~35 DEG C are cultivated 3~5 days, and culture is obtained；Take above-mentioned culture streak inoculation in Sharpe Portugal
On grape sugar agar medium tablet, 30~35 DEG C are cultivated 24~48 hours；If it is feminine gender not have bacterium colony or qualification result on tablet,
Candida albicans is not detected in judgement test sample；
Positive controls：Test liquid 40ml is taken to be added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 500ml Sabouraud dextroses are added
In fluid nutrient medium, Candida albicans mixing is added, 30~35 DEG C are cultivated 3~5 days, and culture is obtained；Above-mentioned culture is taken to draw
Line is inoculated on Sabouraud glucose agar tablet, and 30~35 DEG C are cultivated 24~48 hours；If do not have on tablet bacterium colony or
Qualification result is feminine gender, and Candida albicans is not detected in judgement positive controls；
Negative control group：It takes pH7.0 sterile NaCls-peptone buffer agent 40ml to be added to 43 DEG C and contains 3% polysorbate
80 and 0.3% soybean lecithin pH7.0 sterile NaCls-peptone buffer agent 500ml in, with membrane filter whole mistake
Filter is added in 500ml Sabouraud dextrose fluid nutrient mediums, mixing, and 30~35 DEG C are cultivated 3~5 days, and culture is obtained；It takes above-mentioned
In on Sabouraud glucose agar tablet, 30~35 DEG C are cultivated 24~48 hours for culture streak inoculation；If not having on tablet
It is feminine gender to have bacterium colony or qualification result, and Candida albicans is not detected in judgement negative control group.
Candida albicans inspection result is shown in Table 10.
8 staphylococcus aureus inspection result of table
| Test sample group | Positive controls | Negative control group |
| - | + | - |
9 pseudomonas aeruginosa inspection result of table
| Test sample group | Positive controls | Negative control group |
| - | + | - |
10 Candida albicans inspection result of table
| Test sample group | Positive controls | Negative control group |
| - | + | - |
Claims (6)
1. a kind of microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule, it is characterised in that steps are as follows：
(1) prepared by bacterium solution
Staphylococcus aureus bacteria suspension, pseudomonas aeruginosa bacteria suspension, bacillus subtilis bacteria suspension, white is prepared respectively to read
Pearl bacterium bacteria suspension and aspergillus niger spore suspension；
(2) prepared by test liquid
Nifuratel nystatin expandable vaginal soft capsule test sample 10g is weighed, is shredded and is placed in sterile homogenizing bag with sterile scissors, added
43 DEG C of sterile isopropyl myristates of 80ml are patted 1min with homogenizer, are poured into sterile triangular flask, capsule shells stay in sterile
43 DEG C of pH7.0 sterile NaCls-peptone buffer agents of addition 50ml are rubbed broken with hand handle capsule shells in homogenizing bag, then pour into triangle
After being shaked in bottle, 43 DEG C of pH7.0 sterile NaCls-peptone buffer agent 350ml are added and shake standing 5 minutes, use aseptic injection
Device draws upper layer isopropyl myristate and the solution of test sample mixing discards, then adds sterile digestion instrument needle to take with asepsis injector
Water layer 240-260ml is moved into sterile triangular flask, as 1：40 test liquid；
(3) aerobic bacteria, yeast and mold sum measurement
1. test group：Take 1:40 test liquid 1ml, set 43 DEG C of pH7.0 containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin without
It in bacterium sodium chloride-peptone buffer agent 500ml, is handled through membrane-filter procedure, golden yellow is separately added into when last time filters
Staphylococcus bacteria suspension, pseudomonas aeruginosa bacteria suspension, bacillus subtilis bacteria suspension, Candida albicans bacteria suspension and aspergillus niger
Spore suspension removes filter membrane and is affixed on to set in 30-35 DEG C of incubator on pancreas junket soya peptone agar medium and cultivates 3 days；In addition again will
The filter membrane for being separately added into Candida albicans bacteria suspension and aspergillus niger spore suspension is affixed on Sabouraud glucose agar and sets 20-
It is cultivated 5 days in 25 DEG C of incubators；
2. bacterium solution group：It takes pH7.0 sterile NaCls-peptone buffer agent to replace test liquid, is operated by test group and bacterium solution is added, survey
The bacterium number of fixed added bacterium solution；
3. test sample group：The test liquid prepared is taken, bacterium solution is replaced with pH7.0 sterile NaCls-peptone buffer agent, other are same
Test group operates；It sets in 30-35 DEG C of incubator and cultivates 5 days on pancreas junket soya peptone agar medium；Sabouraud's dextrose agar culture
It sets in 20-25 DEG C of incubator and cultivates 7 days on base；
4. culture after carry out aerobic bacteria, yeast and mold sum measurement,
Test group clump count subtracts the value of test sample group clump count
It is qualified；
(4) inspection of bacterium is controlled
1. the inspection of staphylococcus aureus
Test sample group：Take 1：40 test liquid 40ml are added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 100mlTSB culture mediums is added,
Mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation in mannitol sodium chloride agar
On culture medium flat plate, 30~35 DEG C are cultivated 18~72 hours；If it is feminine gender not have bacterium colony or qualification result on tablet, judgement is for examination
Staphylococcus aureus is not detected in product；
Positive controls：Take 1：40 test liquid 40ml are added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, with membrane filter, all 100mlTSB culture mediums are added in filtering, add
Enter staphylococcus aureus mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation in
On mannitol sodium chloride agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If there is no bacterium colony or identification knot on tablet
Fruit is feminine gender, and staphylococcus aureus is not detected in judgement positive controls；
Negative control group：Take pH7.0 sterile NaCls-peptone buffer agent 40ml be added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and
In pH7.0 sterile NaCls-peptone buffer agent 500ml of 0.3% soybean lecithin, is all filtered, added with membrane filter
Enter 100mlTSB culture mediums, mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation
In on mannitol sodium chloride agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If there is no bacterium colony or identification on tablet
As a result it is feminine gender, staphylococcus aureus is not detected in judgement negative control group；
2. the inspection of pseudomonas aeruginosa
Test sample group：Take 1：40 test liquid 40ml are added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 100mlTSB culture mediums is added,
Mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Take above-mentioned culture streak inoculation in cetyltrimethylammonium
On ammonium agar medium tablet, 30~35 DEG C are cultivated 18~72 hours；If it is feminine gender not have bacterium colony or qualification result on tablet, sentence
Determine test sample and pseudomonas aeruginosa is not detected；
Positive controls：Take 1：40 test liquid 40ml are added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In pH7.0 sterile NaCls-peptone buffer agent 500ml, is all filtered with membrane filter, 100mlTSB culture mediums are added
In, pseudomonas aeruginosa mixing is added, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Above-mentioned culture scribing line is taken to connect
In on cetab agar medium tablet, 30~35 DEG C are cultivated 18~72 hours kind；If there is no bacterium on tablet
It falls or qualification result is feminine gender, pseudomonas aeruginosa is not detected in judgement positive controls；
Negative control group：Take pH7.0 sterile NaCls-peptone buffer agent 40ml be added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and
In pH7.0 sterile NaCls-peptone buffer agent 500ml of 0.3% soybean lecithin, is all filtered, added with membrane filter
Enter in 100mlTSB culture mediums, mixing, 30~35 DEG C are cultivated 18~24 hours, and culture is obtained；Above-mentioned culture scribing line is taken to connect
In on cetab agar medium tablet, 30~35 DEG C are cultivated 18~72 hours kind；If there is no bacterium on tablet
It falls or qualification result is feminine gender, pseudomonas aeruginosa is not detected in judgement negative control group；
3. the inspection of Candida albicans
Test sample group：Take test liquid 40ml be added to 43 DEG C of pH7.0 containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin without
In bacterium sodium chloride-peptone buffer agent 500ml, is all filtered with membrane filter, 500ml Sabouraud dextrose Liquid Cultures are added
In base, mixing, 30~35 DEG C are cultivated 3~5 days, and culture is obtained；Take above-mentioned culture streak inoculation in Sabouraud's dextrose agar
On culture medium flat plate, 30~35 DEG C are cultivated 24~48 hours；If it is feminine gender not have bacterium colony or qualification result on tablet, judgement is for examination
Candida albicans is not detected in product；
Positive controls：Test liquid 40ml is taken to be added to 43 DEG C of pH7.0 for containing 3% polyoxyethylene sorbitan monoleate and 0.3% soybean lecithin
In sterile NaCl-peptone buffer agent 500ml, is all filtered with membrane filter, the training of 500ml Sabouraud dextrose liquid is added
It supports in base, Candida albicans mixing is added, 30~35 DEG C are cultivated 3~5 days, and culture is obtained；Take above-mentioned culture streak inoculation
In on Sabouraud glucose agar tablet, 30~35 DEG C are cultivated 24~48 hours；If there is no bacterium colony or identification knot on tablet
Fruit is feminine gender, and Candida albicans is not detected in judgement positive controls；
Negative control group：Take pH7.0 sterile NaCls-peptone buffer agent 40ml be added to 43 DEG C containing 3% polyoxyethylene sorbitan monoleate and
In pH7.0 sterile NaCls-peptone buffer agent 500ml of 0.3% soybean lecithin, is all filtered, added with membrane filter
Enter in 500ml Sabouraud dextrose fluid nutrient mediums, mixing, 30~35 DEG C are cultivated 3~5 days, and culture is obtained；Take above-mentioned culture
In on Sabouraud glucose agar tablet, 30~35 DEG C are cultivated 24~48 hours for streak inoculation；If there is no bacterium colony on tablet
Or qualification result is feminine gender, Candida albicans is not detected in judgement negative control group.
2. the microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule according to claim 1, feature
It is that the preparation method of staphylococcus aureus bacteria suspension in step (1) is 30~35 DEG C of cultures, 18~24 hours golden yellow of learning from else's experience
It is outstanding containing the bacterium that bacterium number is 50~100cfu that every 1ml is made with 0.9% aseptic sodium chloride solution in color staphylococcus fresh cultured object
Liquid.
3. the microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule according to claim 1, feature
It is that the preparation method of pseudomonas aeruginosa bacteria suspension in step (1) is 30~35 DEG C of verdigris for cultivating 18~24 hours vacations of learning from else's experience
Every 1ml is made containing the bacteria suspension that bacterium number is 50~100cfu with 0.9% aseptic sodium chloride solution in monad fresh cultured object.
4. the microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule according to claim 1, feature
It is that the preparation method of bacillus subtilis bacteria suspension in step (1) is 30~35 DEG C of withered grass buds for cultivating 18~24 hours of learning from else's experience
Every 1ml is made containing the bacteria suspension that bacterium number is 50~100cfu with 0.9% aseptic sodium chloride solution in spore bacillus fresh cultured object.
5. the microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule according to claim 1, feature
Be the preparation method of Candida albicans bacteria suspension in step (1) be learn from else's experience 20~25 DEG C culture 2~3 days Candida albicans it is new
Every 1ml is made containing the bacteria suspension that bacterium number is 50~100cfu with 0.9% aseptic sodium chloride solution in fresh culture.
6. the microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule according to claim 1, feature
Be the preparation method of aspergillus niger spore suspension in step (1) be learn from else's experience 20~25 DEG C culture 5~7 days aspergillus niger fresh training
Object is supported, adds 3~5ml to contain 0.9% aseptic sodium chloride solution of 0.05% polyoxyethylene sorbitan monoleate, spore is eluted, spore suspension is sucked out
To sterile test tube, every 1ml is made containing the spore suspension that spore count is 50~100cfu with 0.9% aseptic sodium chloride solution.
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