CN108048524A - The detection method of microbial limit in a kind of injection activated carbon - Google Patents
The detection method of microbial limit in a kind of injection activated carbon Download PDFInfo
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Abstract
The invention discloses a kind of detection methods of microbial limit in injection activated carbon, state detection method and include the following steps：(1) prepared by microbial inoculum, test liquid；(2) microbial inoculum test group is handled；(3) microbial inoculum test liquid control group is handled；(4) diluent control group is handled；(5) microbial inoculum group is handled；(6) the bacterium colony rate of recovery calculates.Detection method is convenient to carry out, and testing result is accurate, reliable.
Description
Technical field
The present invention relates to microbial limit detection technique field, more particularly, to micro- life in a kind of activated carbon for injection
The detection method of object limit.
Background technology
Activated carbon is auxiliary material more common in injection production.There are many micropore inside activated carbon, there is very big activity
Surface.Have the effects that adsorb heat source, decoloration, drainage.《Chinese Pharmacopoeia》The bright spot of version pharmaceutic adjuvant standard maximum in 2015 it
The reason for one is exactly the standard for dramatically increasing injection pharmaceutic adjuvant, increase injection pharmaceutic adjuvant standard is recent domestic
It is heard of during the key event triggered due to pharmaceutic adjuvant quality problems, it is high that these events, which remind our injection pharmaceutic adjuvants,
The pharmaceutic adjuvant kind of risk, should individually set up standard.《Chinese Pharmacopoeia》Version in 2015 adds activated carbon (injection) mark
Standard, activated carbon is the important way of industrial great transfusion preparation removal heat source at present, and China is an infusion big country, and China is annual
Because unsafe injection causes dead number to remain high.The quality of activated carbon is most important to ensureing injection safe medication.
The content of the invention
In view of the above-mentioned problems existing in the prior art, the applicant provides microorganisms in a kind of injection activated carbon
The detection method of limit.Detection method is convenient to carry out, and testing result is accurate, reliable.
Technical scheme is as follows：
The detection method of microbial limit, the detection method include the following steps in a kind of injection activated carbon：
(1) prepared by microbial inoculum, test liquid；
1. pancreas junket soya peptone agar medium, pancreas junket soya peptone fluid nutrient medium, Sabouraud's dextrose agar training are prepared respectively
Support base, Sabouraud dextrose fluid nutrient medium；The culture medium source is three medicine scientific and technological development company of Beijing, and condition of storage is normal
At temperature is dry；
It is 2. the fresh cultured object of staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa is big in pancreas junket respectively
In beans peptone fluid nutrient medium, when 30~35 DEG C of cultures 18~24 are small；By the fresh cultured object of Candida albicans with 0.9% sterile chlorine
Change sodium solution and the bacteria suspension that every 1ml is less than 100cfu containing bacterium number is respectively prepared, go in Sabouraud dextrose fluid nutrient medium, 20~
When 25 DEG C of cultures 24~48 are small；On the fresh cultured object of inoculated aspergillus niger to Sabouraud's dextrose agar slant medium, 20~25
DEG C culture 5~7 days；
3. 2. staphylococcus aureus that step is cultivated, bacillus subtilis, pseudomonas aeruginosa, Candida albicans
Every 1ml is made containing bacteria suspension of the bacterium number less than 100cfu with 0.9% aseptic sodium chloride solution respectively in cultured products；It is 2. trained in step
Foster aspergillus niger cultured products add in the aseptic sodium chloride solution of the 0.9wt% of 3~5ml polysorbate 80s containing 0.05v%,
Spore is eluted, is washed out in spore suspension to sterile test tube, with the sterile chlorine of the 0.9wt% of the polysorbate 80 containing 0.05v%
Change sodium solution and the spore suspension that every 1ml is less than 100cfu containing spore count is made；
4. by 10g injection activated carbon, sterile NaCl-peptone buffer agent of 1000ml pH 7.0 is added in, is shaken,
It is made 1：100 test liquid.
(2) microbial inoculum test group is handled；
1. staphylococcus aureus test group：100ml test liquids are taken, add in 1ml (10-5) staphylococcus aureus bacterium hangs
Liquid, mixing pipette 1ml immediately, are placed in the sterilized petri dishes of diameter 90mm, and 15~20ml of injection temperature is no more than 45 DEG C of fusings
Pancreas junket soya peptone agar medium, mixing, solidification, it is parallel preparation two pieces of wares, 30~35 DEG C, be inverted culture 3 days；
2. bacillus subtilis test group：100ml test liquids are taken, add in 1ml (10-3) bacillus subtilis bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is placed in the sterilized petri dishes of diameter 90mm, and 15~20ml of injection temperature is no more than the pancreas junket of 45 DEG C of fusings
Soya peptone agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 30~35 DEG C；
3. pseudomonas aeruginosa test group：100ml test liquids are taken, add in 1ml (10-4) pseudomonas aeruginosa bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is put directly in the sterilized petri dishes of footpath 90mm, and 15~20ml of injection temperature is no more than the pancreas junket of 45 DEG C of fusings
Soya peptone agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 30~35 DEG C；
4. Candida albicans test group：100ml test liquids are taken, add in 1ml (10-2) Candida albicans bacteria suspension, mixing, stand
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the Sabouraud dextrose of 45 DEG C of fusings
Agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 20~25 DEG C；
5. aspergillus niger test group：100ml test liquids are taken, add in 1ml (10-2) black-koji mould suspension, mixing pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, and 15~20ml of injection temperature is no more than the Sabouraud's dextrose agar training of 45 DEG C of fusings
Base is supported, mixing, solidification, two pieces of wares of parallel preparation, 20~25 DEG C, inversion is cultivated 5 days.
(3) microbial inoculum test liquid control group is handled；
It 1. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than 45 DEG C of fusings
Pancreas junket soya peptone agar medium, mixing, solidification, 30~35 DEG C, be inverted culture 3 days；
It 2. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than 45 DEG C of fusings
Sabouraud glucose agar, mixing, solidification, 20~25 DEG C, be inverted culture 5 days.
(4) diluent control group is handled；
1. staphylococcus aureus diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken,
Add in 1ml (10-5) staphylococcus aureus bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately,
Inject the pancreas junket soya peptone agar medium that 15~20ml temperature is no more than 45 DEG C of fusings, mixing, solidification, parallel two pieces of preparation
Ware, is inverted culture 3 days by 30~35 DEG C；
2. bacillus subtilis diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added
Enter 1ml (10-3) bacillus subtilis bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects
15~20ml temperature is no more than the pancreas junket soya peptone agar medium of 45 DEG C of fusings, and mixing solidifies, two pieces of wares of parallel preparation, and 30
~35 DEG C, it is inverted culture 3 days；
3. pseudomonas aeruginosa diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added
Enter 1ml (10-4) pseudomonas aeruginosa bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects
15~20ml temperature is no more than the pancreas junket soya peptone agar medium of 45 DEG C of fusings, and mixing solidifies, two pieces of wares of parallel preparation, and 30
~35 DEG C, it is inverted culture 3 days；
4. Candida albicans diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added in
1ml(10-2) Candida albicans bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, and injection 15~
20ml temperature is no more than the Sabouraud glucose agar of 45 DEG C of fusings, and mixing solidifies, two pieces of wares of parallel preparation, and 20~25
DEG C, it is inverted culture 5 days；
5. aspergillus niger diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, adds in 1ml
(10-2) black-koji mould suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects 15~20ml temperature
No more than the Sabouraud glucose agar of 45 DEG C of fusings, mixing, solidification, two pieces of wares of parallel preparation, 20~25 DEG C, inversion is trained
It supports 5 days.
(5) microbial inoculum group is handled；
1. staphylococcus aureus bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-5) golden yellow
Color staphylococcus bacteria suspension, mixing are made 10-7Staphylococcus aureus bacteria suspension (being less than 100cfu containing bacterium number per 1ml) is immediately
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the pancreas junket soya peptone of 45 DEG C of fusings
Agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 30~35 DEG C；
2. bacillus subtilis bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-3) withered grass bud
Spore bacillus bacteria suspension, mixing are made 10-5Bacillus subtilis bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, and 15~20ml of injection temperature is no more than the pancreas junket soya peptone agar training of 45 DEG C of fusings
Base is supported, mixing, solidification, two pieces of wares of parallel preparation, 30~35 DEG C, inversion is cultivated 3 days；
3. pseudomonas aeruginosa bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-4) verdigris vacation
Monad bacteria suspension, mixing are made 10-6Pseudomonas aeruginosa bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, and 15~20ml of injection temperature is no more than the pancreas junket soya peptone agar training of 45 DEG C of fusings
Base is supported, mixing, solidification, two pieces of wares of parallel preparation, 30~35 DEG C, inversion is cultivated 3 days；
4. Candida albicans bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-2) Candida albicans
Bacteria suspension, mixing are made 10-4Candida albicans bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in straight
In the sterilized petri dishes of footpath 90mm, injection 15~20ml temperature is no more than the Sabouraud glucose agars of 45 DEG C of fusings, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 20~25 DEG C；
5. aspergillus niger bacterium solution group：100ml0.9% aseptic sodium chloride solutions are taken, add in 1ml (10-2) black-koji mould suspension, it mixes
It is even, it is made 10-4Black-koji mould suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in the sterile flat of diameter 90mm
In ware, 15~20ml of injection temperature is no more than the Sabouraud glucose agar of 45 DEG C of fusings, mixing, solidification, parallel preparation
Two pieces of wares, are inverted culture 5 days by 20~25 DEG C.
(6) the bacterium colony rate of recovery calculates；
The average colony number of the bacterium colony rate of recovery of diluent control group=diluent control group average colony number/bacterium solution group ×
100%；
The bacterium colony rate of recovery of microbial inoculum test group=(average colony number of test group-test liquid control group is averaged
Clump count)/bacterium solution group average colony number × 100%.
The bacterium solution test group, bacterium solution test liquid control group, diluent control group, bacterium solution group carry out respectively 3 times it is independent
Parallel laboratory test.
The present invention is beneficial to be had technical effect that：
Detection method is convenient to carry out, and testing result is accurate, reliable.
Specific embodiment
With reference to embodiment, the present invention is specifically described.
Embodiment 1
The detection method of microbial limit, the detection method include the following steps in a kind of injection activated carbon：
(1) prepared by microbial inoculum, test liquid；
1. pancreas junket soya peptone agar medium, pancreas junket soya peptone fluid nutrient medium, Sabouraud's dextrose agar training are prepared respectively
Support base, Sabouraud dextrose fluid nutrient medium；The culture medium source is three medicine scientific and technological development company of Beijing, and condition of storage is normal
At temperature is dry；
It is 2. the fresh cultured object of staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa is big in pancreas junket respectively
In beans peptone fluid nutrient medium, when 33 DEG C of cultures 24 are small；By the fresh cultured object of Candida albicans with 0.9% aseptic sodium chloride solution
The bacteria suspension that every 1ml is less than 100cfu containing bacterium number is respectively prepared, goes in Sabouraud dextrose fluid nutrient medium, 23 DEG C of cultures 48 are small
When；On the fresh cultured object of inoculated aspergillus niger to Sabouraud's dextrose agar slant medium, 23 DEG C are cultivated 7 days；
3. 2. staphylococcus aureus that step is cultivated, bacillus subtilis, pseudomonas aeruginosa, Candida albicans
Every 1ml is made containing bacteria suspension of the bacterium number less than 100cfu with 0.9% aseptic sodium chloride solution respectively in cultured products；It is 2. trained in step
Foster aspergillus niger cultured products add in the aseptic sodium chloride solution of the 0.9wt% of 5ml polysorbate 80s containing 0.05v%, by spore
Son elution, washes out in spore suspension to sterile test tube, with the sterile NaCl of the 0.9wt% of the polysorbate 80 containing 0.05v%
The spore suspension that every 1ml is less than 100cfu containing spore count is made in solution；
4. by 10g injection activated carbon, sterile NaCl-peptone buffer agent of 1000ml pH 7.0 is added in, is shaken,
It is made 1：100 test liquid.
(2) microbial inoculum test group is handled；
1. staphylococcus aureus test group：100ml test liquids are taken, add in 1ml (10-5) staphylococcus aureus bacterium hangs
Liquid, mixing pipette 1ml immediately, are placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone of injection 20ml 42 DEG C of fusings of temperature
Agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
2. bacillus subtilis test group：100ml test liquids are taken, add in 1ml (10-3) bacillus subtilis bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar of injection 20ml 42 DEG C of fusings of temperature
Culture medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
3. pseudomonas aeruginosa test group：100ml test liquids are taken, add in 1ml (10-4) pseudomonas aeruginosa bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is put directly in the sterilized petri dishes of footpath 90mm, the pancreas junket soya peptone agar of injection 20ml 42 DEG C of fusings of temperature
Culture medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
4. Candida albicans test group：100ml test liquids are taken, add in 1ml (10-2) Candida albicans bacteria suspension, mixing, stand
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, the Sabouraud's dextrose agar culture of injection 20ml 42 DEG C of fusings of temperature
Base, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C；
5. aspergillus niger test group：100ml test liquids are taken, add in 1ml (10-2) black-koji mould suspension, mixing pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C.
(3) microbial inoculum test liquid control group is handled；
It 1. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soybean of injection 20ml 42 DEG C of fusings of temperature
Peptone agar medium, mixing, solidification, are inverted culture 3 days by 33 DEG C；
It 2. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, the Sharpe grape of injection 20ml 42 DEG C of fusings of temperature
Sugared agar medium, mixing, solidification, are inverted culture 5 days by 23 DEG C.
(4) diluent control group is handled；
1. staphylococcus aureus diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken,
Add in 1ml (10-5) staphylococcus aureus bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately,
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature is injected, mixing, solidification is parallel to prepare two pieces of wares, 33 DEG C, is inverted
Culture 3 days；
2. bacillus subtilis diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added
Enter 1ml (10-3) bacillus subtilis bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture by 33 DEG C
3 days；
3. pseudomonas aeruginosa diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added
Enter 1ml (10-4) pseudomonas aeruginosa bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture by 33 DEG C
3 days；
4. Candida albicans diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added in
1ml(10-2) Candida albicans bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects 20ml temperature
The Sabouraud glucose agar of 42 DEG C of fusings of degree, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C；
5. aspergillus niger diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, adds in 1ml
(10-2) black-koji mould suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, 42 DEG C of injection 20ml temperature
The Sabouraud glucose agar of fusing, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C.
(5) microbial inoculum group is handled；
1. staphylococcus aureus bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-5) golden yellow
Color staphylococcus bacteria suspension, mixing are made 10-7Staphylococcus aureus bacteria suspension (being less than 100cfu containing bacterium number per 1ml) is immediately
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature,
Mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
2. bacillus subtilis bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-3) withered grass bud
Spore bacillus bacteria suspension, mixing are made 10-5Bacillus subtilis bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
3. pseudomonas aeruginosa bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-4) verdigris vacation
Monad bacteria suspension, mixing are made 10-6Pseudomonas aeruginosa bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
4. Candida albicans bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-2) Candida albicans
Bacteria suspension, mixing are made 10-4Candida albicans bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in straight
In the sterilized petri dishes of footpath 90mm, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing solidifies, parallel
Two pieces of wares are prepared, 23 DEG C, are inverted culture 5 days；
5. aspergillus niger bacterium solution group：100ml0.9% aseptic sodium chloride solutions are taken, add in 1ml (10-2) black-koji mould suspension, it mixes
It is even, it is made 10-4Black-koji mould suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in the sterile flat of diameter 90mm
In ware, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing, solidification, two pieces of wares of parallel preparation, 23
DEG C, it is inverted culture 5 days.
(6) the bacterium colony rate of recovery calculates；
The average colony number of the bacterium colony rate of recovery of diluent control group=diluent control group average colony number/bacterium solution group ×
100%；
The bacterium colony rate of recovery of microbial inoculum test group=(average colony number of test group-test liquid control group is averaged
Clump count)/bacterium solution group average colony number × 100%；
Test group bacterium colony rate of recovery result of calculation of the embodiment of the present invention is as shown in table 1.Diluent of the embodiment of the present invention compares
Group bacterium colony rate of recovery result of calculation is as shown in table 2.
Table 1
Table 2
The bacterium colony rate of recovery that the method for the present invention detects it can be seen from table 1,2 data meets 100% or so
The requirement of the rate of recovery 50%~200% in States Pharmacopoeia specifications.
Embodiment 2
The detection method of microbial limit, the detection method include the following steps in a kind of injection activated carbon：
(1) prepared by microbial inoculum, test liquid；
1. pancreas junket soya peptone agar medium, pancreas junket soya peptone fluid nutrient medium, Sabouraud's dextrose agar training are prepared respectively
Support base, Sabouraud dextrose fluid nutrient medium；The culture medium source is three medicine scientific and technological development company of Beijing, and condition of storage is normal
At temperature is dry；
It is 2. the fresh cultured object of staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa is big in pancreas junket respectively
In beans peptone fluid nutrient medium, when 33 DEG C of cultures 24 are small；By the fresh cultured object of Candida albicans with 0.9% aseptic sodium chloride solution
The bacteria suspension that every 1ml is less than 100cfu containing bacterium number is respectively prepared, goes in Sabouraud dextrose fluid nutrient medium, 23 DEG C of cultures 48 are small
When；On the fresh cultured object of inoculated aspergillus niger to Sabouraud's dextrose agar slant medium, 23 DEG C are cultivated 7 days；
3. 2. staphylococcus aureus that step is cultivated, bacillus subtilis, pseudomonas aeruginosa, Candida albicans
Every 1ml is made containing bacteria suspension of the bacterium number less than 100cfu with 0.9% aseptic sodium chloride solution respectively in cultured products；It is 2. trained in step
Foster aspergillus niger cultured products add in the aseptic sodium chloride solution of the 0.9wt% of 5ml polysorbate 80s containing 0.05v%, by spore
Son elution, washes out in spore suspension to sterile test tube, with the sterile NaCl of the 0.9wt% of the polysorbate 80 containing 0.05v%
The spore suspension that every 1ml is less than 100cfu containing spore count is made in solution；
4. by 10g injection activated carbon, sterile NaCl-peptone buffer agent of 1000ml pH 7.0 is added in, is shaken,
It is made 1：100 test liquid.
(2) microbial inoculum test group is handled；
1. staphylococcus aureus test group：100ml test liquids are taken, add in 1ml (10-5) staphylococcus aureus bacterium hangs
Liquid, mixing pipette 1ml immediately, are placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone of injection 20ml 42 DEG C of fusings of temperature
Agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
2. bacillus subtilis test group：100ml test liquids are taken, add in 1ml (10-3) bacillus subtilis bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar of injection 20ml 42 DEG C of fusings of temperature
Culture medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
3. pseudomonas aeruginosa test group：100ml test liquids are taken, add in 1ml (10-4) pseudomonas aeruginosa bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is put directly in the sterilized petri dishes of footpath 90mm, the pancreas junket soya peptone agar of injection 20ml 42 DEG C of fusings of temperature
Culture medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
4. Candida albicans test group：100ml test liquids are taken, add in 1ml (10-2) Candida albicans bacteria suspension, mixing, stand
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, the Sabouraud's dextrose agar culture of injection 20ml 42 DEG C of fusings of temperature
Base, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C；
5. aspergillus niger test group：100ml test liquids are taken, add in 1ml (10-2) black-koji mould suspension, mixing pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C.
(3) microbial inoculum test liquid control group is handled；
It 1. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soybean of injection 20ml 42 DEG C of fusings of temperature
Peptone agar medium, mixing, solidification, are inverted culture 3 days by 33 DEG C；
It 2. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, the Sharpe grape of injection 20ml 42 DEG C of fusings of temperature
Sugared agar medium, mixing, solidification, are inverted culture 5 days by 23 DEG C.
(4) diluent control group is handled；
1. staphylococcus aureus diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken,
Add in 1ml (10-5) staphylococcus aureus bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately,
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature is injected, mixing, solidification is parallel to prepare two pieces of wares, 33 DEG C, is inverted
Culture 3 days；
2. bacillus subtilis diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added
Enter 1ml (10-3) bacillus subtilis bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture by 33 DEG C
3 days；
3. pseudomonas aeruginosa diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added
Enter 1ml (10-4) pseudomonas aeruginosa bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture by 33 DEG C
3 days；
4. Candida albicans diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added in
1ml(10-2) Candida albicans bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects 20ml temperature
The Sabouraud glucose agar of 42 DEG C of fusings of degree, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C；
5. aspergillus niger diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, adds in 1ml
(10-2) black-koji mould suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, 42 DEG C of injection 20ml temperature
The Sabouraud glucose agar of fusing, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C.
(5) microbial inoculum group is handled；
1. staphylococcus aureus bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-5) golden yellow
Color staphylococcus bacteria suspension, mixing are made 10-7Staphylococcus aureus bacteria suspension (being less than 100cfu containing bacterium number per 1ml) is immediately
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature,
Mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
2. bacillus subtilis bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-3) withered grass bud
Spore bacillus bacteria suspension, mixing are made 10-5Bacillus subtilis bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
3. pseudomonas aeruginosa bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-4) verdigris vacation
Monad bacteria suspension, mixing are made 10-6Pseudomonas aeruginosa bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
4. Candida albicans bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-2) Candida albicans
Bacteria suspension, mixing are made 10-4Candida albicans bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in straight
In the sterilized petri dishes of footpath 90mm, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing solidifies, parallel
Two pieces of wares are prepared, 23 DEG C, are inverted culture 5 days；
5. aspergillus niger bacterium solution group：100ml0.9% aseptic sodium chloride solutions are taken, add in 1ml (10-2) black-koji mould suspension, it mixes
It is even, it is made 10-4Black-koji mould suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in the sterile flat of diameter 90mm
In ware, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing, solidification, two pieces of wares of parallel preparation, 23
DEG C, it is inverted culture 5 days.
(6) the bacterium colony rate of recovery calculates；
The average colony number of the bacterium colony rate of recovery of diluent control group=diluent control group average colony number/bacterium solution group ×
100%；
The bacterium colony rate of recovery of microbial inoculum test group=(average colony number of test group-test liquid control group is averaged
Clump count)/bacterium solution group average colony number × 100%；
Test group bacterium colony rate of recovery result of calculation of the embodiment of the present invention is as shown in table 3.Diluent of the embodiment of the present invention compares
Group bacterium colony rate of recovery result of calculation is as shown in table 4.
Table 3
Table 4
The bacterium colony rate of recovery that the method for the present invention detects it can be seen from table 3,4 data meets 100% or so
The requirement of the rate of recovery 50%~200% in States Pharmacopoeia specifications.
Embodiment 3
The detection method of microbial limit, the detection method include the following steps in a kind of injection activated carbon：
(1) prepared by microbial inoculum, test liquid；
1. pancreas junket soya peptone agar medium, pancreas junket soya peptone fluid nutrient medium, Sabouraud's dextrose agar training are prepared respectively
Support base, Sabouraud dextrose fluid nutrient medium；The culture medium source is three medicine scientific and technological development company of Beijing, and condition of storage is normal
At temperature is dry；
It is 2. the fresh cultured object of staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa is big in pancreas junket respectively
In beans peptone fluid nutrient medium, when 33 DEG C of cultures 24 are small；By the fresh cultured object of Candida albicans with 0.9% aseptic sodium chloride solution
The bacteria suspension that every 1ml is less than 100cfu containing bacterium number is respectively prepared, goes in Sabouraud dextrose fluid nutrient medium, 23 DEG C of cultures 48 are small
When；On the fresh cultured object of inoculated aspergillus niger to Sabouraud's dextrose agar slant medium, 23 DEG C are cultivated 7 days；
3. 2. staphylococcus aureus that step is cultivated, bacillus subtilis, pseudomonas aeruginosa, Candida albicans
Every 1ml is made containing bacteria suspension of the bacterium number less than 100cfu with 0.9% aseptic sodium chloride solution respectively in cultured products；It is 2. trained in step
Foster aspergillus niger cultured products add in the aseptic sodium chloride solution of the 0.9wt% of 5ml polysorbate 80s containing 0.05v%, by spore
Son elution, washes out in spore suspension to sterile test tube, with the sterile NaCl of the 0.9wt% of the polysorbate 80 containing 0.05v%
The spore suspension that every 1ml is less than 100cfu containing spore count is made in solution；
4. by 10g injection activated carbon, sterile NaCl-peptone buffer agent of 1000ml pH 7.0 is added in, is shaken,
It is made 1：100 test liquid.
(2) microbial inoculum test group is handled；
1. staphylococcus aureus test group：100ml test liquids are taken, add in 1ml (10-5) staphylococcus aureus bacterium hangs
Liquid, mixing pipette 1ml immediately, are placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone of injection 20ml 42 DEG C of fusings of temperature
Agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
2. bacillus subtilis test group：100ml test liquids are taken, add in 1ml (10-3) bacillus subtilis bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar of injection 20ml 42 DEG C of fusings of temperature
Culture medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
3. pseudomonas aeruginosa test group：100ml test liquids are taken, add in 1ml (10-4) pseudomonas aeruginosa bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is put directly in the sterilized petri dishes of footpath 90mm, the pancreas junket soya peptone agar of injection 20ml 42 DEG C of fusings of temperature
Culture medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
4. Candida albicans test group：100ml test liquids are taken, add in 1ml (10-2) Candida albicans bacteria suspension, mixing, stand
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, the Sabouraud's dextrose agar culture of injection 20ml 42 DEG C of fusings of temperature
Base, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C；
5. aspergillus niger test group：100ml test liquids are taken, add in 1ml (10-2) black-koji mould suspension, mixing pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C.
(3) microbial inoculum test liquid control group is handled；
It 1. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soybean of injection 20ml 42 DEG C of fusings of temperature
Peptone agar medium, mixing, solidification, are inverted culture 3 days by 33 DEG C；
It 2. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, the Sharpe grape of injection 20ml 42 DEG C of fusings of temperature
Sugared agar medium, mixing, solidification, are inverted culture 5 days by 23 DEG C.
(4) diluent control group is handled；
1. staphylococcus aureus diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken,
Add in 1ml (10-5) staphylococcus aureus bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately,
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature is injected, mixing, solidification is parallel to prepare two pieces of wares, 33 DEG C, is inverted
Culture 3 days；
2. bacillus subtilis diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added
Enter 1ml (10-3) bacillus subtilis bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture by 33 DEG C
3 days；
3. pseudomonas aeruginosa diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added
Enter 1ml (10-4) pseudomonas aeruginosa bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects
The pancreas junket soya peptone agar medium of 42 DEG C of fusings of 20ml temperature, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture by 33 DEG C
3 days；
4. Candida albicans diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added in
1ml(10-2) Candida albicans bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects 20ml temperature
The Sabouraud glucose agar of 42 DEG C of fusings of degree, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C；
5. aspergillus niger diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, adds in 1ml
(10-2) black-koji mould suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, 42 DEG C of injection 20ml temperature
The Sabouraud glucose agar of fusing, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 23 DEG C.
(5) microbial inoculum group is handled；
1. staphylococcus aureus bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-5) golden yellow
Color staphylococcus bacteria suspension, mixing are made 10-7Staphylococcus aureus bacteria suspension (being less than 100cfu containing bacterium number per 1ml) is immediately
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature,
Mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
2. bacillus subtilis bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-3) withered grass bud
Spore bacillus bacteria suspension, mixing are made 10-5Bacillus subtilis bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
3. pseudomonas aeruginosa bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-4) verdigris vacation
Monad bacteria suspension, mixing are made 10-6Pseudomonas aeruginosa bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, the pancreas junket soya peptone agar medium of injection 20ml 42 DEG C of fusings of temperature, mixing,
Solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 33 DEG C；
4. Candida albicans bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-2) Candida albicans
Bacteria suspension, mixing are made 10-4Candida albicans bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in straight
In the sterilized petri dishes of footpath 90mm, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing solidifies, parallel
Two pieces of wares are prepared, 23 DEG C, are inverted culture 5 days；
5. aspergillus niger bacterium solution group：100ml0.9% aseptic sodium chloride solutions are taken, add in 1ml (10-2) black-koji mould suspension, it mixes
It is even, it is made 10-4Black-koji mould suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in the sterile flat of diameter 90mm
In ware, the Sabouraud glucose agar of injection 20ml 42 DEG C of fusings of temperature, mixing, solidification, two pieces of wares of parallel preparation, 23
DEG C, it is inverted culture 5 days.
(6) the bacterium colony rate of recovery calculates；
The average colony number of the bacterium colony rate of recovery of diluent control group=diluent control group average colony number/bacterium solution group ×
100%；
The bacterium colony rate of recovery of microbial inoculum test group=(average colony number of test group-test liquid control group is averaged
Clump count)/bacterium solution group average colony number × 100%；
Test group bacterium colony rate of recovery result of calculation of the embodiment of the present invention is as shown in table 5.Diluent of the embodiment of the present invention compares
Group bacterium colony rate of recovery result of calculation is as shown in table 6.
Table 5
Table 6
The bacterium colony rate of recovery that the method for the present invention detects it can be seen from table 5,6 data meets 100% or so
The requirement of the rate of recovery 50%~200% in States Pharmacopoeia specifications.
Claims (9)
1. the detection method of microbial limit in a kind of injection activated carbon, it is characterised in that the detection method includes following step
Suddenly：
(1) prepared by microbial inoculum, test liquid；
(2) microbial inoculum test group is handled；
(3) microbial inoculum test liquid control group is handled；
(4) diluent control group is handled；
(5) microbial inoculum group is handled；
(6) the bacterium colony rate of recovery calculates.
2. detection method according to claim 1, it is characterised in that the detailed process of the step (1) is：
1. prepare respectively pancreas junket soya peptone agar medium, pancreas junket soya peptone fluid nutrient medium, Sabouraud glucose agar,
Sabouraud dextrose fluid nutrient medium；
2. by staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa fresh cultured object respectively in pancreas junket soya peptone
In fluid nutrient medium, when 30~35 DEG C of cultures 18~24 are small；By the fresh cultured object of Candida albicans with 0.9% sterile NaCl
The bacteria suspension that every 1ml is less than 100cfu containing bacterium number is respectively prepared in solution, goes in Sabouraud dextrose fluid nutrient medium, 20~25 DEG C
When culture 24~48 is small；On the fresh cultured object of inoculated aspergillus niger to Sabouraud's dextrose agar slant medium, 20~25 DEG C of trainings
It supports 5~7 days；
3. 2. staphylococcus aureus that step is cultivated, the culture of bacillus subtilis, pseudomonas aeruginosa, Candida albicans
Every 1ml is made containing bacteria suspension of the bacterium number less than 100cfu with 0.9% aseptic sodium chloride solution respectively in product；It is 2. cultivated in step
Aspergillus niger cultured products add in the aseptic sodium chloride solution of the 0.9wt% of 3~5ml polysorbate 80s containing 0.05v%, by spore
Elution is washed out in spore suspension to sterile test tube, and the sterile NaCl with the 0.9wt% of the polysorbate 80 containing 0.05v% is molten
The spore suspension that every 1ml is less than 100cfu containing spore count is made in liquid；
4. by 10g injection activated carbons, sterile NaCl-peptone buffer agent of 1000ml pH 7.0 is added in, shakes, is made
1：100 test liquid.
3. detection method according to claim 1, it is characterised in that the processing method of the step (2) is：
1. staphylococcus aureus test group：100ml test liquids are taken, add in 1ml (10-5) staphylococcus aureus bacteria suspension, it mixes
It is even, 1ml is pipetted immediately, is placed in the sterilized petri dishes of diameter 90mm, and 15~20ml of injection temperature is no more than the pancreas junket of 45 DEG C of fusings
Soya peptone agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 30~35 DEG C；
2. bacillus subtilis test group：100ml test liquids are taken, add in 1ml (10-3) bacillus subtilis bacteria suspension, mixing, stand
1ml is pipetted, is placed in the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the pancreas junket soya peptone of 45 DEG C of fusings
Agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 30~35 DEG C；
3. pseudomonas aeruginosa test group：100ml test liquids are taken, add in 1ml (10-4) pseudomonas aeruginosa bacteria suspension, mixing, stand
1ml is pipetted, is put directly in the sterilized petri dishes of footpath 90mm, 15~20ml of injection temperature is no more than the pancreas junket soya peptone of 45 DEG C of fusings
Agar medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 30~35 DEG C；
4. Candida albicans test group：100ml test liquids are taken, add in 1ml (10-2) Candida albicans bacteria suspension, mixing moves immediately
1ml is taken, is placed in the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the Sabouraud's dextrose agar of 45 DEG C of fusings
Culture medium, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 20~25 DEG C；
5. aspergillus niger test group：100ml test liquids are taken, add in 1ml (10-2) black-koji mould suspension, mixing pipettes 1ml, puts immediately
In the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the Sabouraud glucose agar of 45 DEG C of fusings,
Mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 20~25 DEG C.
4. detection method according to claim 1, it is characterised in that step (3) processing method is：
It 1. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the pancreas of 45 DEG C of fusings
Junket soya peptone agar medium, mixing, solidification, are inverted culture 3 days by 30~35 DEG C；
It 2. taking 1ml test liquids, is placed in the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the sand of 45 DEG C of fusings
Family name's glucose agar medium, mixing, solidification, are inverted culture 5 days by 20~25 DEG C.
5. detection method according to claim 1, it is characterised in that the processing method of the step (4) is：
1. staphylococcus aureus diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added in
1ml(10-5) staphylococcus aureus bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injection 15
~20ml temperature is no more than the pancreas junket soya peptone agar medium of 45 DEG C of fusings, and mixing solidifies, two pieces of wares of parallel preparation, 30~
35 DEG C, it is inverted culture 3 days；
2. bacillus subtilis diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added in
1ml(10-3) bacillus subtilis bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, and injection 15~
20ml temperature is no more than the pancreas junket soya peptone agar medium of 45 DEG C of fusings, and mixing solidifies, two pieces of wares of parallel preparation, and 30~35
DEG C, it is inverted culture 3 days；
3. pseudomonas aeruginosa diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, is added in
1ml(10-4) pseudomonas aeruginosa bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, and injection 15~
20ml temperature is no more than the pancreas junket soya peptone agar medium of 45 DEG C of fusings, and mixing solidifies, two pieces of wares of parallel preparation, and 30~35
DEG C, it is inverted culture 3 days；
4. Candida albicans diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, adds in 1ml
(10-2) Candida albicans bacteria suspension, mixing pipettes 1ml, is placed in the sterilized petri dishes of diameter 90mm immediately, injects 15~20ml
Temperature is no more than the Sabouraud glucose agar of 45 DEG C of fusings, and mixing, solidification is parallel to prepare two pieces of wares, 20~25 DEG C, falls
Put culture 5 days；
5. aspergillus niger diluent control group：Sterile NaCl-peptone buffer agent of 100ml pH 7.0 is taken, adds in 1ml (10-2)
Black-koji mould suspension, mixing pipette 1ml immediately, are placed in the sterilized petri dishes of diameter 90mm, and injection 15~20ml temperature is no more than
The Sabouraud glucose agar of 45 DEG C of fusings, mixing, solidification, two pieces of wares of parallel preparation, are inverted culture 5 days by 20~25 DEG C.
6. detection method according to claim 1, it is characterised in that the processing method of the step (5) is：
1. staphylococcus aureus bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-5) golden yellow grape
Coccus bacteria suspension, mixing are made 10-7Staphylococcus aureus bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes immediately
1ml is placed in the sterilized petri dishes of diameter 90mm, and 15~20ml of injection temperature is no more than the pancreas junket soya peptone agar training of 45 DEG C of fusings
Base is supported, mixing, solidification, two pieces of wares of parallel preparation, 30~35 DEG C, inversion is cultivated 3 days；
2. bacillus subtilis bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-3) bacillus subtilis
Bacteria suspension, mixing are made 10-5Bacillus subtilis bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in
In the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the pancreas junket soya peptone agar medium of 45 DEG C of fusings, mixes
Even, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 30~35 DEG C；
3. pseudomonas aeruginosa bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-4) pseudomonas aeruginosa
Bacteria suspension, mixing are made 10-6Pseudomonas aeruginosa bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in
In the sterilized petri dishes of diameter 90mm, 15~20ml of injection temperature is no more than the pancreas junket soya peptone agar medium of 45 DEG C of fusings, mixes
Even, solidification, two pieces of wares of parallel preparation, are inverted culture 3 days by 30~35 DEG C；
4. Candida albicans bacterium solution group：0.9% aseptic sodium chloride solutions of 100ml are taken, add in 1ml (10-2) Candida albicans bacterium hangs
Liquid, mixing are made 10-4Candida albicans bacteria suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in diameter
In the sterilized petri dishes of 90mm, 15~20ml of injection temperature is no more than the Sabouraud glucose agar of 45 DEG C of fusings, and mixing is coagulated
Gu two pieces of wares of parallel preparation, are inverted culture 5 days by 20~25 DEG C；
5. aspergillus niger bacterium solution group：100ml0.9% aseptic sodium chloride solutions are taken, add in 1ml (10-2) black-koji mould suspension, mixing,
It is made 10-4Black-koji mould suspension (being less than 100cfu containing bacterium number per 1ml) pipettes 1ml immediately, is placed in the sterilized petri dishes of diameter 90mm
In, 15~20ml of injection temperature is no more than the Sabouraud glucose agar of 45 DEG C of fusings, mixing, solidification, parallel preparation two
Block ware, is inverted culture 5 days by 20~25 DEG C.
7. detection method according to claim 1, it is characterised in that the computational methods of the step (6) are：
The average colony number of the bacterium colony rate of recovery of diluent control group=diluent control group average colony number/bacterium solution group ×
100%
The bacterium colony rate of recovery of the microbial inoculum test group=(average colony of the average colony number of test group-test liquid control group
Number)/bacterium solution group average colony number × 100%.
8. detection method according to claim 2, it is characterised in that the culture medium source is that three medicine science and technology of Beijing is opened
Hair company, condition of storage is at air drying.
9. detection method according to claim 1, it is characterised in that the bacterium solution test group, bacterium solution test liquid control group,
Diluent control group, bacterium solution group carry out 3 independent parallel laboratory tests respectively.
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