CN107142303A - Bile tolerance gram-negative bacteria proficiency testing sample and preparation method thereof in medicine - Google Patents
Bile tolerance gram-negative bacteria proficiency testing sample and preparation method thereof in medicine Download PDFInfo
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- CN107142303A CN107142303A CN201710372892.1A CN201710372892A CN107142303A CN 107142303 A CN107142303 A CN 107142303A CN 201710372892 A CN201710372892 A CN 201710372892A CN 107142303 A CN107142303 A CN 107142303A
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Abstract
Bile tolerance gram-negative bacteria proficiency testing sample and preparation method thereof in field of quality control in terms of the invention belongs to microbiologic inhibition tests, more particularly to a kind of medicine.Bile tolerance gram-negative bacteria proficiency testing sample includes target flora and background flora in medicine, the target flora is made up of EHEC, Klebsiella Pneumoniae, enterobacter cloacae, proteus mirabilis, clostridium perfringen, citrobacter freundii, and the background flora is made up of staphylococcus aureus, Bacillus cereus.Uniformity of the present invention, stability meet proficiency testing requirement, can at utmost ensure the stability of test sample, the content of EHEC changes in transport and storage, the total amount of bile tolerance gram-negative bacteria is not interfered with statistical significance yet；The preparation method of sample, technique is simple, and success rate is high.
Description
Technical field
Bile tolerance leather is blue in field of quality control in terms of the invention belongs to microbiologic inhibition tests, more particularly to a kind of medicine
Negative bacterium proficiency testing sample and preparation method thereof.
Background technology
Medicine microbiological Test field is very special.At present, although have accreditation body both at home and abroad and organize the micro- life of medicine
Thing proficiency testing and the large-scale precedent for preparing microorganism proficiency testing sample, but sample is that the only leather containing bile tolerance is blue cloudy
Property bacterium bacterial strain, exist without background bacterium, differed greatly with actual sample, and bile tolerance gram-negative bacteria is temperature sensitive, sample
The stability of product is difficult to meet to require, i.e., bile tolerance gram-negative bacteria standard sample still belongs to blank field.
Bile tolerance gram-negative bacteria refers to the gram-negative bacteria that can be survived and breed in bile acid, including enterobacteriaceae,
Pseudomonas, Aeromonas.Enterobacteriaceae mainly includes EHEC, Klebsiella Pneumoniae, enterobacter cloacae, unusual
Proteus and clostridium perfringen etc..If in the bile tolerance gram-negative bacteria sample prepared, target flora easily changes (numerous
Grow with death etc.), the storage life that may result in sample is short, and the uniformity and stability of sample are poor.Therefore, bacterium is considered
Biochemical characteristic, chooses the metastable flora of property very heavy to the uniformity and stability for ensureing sample as target flora
Will.The process specifications of bile tolerance gram-negative bacteria sample preparation are higher, and the uniformity and stability and jelly of sample
Dry strain, lyophilized process conditions, the use of freeze drying protectant, medium of rehydration etc. have close relationship.
Bile tolerance gram-negative bacteria index in medicine, directly reflects the hygienic quality and potential pathogenic risk of medicine.Such as
Bile tolerance Grain-negative bacterium number can cause patient's direct infection bile tolerance gram-negative bacteria, very more than a threshold quantity in fruit medicine
To there is fatal risk.Therefore, uniformity and all good bile tolerance gram-negative bacteria sample of stability are prepared, resistance to courage can be avoided
The random and uncertainty that salt gram-negative bacteria is examined, truly reflects the ability of testing laboratory.This is to improving laboratory
Quality control level, ensures drug safety, and even breaking International trade practices, raising China medicine international competitiveness all has
Special important meaning.
The content of the invention
The purpose of the present invention is the number of bacteria for overcoming work total in bile tolerance gram-negative bacteria determination sample in transport, storage
The problem of clump count can all change during depositing and test etc. is tested there is provided bile tolerance gram-negative bacteria ability in a kind of medicine
Sample is demonstrate,proved, uniformity, stability meet proficiency testing requirement, and it is a further object to provide the preparation side of the sample
Method, technique is simple, and success rate is high.
The technical scheme that is used to achieve the above object of the present invention is：Bile tolerance gram-negative bacteria proficiency testing in medicine
Sample, it is characterized in that：Including target flora and background flora, the target flora is by EHEC (E.coil), pneumonia gram
The primary bacterium of thunder (Klebsiella pnenmoniae), enterobacter cloacae (Enterobacter cloacae), proteus mirabilis
(Proteus mirabilis), clostridium perfringen (Enterobacter aerogenes) and citrobacter freundii
(Citrobacterfreundii) constitute, the background flora is by staphylococcus aureus (Rhodococcus equi), waxy
Bacillus (Bacillus cereus) constitutes.
The sample is using trehalose, skimmed milk power and sterilized water as matrix, and wherein the volume fraction of trehalose is 12%, taken off
The volume fraction of fat milk powder is 0.5%.
The aimed concn of target flora is 10 in the sample2CFU/mL, the aimed concn of background flora is 103CFU/mL。
The preparation method of bile tolerance gram-negative bacteria proficiency testing sample in a kind of medicine, it is characterized in that：Add including sample
Plus the selection of bacterial strain, the preparation of freeze drying protectant, sample are lyophilized, four steps of the uniformity of sample and stability test, wherein
Sample freeze-drying process is：Bacterial strain recovery passage-increasing bacterium-bacteria suspension prepares-lyophilized and packaging-storage, and detailed process is：
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to recovery
Bacterial strain is identified；
(2) bacterium is increased
Target bacteria group culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL's
The bacterium solution of corresponding single culture is made in freeze drying protectant；
(3) bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of flora2CFU/mL and background
The aimed concn 10 of flora3CFU/mL prepares bacteria suspension, and the bacteria suspension of 6 object bacterias of preparation and the bacterium of 2 background bacterium are hanged respectively
Liquid, respectively by 1:1 volume ratio is mixed to form target flora suspension and background flora suspension, then by target flora suspension and background bacterium
Group's suspension presses 1:1 volume ratio is mixed, and obtains plastc ring, is placed in be stirred continuously down on magnetic stirring apparatus and is dispensed into sample bottle
In, bottle stopper is added, but space is left, reality should not be covered；
(4) freeze and pack
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, is freeze-dried 40~50h, is tied when sample is freeze-dried
Shu Hou, directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
(5) store
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, random selected sample carries out uniformity and stably from whole samples
Property experiment, satisfaction require after discharge to participate in the experiment laboratory carry out contrasting between laboralories.
The freeze drying protectant is the sterilized water containing trehalose and skimmed milk power, and wherein volume fraction is respectively：Marine alga
Sugar 12%, skimmed milk power 0.5%.
The uniformity of the sample and stability test are according to CNAS-GL03《Proficiency testing sample homogeneity and stability
Evaluation guide》Carry out.
The selection of strain of the present invention using EHEC, Klebsiella Pneumoniae, enterobacter cloacae, proteus mirabilis,
Clostridium perfringen and citrobacter freundii as bile tolerance gram-negative bacteria sample target flora, with Staphylococcus aureus
Bacterium, Bacillus cereus are used as background flora.It can be survived and numerous in bile acid it is well known that bile tolerance gram-negative bacteria refers to
The gram-negative bacteria grown, including enterobacteriaceae, pseudomonas, Aeromonas.Large intestine angstrom in bile tolerance gram-negative bacteria
Uncommon bacterium is highly susceptible to the influence of environmental factor (particularly temperature).Comparatively speaking, with Klebsiella Pneumoniae, cloaca intestines bar
Bacterium, proteus mirabilis, clostridium perfringen and citrobacter freundii can at utmost ensure test specimens as target flora
The stability of product, the content of EHEC changes in transport and storage, also will not shadow on statistical significance
Ring the total amount of bile tolerance gram-negative bacteria.
The bile tolerance gram-negative bacteria proficiency testing sample of the present invention has the advantage that characteristic：Microorganism living, quantity
Do not change, biochemical character does not morph, set about from the condition for meeting special transport, pass through studying, surely for special process
Research of qualitative and uniformity etc. forms resistance in the sample preparation technology of a set of perfect proficiency testing, entirely appropriate development medicine
The examination of cholate gram-negative bacteria (qualitative) project, correct evaluating ability the result.
Brief description of the drawings
Fig. 1 is present invention process flow chart.
Fig. 2 is the stability test result figure of sample bile tolerance gram-negative bacteria under the conditions of 4 DEG C.
Fig. 3 is the stability test result figure of sample at different temperatures.
Embodiment
Below in conjunction with the accompanying drawings and specific embodiment is described in further detail to the present invention, but the invention is not limited in tool
Body embodiment.
Embodiment 1
Bile tolerance gram-negative bacteria proficiency testing sample in medicine, including target flora and background flora, the target flora by
EHEC (E.coil), Klebsiella Pneumoniae (Klebsiella pnenmoniae), enterobacter cloacae
(Enterobacter cloacae), proteus mirabilis (Proteus mirabilis), clostridium perfringen
(Enterobacter aerogenes) and citrobacter freundii (Citrobacter freundii) are constituted, the background bacterium
Group is made up of staphylococcus aureus (Staphylococcus aureus), Bacillus cereus (Bacillus cereus).
The sample is using trehalose, skimmed milk power and sterilized water as matrix, and wherein trehalose volume fraction is 12%, degreasing
Milk powder volume fraction is 0.5%.
The aimed concn of target flora is 10 in the sample2CFU/mL, the aimed concn of background flora is 103CFU/mL。
Embodiment 2
As shown in figure 1, in a kind of embodiment 1 in medicine bile tolerance gram-negative bacteria proficiency testing sample preparation method, including
The selection of sample addition bacterial strain, the preparation of freeze drying protectant, lyophilized sample, four steps of the uniformity of sample and stability test
Suddenly, comprise the following steps that：
1st, sample adds the selection of bacterial strain
According to target flora：EHEC (E.coil), Klebsiella Pneumoniae (Klebsiellapnenmoniae), cloaca intestines
Bacillus (Enterobacter cloacae), proteus mirabilis (Proteus mirabilis), clostridium perfringen
(Enterobacter aerogenes) and citrobacter freundii (Citrobacterfreundii), background flora：It is golden yellow
Staphylococcus (Staphylococcus aureus), Bacillus cereus (Bacillus cereus) selection standard bacterial strain, institute
There is reference culture to be purchased from the mechanism that government specifies, and with bacterial strain certificate, it is ensured that the traceability of bacterial strain.
2nd, the preparation of freeze drying protectant
Sample is using trehalose, skimmed milk power and sterilized water as matrix (volume fraction：Trehalose 12%, skimmed milk power 0.5%) match somebody with somebody
Freeze drying protectant processed.
3rd, sample is freezed
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar, it is recovered and is grown at 36 ± 1 DEG C, and to the bacterial strain of recovery
Identified；
(2) bacterium is increased
Target bacteria group culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL's
The bacterium solution of corresponding single culture is made in freeze drying protectant；
(3) bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of flora2CFU/mL and background
The aimed concn 10 of flora3CFU/mL prepares bacteria suspension.To ensure the bacterium containing above aimed concn in obtained final sample
According to object bacteria 10 in strain, the present embodiment2CFU/mL prepares the bacteria suspension of 6 object bacterias respectively, by 1:1 volume ratio is mixed, shape
Into target flora suspension；Background bacterium presses 103CFU/mL prepares the bacteria suspension of 2 background bacterium, by 1:1 volume ratio is mixed, and forms the back of the body
Scape flora suspension；Background flora suspension presses 1 with target flora suspension:1 volume ratio is mixed, and plastc ring is obtained, using than turbid
Method checks bacterial strain content；Being placed on magnetic stirring apparatus to be stirred continuously down takes 1.0mL to be dispensed into sample bottle (cillin bottle), adds bottle
Plug, but space is left, reality should not be covered；
(4) freeze and pack
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, and freeze drier parameter is by following setting：
Chilling rate (Cooling Rate) 0.5 DEG C/min
- 1 DEG C of 15min of early stage cold point (Incipient Freezing Point)
- 40 DEG C of cryogenic temperature (Cooling temperature)
- 29 DEG C of eutectic point (Melting Point Eutectic temperature)
20 DEG C of 120min of heating-up temperature (Heating temperature)
Start freeze drier, machine is directly entered the freezing dry process of sequencing, whole freeze-drying process 40h.
After sample freeze-drying terminates, directly carry out jumping a queue in case；Closing machine.The not tight cillin bottle of plug is rejected,
Sample is vacuum state in cillin bottle；
(5) store
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, and the sample of preservation suitably manage and detect, from whole sample
Random selected sample carries out uniformity and stability test in product, and satisfaction requires that after discharge carries out laboratory monitoring to laboratory of participating in the experiment
Compare.
4th, the uniformity of sample and stability test
Uniformity and stability inspection to object bacteria in sample are the main methods of verification sample preparation process validity.Check
Sample homogeneity and stability are according to CNAS-GL03《Proficiency testing sample homogeneity and estimation of stability guide》Carry out.
Gained sample homogeneity and Detection of Stability method and result are as follows：
12 samples are randomly selected respectively, and using SN/T 1896-2007 methods, the resistance to courage of 2 × 12 parts of samples is tested in repeat condition
Salt gram-negative bacteria.Result data carries out statistical disposition with one-way analysis of variance (ANOV), and statistic procedure and result are as follows：
The variance analysis formula of table 1
In upper table,
The sample homogeneity testing result of table 2
The sample homogeneity of table 3 tests the results of analysis of variance
Conclusion：Under 95% fiducial probability, compared with influence of the other factors to test result, the inhomogeneities of sample is to connect
Receive.
Using two kinds of stability test：One kind is the stability test at short temperature (4 DEG C), another
Kind it is the stability test at high temperature (traffic condition of analog sample), from three temperature spots, respectively 20 DEG C, 36
DEG C and 45 DEG C.Periodic detection sample, tests 3 samples, by 2 × 3 parts of sample knots for the different temperature points different holding time
Average (logarithmic transformed after) and the uniformity test results contrast of fruit, between absolute difference divided by test plan in merit rating
With sane standard deviation S*, it is that principle determines to meet proficiency testing sample requirement under condition of different temperatures most that ratio, which is less than 0.3,
The long holding time.Stability test result is shown in Fig. 2 and Fig. 3.
Embodiment 3
In medicine described in the present embodiment each step of the preparation method of bile tolerance gram-negative bacteria proficiency testing sample with
Identical in embodiment 2, different technical parameters are：Strain cultures select nutrient agar slant medium；During bacteria suspension is prepared,
The concentration of each object bacteria is according to 5 × 10 in target flora suspension2CFU/mL is prepared, the concentration of each background bacterium in background flora suspension
According to 103CFU/mL is prepared；Freeze-drying process 45h.
Embodiment 4
In medicine described in the present embodiment each step of the preparation method of bile tolerance gram-negative bacteria proficiency testing sample with
Identical in embodiment 3, different technical parameters are：Bacteria suspension prepare in, in target flora suspension the concentration of each object bacteria according to
4.5×102CFU/mL is prepared；Freeze-drying process 50h.
Claims (6)
1. bile tolerance gram-negative bacteria proficiency testing sample in medicine, it is characterized in that：It is described including target flora and background flora
Target flora is by EHEC, Klebsiella Pneumoniae, enterobacter cloacae, proteus mirabilis, clostridium perfringen and Freund lemon
Lemon acidfast bacilli is constituted, and the background flora is made up of staphylococcus aureus, Bacillus cereus.
2. bile tolerance gram-negative bacteria proficiency testing sample in medicine according to claim 1, it is characterized in that：The sample
Using trehalose, skimmed milk power and sterilized water as matrix, wherein the volume fraction of trehalose is the 12%, volume fraction of skimmed milk power
For 0.5%.
3. bile tolerance gram-negative bacteria proficiency testing sample in medicine according to claim 1 or 2, it is characterized in that：It is described
The aimed concn of target flora is 10 in sample2CFU/mL, the aimed concn of background flora is 103 CFU/mL。
4. the preparation method of bile tolerance gram-negative bacteria proficiency testing sample, its feature in medicine according to claim 1
It is：Selection, the preparation of freeze drying protectant including sample addition bacterial strain, lyophilized sample, the uniformity of sample and stability test
Four steps, wherein sample freeze-drying process is：Bacterial strain recovery passage-increasing bacterium-bacteria suspension prepares-lyophilized and packaging-storage,
Detailed process is：
（1）Bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to recovery
Bacterial strain is identified；
（2）Increase bacterium
Target bacteria group culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL's
The bacterium solution of corresponding single culture is made in freeze drying protectant；
（3）Bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of flora2CFU/mL and background
The aimed concn 10 of flora3 CFU/mL prepares bacteria suspension, and the bacteria suspension of 6 object bacterias of preparation and the bacterium of 2 background bacterium are hanged respectively
Liquid, respectively by 1:1 volume ratio is mixed to form target flora suspension and background flora suspension, then by target flora suspension and background bacterium
Group's suspension presses 1:1 volume ratio is mixed, and obtains plastc ring, is placed in be stirred continuously down on magnetic stirring apparatus and is dispensed into sample bottle
In, bottle stopper is added, but space is left, reality should not be covered；
（4）Lyophilized and packaging
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, is freeze-dried 40~50h, is tied when sample is freeze-dried
Shu Hou, directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
（5）Storage
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, random selected sample carries out uniformity and stably from whole samples
Property experiment, satisfaction require after discharge to participate in the experiment laboratory carry out contrasting between laboralories.
5. the preparation method of bile tolerance gram-negative bacteria proficiency testing sample, its feature in medicine according to claim 4
It is：The freeze drying protectant is the sterilized water containing trehalose and skimmed milk power, and wherein volume fraction is respectively：Trehalose 12%,
Skimmed milk power 0.5%.
6. the preparation method of bile tolerance gram-negative bacteria proficiency testing sample, its feature in medicine according to claim 4
It is：The uniformity of the sample and stability test are according to CNAS-GL03《Proficiency testing sample homogeneity and estimation of stability
Guide》Carry out.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1706964A (en) * | 2005-05-26 | 2005-12-14 | 卢行安 | Bacterial colony number sample for verifying microbiological capacity of food and its prepn process |
| CN105176823A (en) * | 2015-06-10 | 2015-12-23 | 舟山出入境检验检疫局综合技术服务中心 | Microorganism ability verification salmonella sample preparation method |
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