CN109295158A - A kind of quick sterilization effect evaluation method - Google Patents
A kind of quick sterilization effect evaluation method Download PDFInfo
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- CN109295158A CN109295158A CN201811156770.XA CN201811156770A CN109295158A CN 109295158 A CN109295158 A CN 109295158A CN 201811156770 A CN201811156770 A CN 201811156770A CN 109295158 A CN109295158 A CN 109295158A
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Abstract
The present invention relates to a kind of quick sterilization effect evaluation methods, belong to sterilization technology field.Evaluation method of the present invention, comprising the following steps: 1) bacterium solution is added drop-wise on stainless steel substrates, is dried after smoothening, obtain sterilizing indicating piece；Bacterium sum is not less than 10 on the sterilizing indicating piece6cfu/cm2；2) the sterilizing indicating piece that step 1) obtains is sterilized, the sterilizing indicating piece after sterilizing is impregnated in eluent, eluted, obtain sterilizing eluent；3) the sterilizing eluent that step 2) obtains is mixed with union dyeing reagent and carries out fluorescent staining, bacterium solution after being dyed；The union dyeing reagent includes viable bacteria staining reagent and dead bacterium staining reagent；4) bacterium solution after dyeing that step 3) obtains is filtered, obtains filter membrane containing bacterium；5) fluorescence detection is carried out to the filter membrane containing bacterium that step 4) obtains and sterilization rate is obtained according to dead bacterium and number of viable.The method of the invention can be realized sterilization effect and sterilization rate is fast and accurately evaluated.
Description
Technical field
The present invention relates to sterilization technology fields, and in particular to a kind of quick sterilization effect evaluation method.
Background technique
The evaluation method of sterilization effect is broadly divided into three categories: chemical method, physical method and biological method, wherein biology
Method is the method for the most directly judging sterilization effect.The principle of bioanalysis evaluation sterilization effect will contain microbial spores
Biological indicator is exposed in certain selected bactericidal agent or sterilizing methods, and the gemma for then crossing exposure treatment, which is placed in, to be tieed up
In the culture environment for holding gemma germination and microorganism growth, so that it is determined that the survival rate for the gemma that exposure treatment is crossed.
Bioanalysis detection sterilization effect method particularly may be divided into two classes: pH detection method and viable bacteria culture counting method.PH detection
Method mainly uses bromocresol purple peptone culture medium, and wherein bromocresol purple is exactly common acid-base indicator；Viable bacteria culture
Counting method master conventional medium tryptose soya agar culture medium (TSA) to be used and pancreas peptone soybean broth culture medium
(TSB)。
When pH detection method is according to gemma restoration ecosystem, metabolism generates acidic metabolite and medium pH is caused to become
Change, by adding pH indicator such as bromocresol purple, according to its color change come interpretation result, the microculture time is 24~
48h, pH detection method result are reliable, but time-consuming is too long, cannot achieve quick interpretation；And pH detection method can only judge sterilizing whether at
Function can not obtain accurate sterilization rate.
It is by sample to be tested after dilution appropriate that viable bacteria, which cultivates counting method, and microorganism therein is fully dispersed at single
Cell takes a certain amount of dilution to be inoculated on plate, and by culture, the naked eyes formed by individual cells growth and breeding can
Should represent in raw sample one of the bacterium colony seen, i.e. a bacterium colony is unicellular.Clump count is counted, according to its extension rate and sampling
Inoculum concentration can converse the bacteria containing amount in sample.The method can obtain viable bacteria information, but cumbersome, as a result need to train
Supporting a period of time could obtain, and measurement result is easily influenced by many factors, and temperature is cultivated in the pollution as caused by misoperation
Spend it is improper caused by bacterium death etc..
Existing bioanalysis test sterilization effect accuracy is low, and testing result could need to be obtained after a period of time, nothing
Method realizes quick interpretation.
Summary of the invention
The purpose of the present invention is to provide a kind of quick sterilization effect evaluation methods.Evaluation method energy provided by the invention
Enough solve the problems, such as it is of the existing technology assessment take a long time, accuracy it is not high, realization fast and accurately evaluate sterilization effect
(sterilization rate).
The present invention provides a kind of quick sterilization effect evaluation methods, comprising the following steps:
1) bacterium solution is added drop-wise on stainless steel substrates, is dried after smoothening, obtain sterilizing indicating piece；Bacterium on the sterilizing indicating piece
Sum is not less than 106cfu/cm2；
2) the sterilizing indicating piece that step 1) obtains is sterilized, the sterilizing indicating piece after sterilizing is soaked in eluent
Bubble, elution obtain sterilizing eluent；
3) the sterilizing eluent that step 2) obtains is mixed with union dyeing reagent and carries out fluorescent staining, bacterium solution after being dyed；
The union dyeing reagent includes viable bacteria staining reagent and dead bacterium staining reagent；
4) bacterium solution after dyeing that step 3) obtains is filtered, obtains filter membrane containing bacterium；
5) fluorescence detection is carried out to the filter membrane containing bacterium that step 4) obtains and sterilization rate is obtained according to dead bacterium and number of viable.
Preferably, the step 1) bacterium bag includes bacillus subtilis and/or staphylococcus albus.
Preferably, the concentration of the step 1) bacterium solution is not less than 2.0 × 107cfu/mL。
Preferably, the method for the step 2) sterilizing includes gas surface sterilization.
It preferably, further include the process that bacterium solution is mixed with sterile water after dyeing before the step 4) filtering.
Preferably, step 4) the filtering filter membrane includes low background fluorescence brightness, good contrast, aperture less than 1 μm
Filter membrane.
The present invention provides a kind of quick sterilization effect evaluation methods.Evaluation method of the present invention is carried out based on fluorescent staining
Count plate is realized pair by the production for the indicating piece that sterilizes in conjunction with subsequent elution, the setting of dyeing and fluorescence detection condition
The high evaluation of sterilization effect accuracy, and entire detection process only needs 30min, evaluation process is quick.
Detailed description of the invention
Fig. 1 is quick sterilization effect assessment flow chart provided by the invention；
Fig. 2 is fluorescence detection result schematic diagram before and after the sterilizing that the embodiment of the present invention 1 provides；
Fig. 3 is dyeing effect schematic diagram before and after the sterilizing that the embodiment of the present invention 2 provides；
Fig. 4 according to a certain percentage illustrates sample 1, the mixed staining conditions of sample 2 for what the embodiment of the present invention 2 provided
Figure.
Specific embodiment
The present invention provides a kind of quick sterilization effect evaluation methods, comprising the following steps:
1) bacterium solution is added drop-wise on stainless steel substrates, is dried after smoothening, obtain sterilizing indicating piece；Bacterium on the sterilizing indicating piece
Sum is not less than 106cfu/cm2；
2) the sterilizing indicating piece that step 1) obtains is sterilized, the sterilizing indicating piece after sterilizing is soaked in eluent
Bubble, elution obtain sterilizing eluent；
3) the sterilizing eluent that step 2) obtains is mixed with union dyeing reagent and carries out fluorescent staining, bacterium solution after being dyed；
The union dyeing reagent includes viable bacteria staining reagent and dead bacterium staining reagent；
4) bacterium solution after dyeing that step 3) obtains is filtered, obtains filter membrane containing bacterium；
5) fluorescence detection is carried out to the filter membrane containing bacterium that step 4) obtains and sterilization rate is obtained according to dead bacterium and number of viable.
Appraisal procedure flow chart of the present invention is as shown in Figure 1.The operation that appraisal procedure of the present invention is related to is preferably sterile
Under the conditions of carry out, avoid the influence of miscellaneous bacteria, superclean bench as well known to the skilled person.First bacterium solution is added dropwise by the present invention
It onto stainless steel substrates, is dried after smoothening, obtains sterilizing indicating piece；Bacterium sum is not less than 10 on the sterilizing indicating piece6cfu/cm2。
In the present invention, the bacterium preferably includes the bacterium with greater activity, more preferably includes bacillus subtilis and/or white Portugal
Grape coccus.In the present invention, the concentration of the bacterium solution is preferably not less than 2.0 × 107cfu/mL.In the present invention, described stainless
It is sterile that steel disc preferably needs cleaned and surface sterilizing to ensure before using.The present invention is not special to the area of the stainless steel substrates
It limits, the area of the stainless steel substrates is preferably 0.5~1.5cm2, more preferably 1cm2.When the area of the stainless steel substrates is
1cm2When, the dropwise addition volume of the bacterium solution is preferably 50 μ L.The present invention does not have special restriction to the shape of the stainless steel substrates,
It is preferably circular.The present invention does not have special restriction to the mode dried, and dries method using conventional, is such as placed into
It is dried in safety cabinet, the temperature in the safety cabinet is preferably room temperature.Sterilizing indicating piece of the present invention has uniformity
Well, it easily rinses, interfere low advantage.
After obtaining sterilizing indicating piece, the present invention sterilizes sterilizing indicating piece, and the sterilizing indicating piece after sterilizing is carried out
Elution obtains sterilizing eluent.In the present invention, the method for the sterilizing preferably includes gas surface sterilization, refers to sterilizing
Show that the viable bacteria of on piece sterilizes.The present invention does not have special restriction to gas surface sterilization, using art technology
Routine known to personnel being capable of the gaseous sterilization process that sterilizes of body surface.Appraisal procedure of the present invention can
Realize efficient, the accurate evaluation to gas surface sterilization effect.In the present invention, the eluent preferably includes sterile water.This
Invention is not particularly limited the time of the immersion, and the time of the immersion is preferably 2~3min.In the present invention, described
It is preferably blown and beaten repeatedly in elution process or low-speed oscillation is carried out using oscillator.Time of the present invention to the low-speed oscillation
There is no special restriction, specifically, the time of the low-speed oscillation is preferably 1~2min.The process energy of elution of the present invention
Incorporate most bacterium in sterile water, convenient for the detection of post sterilization rate.Bacterium is total on sterilizing indicating piece of the present invention
Number is not less than 106cfu/cm2, will preferably sterilize when elution of the present invention indicating piece (every 1cm2) it is added to the eluent of 1~5mL
In, guarantee that the bacteria concentration in eluent is not less than 104cfu/mL。
Obtain sterilizing eluent after, the present invention will sterilize eluent mixed with union dyeing reagent progress fluorescent staining, contaminated
Bacterium solution after color；The union dyeing reagent includes viable bacteria staining reagent and dead bacterium staining reagent.In the present invention, the fluorescent staining
Time is 15~25min, more preferably 20min.In the present invention, the method for the fluorescent staining is preferably simultaneously dyeing method,
To dead bacterium and viable bacteria synchronize dye.In the present invention, the viable bacteria staining reagent preferably includes CFDA or calcein；Institute
It states dead bacterium staining reagent and preferably includes PI or EB.The present invention is not particularly limited the dyeing condition of each coloring agent, using ability
The common staining method of each coloring agent known to field technique personnel dyes sterilizing eluent, pays attention to dyeing condition such as
It is protected from light, incubates.
After being dyed after bacterium solution, bacterium solution after dyeing is filtered by the present invention, obtains filter membrane containing bacterium.In the present invention,
Before the filtering, it is also preferable to include the processes that bacterium solution after dyeing is mixed with sterile water.In the present invention, bacterium solution after the dyeing
The effect mixed with sterile water is the waste that (1) avoids union dyeing reagent；(2) plus sterile water can guarantee enough samples to be filtered
This；(3) guarantee filtering uniformity.The volume ratio that the present invention mixes bacterium solution after the dyeing and sterile water is not particularly limited.
Specifically, when taking the bacterium solution of 100ul or 50ul to add union dyeing reagent in proportion, after the completion of dyeing, sterile water is preferably added by bacterium
Liquid is diluted to 5~10ml, it is ensured that has enough bacterium solutions to be filtered, finally filters.In the present invention, the filtering is excellent with filter membrane
The choosing low, filter membrane of good contrast, aperture less than 1 μm including background fluorescence brightness.In the present invention, the filter membrane is preferably justified
Shape.In the present invention, the strain density on the filter membrane containing bacterium is preferably 102~105cfu/mm2, excellent for the bacterium solution of excessive concentration
It is filtered after the few filtering of choosing or dilution, the bacterium solution too low for concentration preferably filters more samples.The present invention is to the filtering
It is not particularly limited with equipment, filtering equipment of the present invention preferably changes membrane filter, more preferably model
Milipore (Mi Libo) SX0001300's changes membrane filter, and filter diameter is preferably 13mm, area 0.7cm2.Work as use
When above-mentioned filter, filtering of the present invention preferably uses milipore (Mi Libo) HTBP013000,0.4 μ of aperture with filter membrane
m。
After obtaining filter membrane containing bacterium, the present invention carries out fluorescence detection to filter membrane containing bacterium and is gone out according to dead bacterium and number of viable
Bacterium rate.In the present invention, the fluorescence detection preferably includes fluorescence microscope or laser scanning system etc. with device.
A kind of quick sterilization effect evaluation method of the present invention is done further in detail combined with specific embodiments below
Thin introduction, technical solution of the present invention include but is not limited to following embodiment.
Embodiment 1
Filter changes membrane filter, filter diameter 13mm, filter area using milipore (Mi Libo) SX0001300's
0.7cm2；Filter membrane uses milipore (Mi Libo) HTBP013000,0.4 μm of aperture.
The preparation of sterilizing indicating piece: concentration is made not less than 2.0 × 10 in culture bacillus subtilis7Cfu/ml's is suspended
Liquid, taking 50ul to be added to stainless steel substrates, (it is sterile that stainless steel substrates need cleaned and surface sterilizing to ensure, stainless steel substrates are circle, diameter
To smoothen on 1cm), it is put into safety cabinet and dries, total number of bacteria is made not less than 106cfu/cm2Sample.With stainless steel substrates system
At indicating piece uniformity it is good, it is easy rinse, interfere it is low.
Sterilization process: ready indicating piece is put into the space of gas surface sterilizing to be carried out on specific position, choosing
Multiple positions are taken, a piece of sterilizing indicating piece is placed in each position；Separately prepare an indicating piece sample to be placed in outside sterilized space,
Same time is stood, parallel control is done.It requires to carry out gaseous sterilization to object space according to disinfecting action.After the completion of sterilizing, take
Sterilize indicating piece and check sample out, carries out subsequent samples elution and processing.
Eluted sample: (1) according to the sample number of sterilizing group and control group, prepare centrifugation of the identical quantity through high-temperature sterilization
Pipe, is added the sterile water of equivalent in each container, and sterile water can be totally submerged stainless steel substrates；(2) it is then respectively put into and goes out
Bacterium group and control group sample impregnate 2~3min, then blow and beat tens of times repeatedly or directly obtained with 1~2min of oscillator low-speed oscillation
Different sterilizing eluents.
Synchronous fluorescence dyes (life or death bacterium is dyed simultaneously): (1) preparing identical quantity centrifuge tube, be separately added into the difference of equivalent
Sterilize eluent；(2) suitable union dyeing reagent, union dyeing reagent are separately added into each container according to ratio of reagents needed for dyeing
In include viable bacteria staining reagent CFDA, dead bacterium staining reagent PI, timing dye, notice that dyeing condition is such as protected from light, incubates, obtain
Bacterium solution after different dyeing.
It filters sample: after dyeing, bacterium solution after different dyeing being mixed with 5~10mL sterile water respectively, filtering is made
Sample view.Strain density on filter membrane containing bacterium is preferably 102~105cfu/mm2。
Detection device: fluorescence microscope.
To ensure that acquisition range covers entire filter membrane when acquiring image, comprehensive detection is carried out to entire filter membrane.It synchronizes glimmering
Light method dyeing after, in the visual field green fluorescence point be viable bacteria, red fluorescence point be dead bacterium, calculate separately the i.e. dead bacterium of different characteristic point and
The quantity of viable bacteria determines life or death bacterium ratio in sample, obtains accurate sterilization effect.Sterilizing before and sterilizing after fluorescence detection effect
Figure is as shown in Figure 2, wherein Fig. 2 .a is image before sterilizing, and green point represents viable bacteria in image, and red dot represents dead bacterium, is surpassed in the image
Crossing 90% is viable bacteria, and the image after the same sample sterilizing of Fig. 2 .b, is at this time almost dead bacterium entirely in image, before sterilizing
Image can be read to determine sterilization effect fastly afterwards.
Embodiment 2
Filter changes membrane filter, filter diameter 13mm, filter area using milipore (Mi Libo) SX0001300's
0.7cm2；Filter membrane uses milipore (Mi Libo) HTBP013000,0.4 μm of aperture.
The preparation of sterilizing indicating piece: concentration is made not less than 2.0 × 10 in culture bacillus subtilis7Cfu/ml's is suspended
Liquid, taking 50ul to be added to stainless steel substrates, (it is sterile that stainless steel substrates need cleaned and surface sterilizing to ensure, stainless steel substrates are circle, diameter
To smoothen on 1cm), it is put into safety cabinet and dries, total number of bacteria is made not less than 106cfu/cm2Sample.With stainless steel substrates system
At indicating piece uniformity it is good, it is easy rinse, interfere it is low.
Sterilization process: ready indicating piece is put into the space of gas surface sterilizing to be carried out on specific position, choosing
Multiple positions are taken, a piece of sterilizing indicating piece is placed in each position；Separately prepare an indicating piece sample to be placed in outside sterilized space,
Same time is stood, parallel control is done.It requires to carry out gaseous sterilization to object space according to disinfecting action.After the completion of sterilizing, take
Sterilize indicating piece and check sample out, carries out subsequent samples elution and processing.
Eluted sample: (1) according to the sample number of sterilizing group and control group, prepare centrifugation of the identical quantity through high-temperature sterilization
Pipe, is added the sterile water of equivalent in each container, and sterile water can be totally submerged stainless steel substrates；(2) it is then respectively put into and goes out
Bacterium group and control group sample impregnate 2~3min, then blow and beat tens of times repeatedly or directly obtained with 1~2min of oscillator low-speed oscillation
Different sterilizing eluents.
Synchronous fluorescence dyes (life or death bacterium is dyed simultaneously): (1) preparing identical quantity centrifuge tube, be separately added into the difference of equivalent
Sterilize eluent；(2) the union dyeing reagent of equivalent is separately added into each container again, includes viable bacteria staining reagent in union dyeing reagent
Calcein, dead bacterium staining reagent EB, timing are dyed, notice that dyeing condition is such as protected from light, incubates, obtain bacterium after different dyeing
Liquid.
It filters sample: after dyeing, bacterium solution after different dyeing being mixed with 5~10mL sterile water respectively, filtering is made
Sample view.Strain density on filter membrane containing bacterium is preferably 102~105cfu/mm2。
Detection device: laser scanning system.
To ensure that acquisition range covers entire filter membrane when acquiring image, comprehensive detection is carried out to entire filter membrane.It synchronizes glimmering
Light method dyeing after, in the visual field green fluorescence point be viable bacteria, red fluorescence point be dead bacterium, calculate separately the i.e. dead bacterium of different characteristic point and
The quantity of viable bacteria determines life or death bacterium ratio in sample, obtains accurate sterilization effect.Fig. 3 is the dyeing effect signal of sterilizing front and back
Scheme, wherein Fig. 3 .1 is preferable bacterium solution (sample 1) staining conditions of activity before sterilizing, and Fig. 3 .2 is after sterilizing almost without viable bacteria
Sample (sample 2) staining conditions；Fig. 4 be according to a certain percentage by sample 1, the mixed staining conditions schematic diagram of sample 2,
In, ratio mixed by sample 1,2 is respectively 1:2,1:1,2:1, viable bacteria ratio and mixed proportion after dyeing in .1~4.3 Fig. 4
Unanimously.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of quick sterilization effect evaluation method, comprising the following steps:
1) bacterium solution is added drop-wise on stainless steel substrates, is dried after smoothening, obtain sterilizing indicating piece；Bacterium sum on the sterilizing indicating piece
Not less than 106cfu/cm2；
2) the sterilizing indicating piece that step 1) obtains is sterilized, the sterilizing indicating piece after sterilizing is impregnated in eluent, is washed
It is de-, obtain sterilizing eluent；
3) the sterilizing eluent that step 2) obtains is mixed with union dyeing reagent and carries out fluorescent staining, bacterium solution after being dyed；It is described
Union dyeing reagent includes viable bacteria staining reagent and dead bacterium staining reagent；
4) bacterium solution after dyeing that step 3) obtains is filtered, obtains filter membrane containing bacterium；
5) fluorescence detection is carried out to the filter membrane containing bacterium that step 4) obtains and sterilization rate is obtained according to dead bacterium and number of viable.
2. evaluation method according to claim 1, which is characterized in that the step 1) bacterium bag include bacillus subtilis and/
Or staphylococcus albus.
3. evaluation method according to claim 1, which is characterized in that the concentration of the step 1) bacterium solution not less than 2.0 ×
107cfu/mL。
4. evaluation method according to claim 1, which is characterized in that the method for the step 2) sterilizing includes gas surface
Sterilization.
5. evaluation method according to claim 1, which is characterized in that further include after dyeing before the step 4) filtering
The process that bacterium solution is mixed with sterile water.
6. evaluation method according to claim 1, which is characterized in that step 4) the filtering filter membrane includes background fluorescence
Brightness is low, the filter membrane of good contrast, aperture less than 1 μm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110699241A (en) * | 2019-10-28 | 2020-01-17 | 军事科学院系统工程研究院卫勤保障技术研究所 | Automatic disinfection effect rapid evaluation device and method |
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| CN1140470A (en) * | 1994-02-15 | 1997-01-15 | 美国3M公司 | Rapid read-out biological indicator |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110699241A (en) * | 2019-10-28 | 2020-01-17 | 军事科学院系统工程研究院卫勤保障技术研究所 | Automatic disinfection effect rapid evaluation device and method |
| CN110699241B (en) * | 2019-10-28 | 2024-08-20 | 军事科学院系统工程研究院卫勤保障技术研究所 | Automatic disinfection effect rapid evaluation device and method |
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