CN100507524C - Anti-interference rapid detection and reagent for microbe - Google Patents
Anti-interference rapid detection and reagent for microbe Download PDFInfo
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- CN100507524C CN100507524C CNB2004100267945A CN200410026794A CN100507524C CN 100507524 C CN100507524 C CN 100507524C CN B2004100267945 A CNB2004100267945 A CN B2004100267945A CN 200410026794 A CN200410026794 A CN 200410026794A CN 100507524 C CN100507524 C CN 100507524C
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Abstract
A fast detection method of microbe anti- interference includes using large sample method or MPN method to enrich bacteria for microbe culture media sample, processing prepared sample with cell ATP release agent Ec and adding luciferase - luciferin reagent and deinhibitor for ATP luminous detection, confirming that sample is bacteria carrier positive if sample luminous pulse counts CPMs is greater than control sample solution CPMck, checking MPN table to obtain bacteria content in sample. The method can raise detection sensitivity by 1000 times comparing to normal method.
Description
[technical field]
The invention belongs to detection, measure method of microorganism, relate in particular to a kind of method of utilizing bioluminescence the less sample of bacteria containing amount to be carried out microbial rapid detection.
[background technology]
At present, as the most large conventional sense project of microorganism---the detection of bacterium, mould and saccharomycete quantity, still continue to use the agar plate count method of setting up by Pasteur before more than 100 year.Traditional microorganism detection method depends on special microbiological culture media and comes the microorganism that survives in the separation and Culture sample, and these method sensitivities, directly perceived can provide micro organism quantity and kinds of information in the food simultaneously.Yet conventional method needs 1～5 day time from beginning to cultivate macroscopic bacterium colony, and complex operation, is confined to the laboratory, and technical level of operators is required height, occurs artificial error easily; And up to now as the microorganism in the physical environment, can carry out it is generally acknowledged of ARTIFICIAL CULTURE has only 5～10%, does not have independent a kind of nutrient culture media or a cover physics, electrochemical conditions can satisfy the physiological requirement of all microorganisms in the sample; Simultaneously, big, time-consuming, the effort of the preparation of nutrient culture media, dull and stereotyped cultivation, colony counting and biochemical identification workload provides historic information, can not reach enterprise's quality of production control, the health supervision detection requirement of acquisition microorganism information fast.In recent years, along with the progress of science and technology and the application of automatic technology, many improvement that classic method is done on sample preparation, dull and stereotyped cultivation, colony counting and identification systems have made operation simpler and easy and convenient, have also reduced cost simultaneously and have reduced workload.But occuping cultured method need be cost with time still, can't obtain the result fast, can not satisfy food industry production, the on-the-spot demand that detects of environmental sanitary inspection far away.Therefore, the technology of microbial rapid detection and instrument are just taken advantage of a situation and are given birth to, and wherein the online method for rapid inspecting animalcule of ATP bioluminescence is to think the new technology of the online detection of most possible realization micro organism quantity at present.
The ATP bioluminescence technique was proposed by the scientists (Chappelle and Levin etc.) of NASA (NASA) the sixties in 20th century, and its principle is a firefly luciferase (Luciferase) with fluorescein (D-Luciferin), atriphos (ATP) and 0
2Be substrate, at Mg
2+When existing, chemical energy can be converted into luminous energy.Reaction equation is expressed as:
ATP is the essential substrate of luciferase catalytic luminescence, is again the energy source of all biological vital movements, and in the enzymatic luminescence-producing reaction of fluorescein, ATP is in certain concentration range, and its concentration and luminous intensity are linear dependence.D ' Eustachio and Levin studies show that, the bacterium of each growth period all has the ATP content than constant level, therefore, extracts the ATP of bacterium, utilize biloluminescence method to measure the bacteria containing amount that can extrapolate behind the content of ATP in the sample, whole process only is tens minutes.Because biloluminescence method need not incubation, and is easy and simple to handle, highly sensitive, can obtain the result in several minutes, Sharpe etc. (1970) at first use this method and detect the microorganism that exists in the food, but high-caliber non-microorganism cell ATP has reduced sensitivity.However, many scientific research personnel still have keen interest to the microbial contamination that this method is used for assessing in the food, and have made very big effort for this reason.Until the nineties in 20th century is early stage, the ATP bioluminescence technique just really is applied to sanitary quality control (Giffiths, 1993,1995 of food industry; Kyriakides and Patel, 1994) and environmental monitoring.The most successful application is that the sanitary condition of producing preceding production line of beginning and environment detects, and this method can obtain the testing result of equipment surface clean condition in 2 minutes, have the incomparable advantage of other microorganism detection method.Simultaneously, the ATP biloluminescence method also is applied to the microorganism detection of starting material, finished product.Application such as Bautista, JoaoNiza-Ribeiro ATP biloluminescence method detects the content of microorganisms in poultry, thick Ruzhong respectively, in several minutes, obtain the result judging the contamination by micro situation, they think this method compare with colony counting method fast, reliable, accurate.Russell, Sinell etc. think that the ATP biloluminescence method is the monitoring of hygiene method of unique suitable HACCP, are the bases of carrying out HACCP, also are to detect the fastest method of microorganism at present simultaneously.More external companies develop complete bioluminescent detection device according to noctilcent principle and kit is used for the micro organism quantity fast measuring, this detection system is made up of bioluminescent detection instrument and mensuration kit two large divisions, and the detectability of its micro organism quantity is generally bacterium 10
5～6Cells/mL, the highest sensitivity also only reaches 10
3Cells/mL, content of microorganisms is 10
3The above sample of cells/mL can detect the result rapidly in tens minutes.Many so far countries with the ATP biloluminescence method as the on-the-spot effective means that detects of food industry production and environmental sanitary inspection, and domesticly still be in the starting stage.
The same with food industry production and environmental sanitary inspection, the microorganism detection of food and drink product is the important application object of biloluminescence method equally, but there is major defect in this respect in the ATP biloluminescence method, and bacterial concentration is minimum in the sample is no less than 10 because this method requires
3Cells/mL, this sensitivity does not often reach hygienic requirement.When the sample content of molds less than 100cells/mL, even during, directly use biloluminescence method microbial rapid detection technology then can not detect wherein bacteria containing amount effectively less than 1cell/mL.Simultaneously, when the microorganism that exists in the sample was the gemma of dormant state or spore, ATP content was extremely low, does not reach the sensitivity of luminous detection.In addition, in production runes such as food industry, using antiseptic and sanitizer widely, microorganism is subjected to the influence of antiseptic and sanitizer in the sample, is in to suppress or injured state, and ATP content is also extremely low, the requirement that does not reach luminous detection equally.Therefore, though the ATP biloluminescence method is sensitive, fast, because above-mentioned weak point makes its application be subjected to very big restriction.
[summary of the invention]
The objective of the invention is to overcome the under-sensitive problem that present ATP biloluminescence method exists, some antiseptics that exist in the solution sample or the interference problem of residual disinfectancy agent, a kind of increase sensitivity, jamproof method for rapid inspecting animalcule are provided, improve the sensitivity that detects and shorten the required time of detecting.
The microbial rapid detection technology that is provided among the present invention is made up of two parts, the one, microbiological culture media carries out the liquid large sample method to the sample that interfering materials such as antiseptic or sanitizer are arranged or approximate number (MPN) method increases bacterium in disturbing with resisting, and makes the microorganism recovery of damaged or gemma, the spore germination of dormant state; The 2nd, Ec handles sample with the cell ATP releasing agent, discharges the microbial cell ATP in the sample, carries out luminous detection then.
The present invention can shorten detection time greatly, bacterium can be finished detection in 6 hours, yeast was finished detection in 12 hours, mould was finished detection in 24 hours, increase substantially detection sensitivity simultaneously, wherein bioluminescence liquid large sample method can reach 10cells/100mL, and approximate number (MPN) method of bioluminescence can reach 30cells/100mL, than 1000 times of the minimum raisings of the detection sensitivity of biloluminescence method.
The concrete operation method of micro organism quantity method for quick of the present invention:
1) anti-interference bacterium and fungi liquid large sample method and MPN method fast measuring
(1) anti-interference bacterium and fungi liquid large sample method fast measuring
The sterility that this method is suitable for containing the sample of interfering materials such as antiseptic, sanitizer detects, contain the different of the classification of interfering material and test item according to sample, select anti-anticorrosion formulation, anti-sterilization formulation and anti-ozone type bacterium or fungi liquid nutrient culture media respectively:
A. culture medium preparation: with reference to anti-interference bacterium and fungi liquid nutrient culture media explanation preparation 50mL/ bottle three feed liquid body nutrient culture media, divide to install in the 250mL triangular flask, and prepare other matching product, it is standby to sterilize;
B. the cultivation of microorganism: in superclean bench, scattered 100mL (g) sample joins in the 250mL triangular flask that cooled 50mL/ bottle three material nutrient culture media are housed with mixing also, shake up and mix back bacterium cultivation 3～6h under 35-37 ℃, fungi is cultivated 10～24h down at 30-32 ℃;
C. microbial cell ATP extracting method: get the 10mL nutrient solution respectively, in 10000r/min centrifugal 5 minutes, remove supernatant, precipitation adds 1mL microbial cell ATP releasing agent Ec, effect 1～5min;
D.ATP bioluminescence assay method: draw 0.1mL sample ATP extract in luminotron, add to separate in right amount and press down agent and 25mmol/L Tricine damping fluid, making volume is 0.9mL, add 0.1mL luciferase-luciferase reagent again, shake up at once, place 25 ℃ of bioluminescent detection instrument to carry out the led pulse counting;
That does e. that standard A TP detection and nutrient culture media and sample mix do not do to cultivate detects liquid in the same old way;
F. the result judges: as sample led pulse counting CPM
SampleTo liquid CPM in the same old way
CK, be the sample positive of carrying disease germs.
(2) anti-interference bacterium and fungi MPN method fast measuring
The low bacterium of determining to contain interfering materials such as antiseptic, sanitizer is counted the micro organism quantity of sample, can adopt approximate number method (MPN method) that sample is detected.Contain the different of the classification of interfering material and test item according to sample, select anti-anticorrosion formulation, anti-sterilization formulation and anti-ozone type bacterium or fungi liquid nutrient culture media respectively:
A. culture medium preparation: prepare anti-interference bacterium and fungi liquid nutrient culture media (need not to add little voltage regulator tube in test tube) by GB coliform quantity multitube fermentation method, and prepare other matching product, it is standby to sterilize;
B. the cultivation of microorganism: in superclean bench, scattered sample joins in the mensuration system by GB coliform quantity multitube fermentation method with mixing also, shake up and mix back bacterium cultivation 3～6h under 35-37 ℃, fungi is cultivated 10～24h down at 30-32 ℃;
C. microbial cell ATP extracting method: each manages nutrient solution, and in 10000r/min centrifugal 5 minutes, remove supernatant, precipitation adds 1mL microbial cell ATP releasing agent Ec, effect 1～5min;
D.ATP bioluminescence assay method: draw 0.1mL sample ATP extract in luminotron, add to separate in right amount and press down agent and 25mmol/L Tricine damping fluid, making volume is 0.9mL, add 0.1mL luciferase-luciferase reagent again, shake up at once, place 25 ℃ of bioluminescent detection instrument to carry out the led pulse counting;
That does e. that standard A TP detection and nutrient culture media and sample mix do not do to cultivate detects liquid in the same old way;
F. the result represents: as sample led pulse counting CPM
SampleTo liquid CPM in the same old way
CK, being the sample positive of carrying disease germs, the positive pipe of record sample number is looked into the MPN table, promptly gets the result.
2) common bacteria and fungi liquid large sample method and MPN method fast measuring
(1) common bacteria and fungi liquid large sample method fast measuring
The sterility that this method is suitable for not containing the sample of interfering materials such as antiseptic, sanitizer detects.
A. culture medium preparation:, divide to install in the 250mL triangular flask, and prepare other matching product, then sterilization with reference to common bacteria and fungi liquid nutrient culture media explanation preparation 50mL/ bottle three feed liquid body nutrient culture media;
B. the cultivation of microorganism: in 100 grades of superclean benches, scattered 100mL (g) sample joins in the 250mL triangular flask that cooled 50mL/ bottle three material nutrient culture media are housed with mixing also, shake up and mix back bacterium cultivation 3～6h under 35-37 ℃, fungi is cultivated 10～24h down at 30-32 ℃;
C. microbial cell ATP extracting method: get the 10mL nutrient solution respectively, in 10000r/min centrifugal 5 minutes, remove supernatant, precipitation adds 1mL microbial cell ATP releasing agent Ec, effect 1～5min;
D.ATP bioluminescence assay method: draw 0.1mL sample ATP extract in luminotron, add to separate in right amount and press down agent and 25mmol/L Tricine damping fluid, making volume is 0.9mL, add 0.1mL luciferase-luciferase reagent again, shake up at once, place 25 ℃ of bioluminescent detection instrument to carry out the led pulse counting;
That does simultaneously that standard A TP detection and nutrient culture media and sample mix do not do to cultivate detects liquid in the same old way;
E. the result judges: as sample led pulse counting CPM
SampleTo liquid CPM in the same old way
CK, be the sample positive of carrying disease germs.
(2) approximate number method (MPN method) fast measuring
Determine that low bacterium counts the micro organism quantity of sample, can adopt approximate number method (MPN method), sample is detected.
A. culture medium preparation:, and prepare other matching product, sterilization then by GB coliform quantity multitube fermentation method preparation common bacteria and fungi liquid nutrient culture media (need not in test tube, to add little voltage regulator tube);
B. the cultivation of microorganism: in superclean bench, scattered sample joins in the mensuration system by GB coliform quantity multitube fermentation method with mixing also, shake up and mix back bacterium cultivation 3～6h under 35-37 ℃, fungi is cultivated 10～24h down at 30-32 ℃;
C. microbial cell ATP extracting method: each manages nutrient solution, and in 10000r/min centrifugal 5 minutes, remove supernatant, precipitation adds 1mL microbial cell ATP releasing agent Ec, effect 1～5min;
D.ATP bioluminescence assay method: draw 0.1mL sample ATP extract in luminotron, add to separate in right amount and press down agent and 25mmol/L Tricine damping fluid, making volume is 0.9mL, add 0.1mL luciferase-luciferase reagent again, shake up at once, place 25 ℃ of bioluminescent detection instrument to carry out the led pulse counting;
That does simultaneously that standard A TP detection and nutrient culture media and sample mix do not do to cultivate detects liquid in the same old way;
E. the result represents: as sample led pulse counting CPM
SampleTo liquid CPM in the same old way
CK, be the sample positive of carrying disease germs; The positive pipe of record sample number is looked into the MPN table, promptly gets the result.
Among the present invention, sample is cultivated in advance by anti-interference bacterium, fungi liquid nutrient culture media or common bacteria, fungi liquid nutrient culture media, increased bioluminescent detection sensitivity.
The fluid nutrient medium that the present invention is mentioned is used to increase bacterium is to have the bacterium of opposing antiseptic, sanitizer, ozone jamming performance and eight kinds of nutrient culture media such as nutrient broth medium that fungi liquid nutrient culture media, common bacteria or fungal culture are used and mould medium, produce by Guangdong Microbes Inst subordinate Huankai Microbes Tech Co., Ltd., Guangdong, all can buy in the said firm, its working concentration sees the following form 1:
Various fluid nutrient medium and the working concentrations that are used to increase bacterium of table 1
Anti-anticorrosion formulation, anti-sterilization formulation and anti-ozone type liquid full-page proof detect nutrient culture media can eliminate antiseptic sorbic acid and potassium sorbate, benzoic acid and Sodium Benzoate respectively, the interference (table 2) of sanitizer chlorine dioxide, Peracetic acid, sodium hypochlorite and hydrogen peroxide and ozone and chlorine residue, when detection contains the sample of interfering material, compare with the common liq nutrient culture media, can improve the sensitivity of detection greatly.
Interfering material and concentration thereof that the anti-interference microbial liquid nutrient culture media of table 2 can be eliminated
The mentioned microbial cell ATP releasing agent Ec of the present invention contains:
1～30g/L?TritonX-100
0.1～5.0g/L hexadecane trimethyl ammonium bromide (CTAB)
0.1～3.0g/L dimethyl sulfoxide (DMSO)
0.01～0.1g/L ethylenediamine tetraacetic acid (EDTA)
0.01～0.1g/L magnesium sulphate (MgSO
4)
Manufacturing process is: add 1～30g TritonX-100,0.1～5.0g CTAB, 0.1～3.0g DMSO, 0.01～0.1g EDTA, 0.01～0.1g MgSO in every liter of aseptic ultrapure water
4, heating makes its dissolving, and it is even to shake, and all reagent adopt to be analyzed purely, finishes the extraction of microbial cell ATP at ambient temperature in 1～5min, easy, quick.
The mentioned sample preparation of the present invention separates that to press down agent be to include glucose unit to be respectively 6,7,8 cyclodextrin isocyclic compound, and concrete manufacturing process is: get 0.1～15.0g and analyze pure cyclodextrin and be dissolved in pH 7.8 Tricine damping fluids and (include 50mmol/L Tricine, 10mmol/L MgSO
4, 1mmol/L EDTA, 1mmol/L DTT) in, making final volume is 1000mL, divides the bottle be filled to 5 sterilizations after the 0.22 μ m membrane filtration degerming, standby in 4 ℃ of refrigerator storage.This is separated but agent can be got rid of the interference of materials such as kation, negative ion and zwitterionic surfactant to analyzing.
[embodiment]
Embodiment 1: bacterial liquid full-page proof fast measuring in the bottled, potable natural mineral water
Because of the bottled, potable natural mineral water is used the ozone disinfection usually, therefore select for use anti-ozone type bacterial liquid full-page proof to detect nutrient culture media:
A. culture medium preparation: detect nutrient culture media explanation preparation 50mL/ bottle three feed liquid body nutrient culture media with reference to anti-ozone type bacterial liquid full-page proof, divide to install in the 250mL triangular flask, and prepare other matching product, then sterilization;
B. the cultivation of microorganism: in 100 grades of superclean benches, 100mL sample to be checked is joined in the 250mL triangular flask that cooled 50mL/ bottle three material nutrient culture media are housed, shake up and mix back cultivation 6h under 35-37 ℃;
C. microbial cell ATP extracting method: get the 10mL nutrient solution, in 10000r/min centrifugal 5 minutes, remove supernatant, precipitation adds 1mL microbial cell ATP releasing agent Ec, effect 1～5min;
D.ATP bioluminescence assay method: draw 0.1mL sample ATP extract in luminotron, add to separate in right amount and press down agent and 25mmol/L Tricine damping fluid, making volume is 0.9mL, add 0.1mL luciferase-luciferase reagent again, shake up at once, place 25 ℃ of bioluminescent detection instrument to carry out the led pulse counting;
That does e. that standard A TP detection and nutrient culture media and sample mix do not do to cultivate detects liquid in the same old way;
F. led pulse count results: sample led pulse counting CPM
Sample=13450
To liquid CPM in the same old way
CK=1103
The result judges: sample led pulse counting CPM
SampleTo liquid CPM in the same old way
CK, be the sample positive of carrying disease germs.
Example 2: the MPN method fast measuring of the saccharomycete of fruit drink and mould
Fruit drink is added with antiseptic usually extending the shelf life, but its bacteria containing amount of product that harsh output is come is very low, and the low bacterium that therefore will determine to contain antiseptic is counted the micro organism quantity of sample, can adopt approximate number method (MPN method), and sample is detected:
A. culture medium preparation; Detect nutrient culture media (need not in test tube, to add little voltage regulator tube) by the anti-anticorrosion formulation fungi liquid full-page proof of GB coliform quantity multitube fermentation method (9 pipe method) preparation, every pipe 10mL, and prepare other matching product, sterilization then;
B. the cultivation of microorganism: in 100 grades of superclean benches, scattered sample joins in the mensuration system by GB coliform quantity multitube fermentation method with mixing also, shakes up to mix back cultivation 12h under 30-32 ℃;
C. microbial cell ATP extracting method: each manages nutrient solution, and in 10000r/min centrifugal 5 minutes, remove supernatant, precipitation adds 1mL microbial cell ATP releasing agent Ec, effect 1～5min;
D.ATP bioluminescence assay method: draw 0.1mL sample ATP extract in luminotron, add to separate in right amount and press down agent and 25mmol/L Tricine damping fluid, making volume is 0.9mL, add 0.1mL luciferase-luciferase reagent again, shake up at once, place 25 ℃ of bioluminescent detection instrument to carry out the led pulse counting;
That does e. that standard A TP detection and nutrient culture media and sample mix do not do to cultivate detects liquid in the same old way;
F. the result represents: sample led pulse counting CPM
SampleTo liquid CPM in the same old way
CK, be the sample positive of carrying disease germs; The positive pipe of record sample number is looked into the MPN table, promptly gets the result, and the sample bacteria containing amount is 460MPN/100mL.
Table 3 luminous detection result
Claims (6)
1, the anti-interference method for quick of a kind of microorganism, what contain the classification of interfering material and test item at first per sample differently selects for use anti-interference or sample is carried out the liquid large sample method to the common micro-organisms nutrient culture media or the most approximate counting method increases bacterium; With cell ATP releasing agent Ec sample is handled then, added again and utilize the luminous detection instrument to carry out the led pulse counting after an amount of separating presses down agent and luciferase-luciferase reagent;
Wherein, sample being carried out the liquid large sample method is undertaken by following step:
A. culture medium preparation: contain the different of the classification of interfering material and test item according to sample, select anti-anticorrosion formulation, anti-sterilization formulation and anti-ozone type bacterium or fungi liquid nutrient culture media respectively; To the sample that do not contain antiseptic, sanitizer interfering material different choice common bacteria or fungi liquid nutrient culture media according to test item; Then select the mould fluid nutrient medium when surveying mould for use; By explanation preparation 50mL/ bottle three feed liquid body nutrient culture media, divide to install in the 250mL triangular flask, and prepare other matching product, it is standby to sterilize;
B. the cultivation of microorganism: in superclean bench, scattered 100mL sample joins in the 250mL triangular flask that cooled 50mL/ bottle three material nutrient culture media are housed with mixing also, shake up and mix back bacterium cultivation 3～6h under 35-37 ℃, fungi is cultivated 10～24h down at 30-32 ℃;
C. microbial cell ATP extracting method: get the 10mL nutrient solution respectively, in 10000r/min centrifugal 5 minutes, remove supernatant, precipitation adds 1mL microbial cell ATP releasing agent Ec, effect 1～5min;
D.ATP bioluminescence assay method: draw 0.1mL sample ATP extract in luminotron, add to separate in right amount and press down agent and 25mmol/L Tricine damping fluid, making volume is 0.9mL, add 0.1mL luciferase-luciferase reagent again, shake up at once, place the bioluminescent detection instrument to carry out the led pulse counting;
E. do that standard A TP detection and nutrient culture media and sample mix do not do to cultivate to the luminous detection of liquid in the same old way;
F. the result judges: as sample led pulse counting CPM
SampleTo liquid CPM in the same old way
CK, be the sample positive of carrying disease germs;
Sample is carried out the MPN method to be increased bacterium and is undertaken by following step:
A. culture medium preparation: contain the different of the classification of interfering material and test item according to sample, select anti-anticorrosion formulation, anti-sterilization formulation and anti-ozone type bacterium or fungi liquid nutrient culture media respectively; To the sample that do not contain antiseptic, sanitizer interfering material different choice common bacteria or fungi liquid nutrient culture media according to test item; Then select the mould fluid nutrient medium when surveying mould for use; By GB coliform quantity multitube fermentation method obtaining liq nutrient culture media, need not in test tube, to add little voltage regulator tube, and prepare other matching product, it is standby to sterilize;
B. the cultivation of microorganism: in superclean bench, scattered sample joins in the mensuration system by GB coliform quantity multitube fermentation method with mixing also, shake up and mix back bacterium cultivation 3～6h under 35-37 ℃, fungi is cultivated 10～24h down at 30-32 ℃;
C. microbial cell ATP extracting method: each manages nutrient solution, and in 10000r/min centrifugal 5 minutes, remove supernatant, precipitation adds 1mL microbial cell ATP releasing agent Ec, effect 1～5min;
D.ATP bioluminescence assay method: draw 0.1mL sample ATP extract in luminotron, add to separate in right amount and press down agent and 25mmol/L Tricine damping fluid, making volume is 0.9mL, add 0.1mL luciferase-luciferase reagent again, shake up at once, place the bioluminescent detection instrument to carry out led pulse counting, do simultaneously that standard A TP detection and nutrient culture media and sample mix do not do to cultivate to the luminous detection of liquid in the same old way;
E. the result represents: as sample led pulse counting CPM
SampleTo liquid CPM in the same old way
CK, be the sample positive of carrying disease germs; The positive pipe of record sample number is looked into the MPN table, gets the sample bacteria containing amount;
It is characterized in that ATP releasing agent Ec includes:
1～30g/L?TritonX-100
0.1～5.0g/L hexadecane trimethyl ammonium bromide
0.1～3.0g/L dimethyl sulfoxide
0.01～0.1g/L ethylenediamine tetraacetic acid
0.01～0.1g/L?MgSO
4。
2, the described detection method of claim 1, the manufacturing process of ATP releasing agent Ec is: add 1～30gTritonX-100,0.1～5.0g hexadecane trimethyl ammonium bromide, 0.1～3.0g dimethyl sulfoxide, 0.01～0.1g ethylenediamine tetraacetic acid, 0.01～0.1g MgSO in every liter of aseptic ultrapure water
4, heating makes its dissolving, and it is even to shake.
3, claim 1 or 2 described detection methods are separated and are pressed down agent and contain the ring compound that glucose unit is respectively 6,7 or 8 cyclodextrin.
4, the described detection method of claim 3, separate the manufacturing process that presses down agent and be: get 0.1～15.0g cyclodextrin and be dissolved in that to make final volume in the pH7.8Tricine damping fluid be 1000mL, wherein damping fluid includes 50mmol/L Tricine, 10mmol/L MgSO
4, 1mmol/L ethylenediamine tetraacetic acid, 1mmol/L dithiothreitol (DTT).
5, the described detection method of claim 1, common bacteria or fungi liquid nutrient culture media are nutrient broth medium, working concentration is: 22-24g/L; The working concentration of anti-anticorrosion formulation bacteria culture media is: 22-28g/L; The working concentration of anti-sterilization formulation bacteria culture media is: 20-24g/L; The working concentration of anti-ozone type bacteria culture media is: 19-23g/L; The working concentration of mould fluid nutrient medium is: 14-18g/L; Anti-anticorrosion formulation fungi culture medium working concentration is: 21-25g/L; Anti-sterilization formulation fungi culture medium working concentration is: 17-21g/L; Anti-ozone type fungi culture medium working concentration is: 16-20g/L.
6, the described detection method of claim 2, common bacteria or fungi liquid nutrient culture media are nutrient broth medium, working concentration is: 22-24g/L; The working concentration of anti-anticorrosion formulation bacteria culture media is: 22-28g/L; The working concentration of anti-sterilization formulation bacteria culture media is: 20-24g/L; The working concentration of anti-ozone type bacteria culture media is: 19-23g/L; The working concentration of mould fluid nutrient medium is: 14-18g/L; Anti-anticorrosion formulation fungi culture medium working concentration is: 21-25g/L; Anti-sterilization formulation fungi culture medium working concentration is: 17-21g/L; Anti-ozone type fungi culture medium working concentration is: 16-20g/L.
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| CN110274896B (en) * | 2019-03-15 | 2021-11-30 | 李文杰 | ATP bioluminescence lgCB-lgIBMethod for evaluating fungus killing effect of liquid disinfectant by standard yeast method |
| CN110272945A (en) * | 2019-03-15 | 2019-09-24 | 李文杰 | ATP bioluminescence lgCA-lgIAThe method of calibration curve method detection anti-bacteria ceramic anti-fungal property |
-
2004
- 2004-04-08 CN CNB2004100267945A patent/CN100507524C/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| ATP生物发光法－一种新的快速微生物检测技术. 朱全国，胡楚民.现代商检科技，第3卷第3期. 1993 |
| ATP生物发光法－一种新的快速微生物检测技术. 朱全国，胡楚民.现代商检科技，第3卷第3期. 1993 * |
| 抗干扰微生物培养基的抗干扰性能研究. 吴清平，蔡芷荷，张菊梅等.微生物学通报，第26卷第3期. 1999 |
| 抗干扰微生物培养基的抗干扰性能研究. 吴清平，蔡芷荷，张菊梅等.微生物学通报，第26卷第3期. 1999 * |
| 抗干扰微生物检测技术研究. 吴清平，蔡芷荷，张菊梅等.微生物学通报，第27卷第2期. 2000 |
| 抗干扰微生物检测技术研究. 吴清平，蔡芷荷，张菊梅等.微生物学通报，第27卷第2期. 2000 * |
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