CN103940812A - Method for rapidly detecting coliforms by means of spectrophotometry and application of method - Google Patents
Method for rapidly detecting coliforms by means of spectrophotometry and application of method Download PDFInfo
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- 238000002798 spectrophotometry method Methods 0.000 title claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 41
- 239000007788 liquid Substances 0.000 claims abstract description 40
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 29
- 239000008101 lactose Substances 0.000 claims abstract description 29
- 238000000855 fermentation Methods 0.000 claims abstract description 22
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- ABIUHPWEYMSGSR-UHFFFAOYSA-N bromocresol purple Chemical compound BrC1=C(O)C(C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C=C(Br)C(O)=C(C)C=2)=C1 ABIUHPWEYMSGSR-UHFFFAOYSA-N 0.000 claims abstract description 13
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Abstract
The invention relates to a method for rapidly detecting coliforms by means of spectrophotometry. By utilizing the characteristic that coliforms can be utilized to ferment lactose at 37 DEG C to produce acid and gas and taking a self-made culture medium as the main role, bromcresol purple serving as an indicator and triphenyltetrazoliumchloride (TTC) solutions are added, after coliforms is fermented and cultured, red precipitates can be produced and culture liquid turns from purple into yellow; the characteristic that the change degree of the color of the fermentation liquid is in positive correlation with the quantity of coliforms contained in the solutions is utilized, the content of coliforms in sample liquid to be detected can be obtained. Compared with an existing national standard method, the method has the advantages that the detection time is greatly shortened, the quantitative value of coliforms in the sample can be detected when coliforms are cultured for 60-400 minutes after sample adding, the trial range is wide, the requirement on short detection time required by various detection fields is met, and the method has good application prospect in the field of rapid detection in the future.
Description
Technical field
The invention belongs to microorganism detection technical field, relate to the detection of coliform, especially a kind of method and application of spectrophotometric method fast detecting coliform.
Background technology
Coliform refers at 37 DEG C can lactose fermenters, produce sour aerogenesis, aerobic and amphimicrobian Gram-negative sporeless bacterium, is mainly derived from human and animal excreta.The content of coliform in sample is the important indicator of inspection food sanitation safe.Conventional detection method has traditional zymotic method, Rapid paper strip method, filter membrane method, zymolyte method, polymerase chain method, fluorescence in situ hybridization at present, but these known detection methods still do not need 24h-72h not etc. detection time, for the production testing such as industrial waste water, food, coliform sense cycle is longer, and causing a large amount of samples to be detained cannot process in time.In order to solve existing methods deficiency, shorten detection time, the work efficiency of checking in increase water quality detection, food production, the method for quick of seeking coliform is very necessary.
At present, for example filter membrane method, zymolyte method of coliform detection technique in prior art, there is the defect that cost is higher, operation is comparatively loaded down with trivial details in molecular Biological Detection method; In addition, paper disk method is also that most fields detects comparatively conventional method at present, but scraps of paper nutrient culture media exists the shortcoming that needs low temperature to preserve, and is not suitable for field work.
By retrieval, find one section of following patent publication us relevant to patented claim of the present invention:
A kind of method for quick (CN101914609A) of coliform, the present invention relates to a kind of method for quick of coliform.Utilize coliform cell meeting aerogenesis in short time incubation, can produce acid by lactose fermenters, coliform cell is combined with Yihong methylene blue, and atropurpureus or aubergine precipitation that Yihong methylene blue Mixed culture solution is generated, count by biological microscope microscopy, atropurpureus or aubergine precipitum are passed through to the image number that Gram’s staining presents negative bacterium, count the rapid detection method for coliform group bacteria of coliform quantity; Or due to atropurpureus or the mauve precipitation of the generation of Yihong methylene blue Mixed culture solution, and Yihong methylene blue Mixed culture solution colour is changed, the degree characteristic relevant to coliform cell quantity of utilizing Yihong methylene blue Mixed culture solution colour to change, the rapid detection method for coliform group bacteria of identification coliform cell number, the inventive method is applicable to the fast detecting of coliform in biological educational circles, the world of medicine, food circle and various article.
Through contrast, the present invention is that from the difference that above-mentioned patent publication exists medium component is different, Liquid Culture based component is through improveing to shorten incubation time, variable color reason difference, detect special nanometer value difference, therefore mark Qu Butong, and be that GB admits that paper disk method indicator composition used (containing bromcresol purple and TTC) detects for spectrophotometric method after fluid nutrient medium for the first time, there are the different of essence from above-mentioned patent publication in patented claim of the present invention therefore.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, provide that a kind of method is simple, the method for the spectrophotometric method fast detecting coliform of coliform quantity in can fast detecting sample, the method can be applied to the fast detecting of the coliform of water quality monitoring, biology, food, health aspect.
To achieve these goals, technical scheme of the present invention is as follows:
A method for spectrophotometric method fast detecting coliform, step is as follows:
(1) solution preparation:
Lactose fermentation detects nutrient solution: 50-90 μ L2%(mass concentration) bromcresol purple solution, 4g tryptone, 0.5g lactose, 0.5gD-sweet mellow wine, 0.75gNaCl, 0.02g sodium lauryl sulfate, 0.01g NaTDC, 100mL distilled water, pH6.8-7.2,115 DEG C, stand-by after 15min sterilizing;
TTC solution: TTC adds sterilized water to make 0.5-3%(mass concentration) solution, keeps in Dark Place, stand-by;
(2) testing process:
1. the preparation of the even liquid of sample:
Sample to be checked is got in sterile working, 8000r/min～10000r/min homogeneous 1min～2min, and making volume ratio is the even liquid of sample of 1:10, or quality: the even liquid of sample that volume (g:L) is 1:10;
Or, if when liquid sample detection limit≤10cfu/mL to be checked, directly detect by stoste;
2. filter:
With the even liquid of middling speed quantitative filter paper filtered sample, obtain filtrate; In filtrate: the ratio that the volume ratio of concentrated liquid to be checked is 5-10:1 film filter filter liquor, obtains concentrated liquid to be checked; Getting concentrated liquid to be checked contains in lactose fermentation detection nutrient solution for the ratio of 1:5-10 adds by volume, simultaneously, liquid to be checked in concentrated: the volume ratio mL of TTC solution: the ratio that μ L is 1:30-80 is got TTC solution and detected in nutrient solution to lactose fermentation, is placed in 37 DEG C of cultivations;
3. the drafting of typical curve:
Cultivate after 60-400min, detect in nutrient culture media and have change color and sink to the bottom material production with redness, get the nutrient solution in detector tube, through centrifugal treating fermentation liquor, 5000-7000r/min is centrifugal, centrifugal 2min-5min, getting supernatant is placed in quartz colorimetric utensil spectrophotometer and detects, measure the light absorption value of the special nanometers of 360nm-500nm, set up the corresponding linear relationship between different content coliform mixed-culture medium color and coliform content, drawing standard curve;
4. the detection of sample to be checked:
While detecting sample, by step 1.-method 3. processes and cultivates sample to be checked, nutrient solution has variable color and deposited phenomenon, press abovementioned steps by after medium centrifugal, get supernatant and measure light absorption value at 360nm-500nm place, record data, compare with 3. Plays curve, determine contained coliform group count in unknown sample according to comparing result, finally draw contained coliform numerical value in sample to be checked.
And, described step (2) 3. Plays curve regression equation be:
lgY=14.89X-1.33
R
2=0.991
In formula: Y: the expectation content of coliform in unknown sample
The OD value at X:360nm-500nm place
R: related coefficient.
Application in the fast detecting aspect of the coliform of the method for spectrophotometric method fast detecting coliform as above aspect water quality monitoring, biology, food, health.
Advantage of the present invention and good effect are:
1, the inventive method is than existing national standard method, on detection time, there is significantly reduction, after application of sample, cultivate 60min-400min and can detect the coliform numerical value in sample, scope on probation is wider, meet numerous detection field requirement short demands detection time, had good application prospect in following fast detecting field.
2, the inventive method operation is comparatively easy, and culture medium cost is lower, does not need low temperature to preserve, easy to use.
Brief description of the drawings
Fig. 1 is the graph of a relation of coliform content logarithm value of the present invention and absorbance.
Embodiment
Below in conjunction with embodiment, the present invention is further described; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The method using in the present invention, if no special instructions, is the conventional method of this area; The reagent using in the present invention, if no special instructions, is the conventional reagent of this area; Each raw material relative content in the present invention if no special requirements, is mass volume ratio.
Integral Thought of the present invention and detection principle are as follows:
The present invention utilizes coliform can produce acid, biogas production characteristics by lactose fermenters at 37 DEG C, taking self-control nutrient culture media as main, wherein add the colourless TTC of indicator bromcresol purple and TTC() solution, using bromcresol purple and TTC as indicator, bromcresol purple and TTC are added in lactose fermentation nutrient culture media, be mixed with mixed-culture medium, utilize coliform lactose fermenters to produce acid and can make bromcresol purple flavescence, meanwhile, due to the effect of hydrogenlyase, can make formic acid decompose and produce CO
2and H
2red precipitation TTF(tri-Ben formazan is produced in the hydrogen of taking off in this process and TTC reaction), therefore, in test tubes, nutrient solution is understood flavescence and generates with red precipitate, after reaction finishes, and centrifuging and taking supernatant, measure in specific nanometers with spectrophotometer, after image data, the nutrient solution change color degree Criterion curve causing according to variable concentrations coliform content, after this sample to be checked of unknown coliform content is calculated coliform content according to typical curve.The inventive method is applicable to fast detecting, the analysis of coliform in water quality monitoring, biology, health, food etc.
The lactose fermentation using in the present invention detects being formulated as follows of nutrient solution:
50-90 μ L2%(mass concentration) bromcresol purple solution, 4g tryptone, 0.5g lactose, 0.5gD-sweet mellow wine, 0.75gNaCl, 0.02g sodium lauryl sulfate, 0.01g NaTDC, 100mL distilled water, pH6.8-7.2,115 DEG C, stand-by after 15min sterilizing;
Being formulated as follows of the TTC solution using in the present invention:
TTC adds sterilized water to make 0.5-3%(mass concentration) solution, keeps in Dark Place, stand-by.
Embodiment 1
A method for spectrophotometric method fast detecting coliform, step is as follows:
(1) solution preparation:
Lactose fermentation detects nutrient solution: 50 μ L2%(mass concentrations) bromcresol purple solution, 4g tryptone, 0.5g lactose, 0.5gD-sweet mellow wine, 0.75gNaCl, 0.02g sodium lauryl sulfate, 0.01g NaTDC, 100mL distilled water, pH6.8,115 DEG C, stand-by after 15min sterilizing;
TTC solution: TTC adds sterilized water to make 3%(mass concentration) solution, keeps in Dark Place, stand-by.
(2) testing process:
1. the preparation of the even liquid of sample:
Sample 25g to be checked or 25mL are got in sterile working, pack into containing (built-in some beaded glasses) in the triangular flask of 225mL stroke-physiological saline solution, 8000r/min～10000r/min homogeneous 1min～2min, make the even liquid of sample when liquid sample detection limit to be checked lower (≤10cfu/mL) (if, also can directly detect by stoste) of volume ratio 1:10;
2. filter:
With the even liquid of middling speed quantitative filter paper filtered sample, repeatedly rinse once, get 5mL filtrate and concentrate out 1mL bacterium liquid with film filter, getting concentrated liquid to be checked adds in the test tubes that contains 9mL lactose fermentation detection nutrient solution, simultaneously, pipette 30 μ LTTC solution in lactose test tubes with pipettor, be placed in 37 DEG C of cultivations, cultivate 360min;
3. the detection of sample to be checked:
With the sample making typical curve of known coliform content.While detecting sample, by step 1.-method 2. processes and cultivates sample to be checked, nutrient solution has variable color and deposited phenomenon, gets the nutrient solution in detector tube by abovementioned steps, through centrifugal treating fermentation liquor, 7000r/min is centrifugal, and centrifugal 5min gets supernatant and measures light absorption value at 360nm-500nm place, record data, compare with typical curve, determine contained coliform group count in unknown sample according to comparing result, finally draw contained coliform numerical value in unknown sample.
Embodiment 2
A method for spectrophotometric method fast detecting coliform, step is as follows:
(1) solution preparation:
Lactose fermentation detects nutrient solution: 90 μ L2%(mass concentrations) bromcresol purple solution, 4g tryptone, 0.5g lactose, 0.5gD-sweet mellow wine, 0.75gNaCl, 0.02g sodium lauryl sulfate, 0.01g NaTDC, 100mL distilled water, pH7.2,115 DEG C, stand-by after 15min sterilizing;
TTC solution: TTC adds sterilized water to make 1.5%(mass concentration) solution, keeps in Dark Place, stand-by.
(2) testing process:
1. the preparation of the even liquid of sample:
Sample 25g to be checked or 25mL are got in sterile working, pack into containing (built-in some beaded glasses) in the triangular flask of 225mL stroke-physiological saline solution, 8000r/min～10000r/min homogeneous 1min～2min, make the even liquid of sample (if liquid sample detection limit to be checked lower≤when 10cfu/mL, also can directly detect by stoste) of volume ratio 1:10;
2. filter:
With the even liquid of middling speed quantitative filter paper filtered sample, repeatedly rinse once, get 9mL filtrate and concentrate out 1mL bacterium liquid with film filter, getting concentrated liquid to be checked adds in the test tubes that contains 9mL lactose fermentation detection nutrient solution, simultaneously, pipette 80 μ LTTC solution in lactose test tubes with pipettor, be placed in 37 DEG C of cultivations, cultivate 300min;
3. the detection of sample to be checked:
With the sample making typical curve of known coliform content.Detect when sample, by step 1.-method 2. processes and cultivates sample to be checked, nutrient solution has variable color and deposited phenomenon, get the nutrient solution in detector tube by abovementioned steps, through centrifugal treating fermentation liquor, 7000r/min is centrifugal, centrifugal 5min, get supernatant and measure light absorption value at 360nm-500nm place, record data, compare with typical curve, determine contained coliform group count in unknown sample according to comparing result, finally draw contained coliform numerical value in unknown sample.
Embodiment 3
A method for spectrophotometric method fast detecting coliform, step is as follows:
(1) solution preparation:
Lactose fermentation detects nutrient solution: 60 μ L2%(mass concentrations) bromcresol purple solution, 4g tryptone, 0.5g lactose, 0.5gD-sweet mellow wine, 0.75gNaCl, 0.02g sodium lauryl sulfate, 0.01g NaTDC, 100mL distilled water, pH6.9,115 DEG C, stand-by after 15min sterilizing;
TTC solution: TTC adds sterilized water to make 2%(mass concentration) solution, keeps in Dark Place, stand-by.
(3) testing process:
1. the preparation of the even liquid of sample:
Sample 25g to be checked or 25mL are got in sterile working, pack into containing (built-in some beaded glasses) in the triangular flask of 225mL stroke-physiological saline solution, 8000r/min～10000r/min homogeneous 1min～2min, make the even liquid of sample (if liquid sample detection limit to be checked lower≤when 10cfu/mL, also can directly detect by stoste) of volume ratio 1:10;
2. filter:
With the even liquid of middling speed quantitative filter paper filtered sample, repeatedly rinse once, get 10mL filtrate and concentrate out 1mL bacterium liquid with film filter, getting concentrated liquid to be checked adds in the test tubes that contains 9mL lactose fermentation detection nutrient solution, simultaneously, pipette 50 μ LTTC solution in lactose test tubes with pipettor, be placed in 37 DEG C of cultivations, cultivate 300min;
3. the detection of sample to be checked:
With the sample making typical curve of known coliform content.While detecting sample, by step 1.-method 2. processes and cultivates sample to be checked, nutrient solution has variable color and deposited phenomenon, gets the nutrient solution in detector tube by abovementioned steps, through centrifugal treating fermentation liquor, 7000r/min is centrifugal, and centrifugal 5min gets supernatant and measures light absorption value at 360nm-500nm place, record data, compare with typical curve, determine contained coliform group count in unknown sample according to comparing result, finally draw contained coliform numerical value in unknown sample.
The method for making step of Plays curve of the present invention is as follows:
The drafting of typical curve:
Add with the sample of known coliform content in the test tubes of lactose fermentation detection nutrient solution, cultivate after 60-400min, detect in nutrient culture media and have change color and sink to the bottom material production with redness, get the nutrient solution in detector tube, through centrifugal treating fermentation liquor, 5000-7000r/min is centrifugal, centrifugal 2min-5min, getting supernatant is placed in quartz colorimetric utensil spectrophotometer and detects, measure the light absorption value (being OD value) of the special nanometers of 360nm-500nm, set up the corresponding linear relationship between different content coliform mixed-culture medium color and coliform content, drawing standard curve.
The testing result of three embodiment of the present invention average after draw typical curve, result as shown in Figure 1, as can be seen from the figure:
The regression equation of standard spectrophotometric value is:
lgY=14.89X-1.33
R
2=0.991
In formula: Y: the expectation content of coliform in unknown sample
The OD value at X:360nm-500nm place
R: related coefficient.
The coherent detection result of the method for spectrophotometric method fast detecting coliform of the present invention:
Use detection method of the present invention to detect the coliform in the coliform in non-polluted egg, non-Fermented bean product and the coliform in biscuit, testing result is in table 1, table 2, table 3.
Coliform testing result in table 1 non-polluted egg
Coliform testing result in the non-Fermented bean product of table 2
The present invention is taking bromcresol purple and TTC as indicator, change and produce two comparatively obvious phenomenons of red precipitate as judging that coliform produces the standard of sour aerogenesis by solution colour, meanwhile, predict coliform content in unknown sample by solution colour intensity of variation.In embodiment, add in 9mL test tubes with concentrating sample 1mL bacterium liquid, cultivate after 60min-400min for 37 DEG C, at 360nm-500nm wavelength, place carries out light absorption value measurement, draw and detect after the contained coliform quantity of sample according to typical curve, contrast National Standard Method testing result, find that testing result of the present invention all in the credible scope of National Standard Method testing result, illustrates that detection method of the present invention has certain credibility compared with National Standard Method, can be used as effective detection method that coliform detects.Because National Standard Method detecting step is loaded down with trivial details, detection time is longer, the inventive method not only has significantly reduction easy and simple to handle, detection time, and scope on probation is wider, greatly meet the various demands of commercial production and small-size laboratory, estimated to have good application prospect in following fast detecting field.
Claims (3)
1. a method for spectrophotometric method fast detecting coliform, is characterized in that: step is as follows:
(1) solution preparation:
Lactose fermentation detects nutrient solution: 50-90 μ L2%(mass concentration) bromcresol purple solution, 4g tryptone, 0.5g lactose, 0.5gD-sweet mellow wine, 0.75gNaCl, 0.02g sodium lauryl sulfate, 0.01g NaTDC, 100mL distilled water, pH6.8-7.2,115 DEG C, stand-by after 15min sterilizing;
TTC solution: TTC adds sterilized water to make 0.5-3%(mass concentration) solution, keeps in Dark Place, stand-by;
(2) testing process:
1. the preparation of the even liquid of sample:
Sample to be checked is got in sterile working, 8000r/min～10000r/min homogeneous 1min～2min, and making volume ratio is the even liquid of sample of 1:10, or quality: the even liquid of sample that volume (g:L) is 1:10;
Or, if when liquid sample detection limit≤10cfu/mL to be checked, directly detect by stoste;
2. filter:
With the even liquid of middling speed quantitative filter paper filtered sample, obtain filtrate; In filtrate: the ratio that the volume ratio of concentrated liquid to be checked is 5-10:1 film filter filter liquor, obtains concentrated liquid to be checked; Getting concentrated liquid to be checked contains in lactose fermentation detection nutrient solution for the ratio of 1:5-10 adds by volume, simultaneously, liquid to be checked in concentrated: the volume ratio mL of TTC solution: the ratio that μ L is 1:30-80 is got TTC solution and detected in nutrient solution to lactose fermentation, is placed in 37 DEG C of cultivations;
3. the drafting of typical curve:
Cultivate after 60-400min, detect in nutrient culture media and have change color and sink to the bottom material production with redness, get the nutrient solution in detector tube, through centrifugal treating fermentation liquor, 5000-7000r/min is centrifugal, centrifugal 2min-5min, getting supernatant is placed in quartz colorimetric utensil spectrophotometer and detects, measure the light absorption value of the special nanometers of 360nm-500nm, set up the corresponding linear relationship between different content coliform mixed-culture medium color and coliform content, drawing standard curve;
4. the detection of sample to be checked:
While detecting sample, by step 1.-method 3. processes and cultivates sample to be checked, nutrient solution has variable color and deposited phenomenon, press abovementioned steps by after medium centrifugal, get supernatant and measure light absorption value at 360nm-500nm place, record data, compare with 3. Plays curve, determine contained coliform group count in unknown sample according to comparing result, finally draw contained coliform numerical value in sample to be checked.
2. the method for spectrophotometric method fast detecting coliform according to claim 1, is characterized in that: described step (2) 3. Plays curve regression equation be:
lgY=14.89X-1.33
R
2=0.991
In formula: Y: the expectation content of coliform in unknown sample
The OD value at X:360nm-500nm place
R: related coefficient.
3. the application in the fast detecting aspect of the coliform of the method for spectrophotometric method fast detecting coliform as claimed in claim 1 or 2 aspect water quality monitoring, biology, food, health.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410131441.5A CN103940812B (en) | 2014-04-02 | 2014-04-02 | A kind of spectrophotography quickly detects method and the application of coliform |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410131441.5A CN103940812B (en) | 2014-04-02 | 2014-04-02 | A kind of spectrophotography quickly detects method and the application of coliform |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105754849A (en) * | 2016-03-31 | 2016-07-13 | 吉林农业大学 | Total bacterial count testing slip and preparation method and application thereof |
| CN108663361A (en) * | 2018-05-22 | 2018-10-16 | 福州大学 | A kind of method of biomass in quick measurement liquid state fermentation liquid |
| CN110987834A (en) * | 2019-11-26 | 2020-04-10 | 佛山欧神诺陶瓷有限公司 | Method for detecting antibacterial performance of ceramic |
| CN111172230A (en) * | 2020-03-19 | 2020-05-19 | 天津科技大学 | Coliform group short-term test paper piece |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754849A (en) * | 2016-03-31 | 2016-07-13 | 吉林农业大学 | Total bacterial count testing slip and preparation method and application thereof |
| CN105754849B (en) * | 2016-03-31 | 2018-02-16 | 吉林农业大学 | A kind of total plate count test piece, preparation method and applications |
| CN108663361A (en) * | 2018-05-22 | 2018-10-16 | 福州大学 | A kind of method of biomass in quick measurement liquid state fermentation liquid |
| CN108663361B (en) * | 2018-05-22 | 2020-12-25 | 福州大学 | Method for rapidly determining biomass in liquid fermentation broth |
| CN110987834A (en) * | 2019-11-26 | 2020-04-10 | 佛山欧神诺陶瓷有限公司 | Method for detecting antibacterial performance of ceramic |
| CN111172230A (en) * | 2020-03-19 | 2020-05-19 | 天津科技大学 | Coliform group short-term test paper piece |
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| CN103940812B (en) | 2016-07-13 |
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