CN103940812B - A kind of spectrophotography quickly detects method and the application of coliform - Google Patents
A kind of spectrophotography quickly detects method and the application of coliform Download PDFInfo
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Abstract
The present invention relates to a kind of method that spectrophotography quickly detects coliform, utilize coliform can produce acid, biogas production characteristics with lactose fermenters at 37 DEG C, based on self-made medium, wherein add indicator bromocresol purple and TTC solution, red precipitate can not only be produced after coliform fermentation culture, and culture fluid can be made to there occurs the color change being become yellow from purple, the intensity of variation coliform quantity contained with solution utilizing this fermentation liquid color becomes positively related characteristic, can draw the content of coliform in measuring samples liquid.The inventive method is compared to existing national standard method, the detection time has and has significantly reduced, after sample-adding, cultivate 60min 400min can detect the coliform numerical value in sample, scope on probation is wider, meet numerous detection field requirement detection time short demand, have preferable application prospect in following field of fast detection.
Description
Technical field
The invention belongs to technical field of microbial detection, relate to the detection of coliform, especially a kind of spectrophotography
Quickly detect method and the application of coliform.
Background technology
Coliform refer at 37 DEG C can lactose fermenters, produce acid aerogenesis, aerobic and amphimicrobian Gram-negative without
Bacillus cereus, is mainly derived from human and animal excreta.Coliform content in the sample is the important finger of inspection food sanitation safe
Mark.At present conventional detection method has traditional fermentation process, Rapid paper strip method, filter membrane method, zymolyte method, polymerase chain method, glimmering
Light hybridization in situ, but these known detection methods detect the time and still need to 24h-72h, raw for industrial wastewater, food etc.
For producing detection, the coliform detection cycle is longer, causes a large amount of sample retention to process in time.In order to solve existing method
Deficiency, shorten the detection time, in increase water quality detection, food production check work efficiency, seek the quick of coliform
Detection method is the most necessary.
At present, coliform detection technique such as filter membrane method, zymolyte method in prior art, molecular biological assay is deposited
Relatively costly, operate relatively complicated defect；It addition, paper disk method is also the side that most fields detection is more commonly used at present
Method, but there is the shortcoming needing cryopreservation in scraps of paper culture medium, is not suitable for field work.
By retrieval, find a following patent publication us relevant to present patent application:
The method for quick (CN101914609A) of a kind of coliform, the present invention relates to the quick of a kind of coliform
Detection method.Utilize coliform cell meeting aerogenesis in short time incubation, acid can be produced with lactose fermenters, make coliform
Cell is combined with Yihong methylene blue, and makes atropurpureus or the aubergine precipitation of Yihong methylene blue mixed culture solution generation, by biology
Sediments microscope inspection counts, and through Gram’s staining, atropurpureus or aubergine precipitum are presented the image of negative antibacterial
Number, counts the rapid detection method for coliform group bacteria of coliform quantity；Or owing to methylene blue mixed culture solution in Yihong generates
Atropurpureus or mauve precipitation, and make the mixed culture solution colour change of Yihong methylene blue, utilize Yihong methylene blue mixed culture
The degree characteristic relevant to coliform cell quantity of solution colour change, the coliform of identification coliform cell number is fast
Speed detection method, the inventive method is suitable for the quick inspection of coliform in biological educational circles, the world of medicine, food circle and various article
Survey.
Through contrast, the difference that the present invention exists from above-mentioned patent publication is that medium component is different, and liquid is trained
Supporting based component through improvement to shorten incubation time, tarnish reasons is different, detects special nanometer value different, therefore marks Qu Butong,
And be that GB recognizes indicator composition used by paper disk method (containing bromocresol purple and TTC) light splitting after fluid medium for the first time
Photometry detects, and therefore present patent application exists the different of essence from above-mentioned patent publication.
Summary of the invention
In place of it is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of method is simple, can quickly detect sample
The method that the spectrophotography of the coliform quantity in product quickly detects coliform, the method can apply to water quality prison
The quick detection of the coliform in terms of survey, biology, food, health.
To achieve these goals, technical scheme of the present invention is as follows:
A kind of spectrophotography quickly detects the method for coliform, and step is as follows:
(1) solution preparation:
Lactose fermentation detection culture fluid: 50-90 μ L2%(mass concentration) bromocresol purple solution, 4g tryptone, 0.5g breast
Sugar, 0.5gD-mannitol, 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 and makes 0.5-3%(mass concentration) solution, keeps in Dark Place, stand-by；
(2) detect process:
1. the preparation of the even liquid of sample:
Sterile working takes measuring samples, 8000r/min～10000r/min homogenizing 1min～2min, and making volume ratio is 1:
The even liquid of sample of 10, or quality: volume (g:L) is the even liquid of sample of 1:10；
Or, if during liquid measuring samples detection limit≤10cfu/mL, directly detected by stock solution；
2. filter:
With the middling speed even liquid of quantitative filter paper filtered sample, obtain filtrate；By filtrate: the volume ratio of the liquid to be checked of concentration is 5-10:1
Ratio film filter filter liquor, obtain the liquid to be checked of concentration；Take the liquid to be checked of concentration by volume for the ratio of 1:5-10
Add containing in lactose fermentation detection culture fluid, meanwhile, by the liquid to be checked of concentration: volume ratio mL of TTC solution: μ L is 1:30-
The ratio of 80 takes TTC solution and detects in culture fluid to lactose fermentation, is placed in 37 DEG C of cultivations；
3. the drafting of standard curve:
After cultivating 60-400min, detection culture medium have color change and sinks to the bottom material production with redness, taking detection pipe
In culture fluid, process fermentation liquid by centrifugation, 5000-7000r/min is centrifuged, centrifugal 2min-5min, takes supernatant and is placed in quartz
Cuvette detects with spectrophotometer, measures the light absorption value of the special nanometers of 360nm 500nm, set up different content
Homologous pair sexual relationship between coliform mixed-culture medium color and coliform content, draws standard curve；
4. the detection of measuring samples:
During detection sample, by step 1.-method 3. carries out processing and cultivate measuring samples, culture fluid has variable color and precipitation
Phenomenon, by abovementioned steps by after medium centrifugal, takes supernatant and measures light absorption value at 360nm 500nm, record data, with
3. standard curve is compared, and determines contained coliform group count in unknown sample according to comparing result, finally draws and treat sample
Contained coliform numerical value in product.
And, described step the most 3. standard curve regression equation be:
lgY=14.89X-1.33
R2=0.991
In formula: Y: the predictive content of coliform in unknown sample
OD value at X:360nm 500nm
R: correlation coefficient.
Spectrophotography as above quickly detects the method for coliform in water quality monitoring, biology, food, health side
Application in the quick context of detection of the coliform in face.
Advantages of the present invention and good effect be:
1, the inventive method is compared to existing national standard method, has had and significantly reduce on the detection time, cultivates after sample-adding
60min-400min can detect the coliform numerical value in sample, and scope on probation is relatively wide, meets numerous detection field requirement
The demand that the detection time is short, has preferable application prospect in following field of fast detection.
2, the inventive method operation is relatively simple, and culture medium cost is relatively low, is not required to cryopreservation, easy to use.
Accompanying drawing explanation
Fig. 1 is coliform content logarithm value and the graph of a relation of absorbance of the present invention.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further described；Following embodiment is illustrative, is not determinate,
Protection scope of the present invention can not be limited with following embodiment.
Method used in the present invention, if no special instructions, is the conventional method of this area；Used in the present invention
Reagent, if no special instructions, be the conventional reagent of this area；Each raw material relative content in the present invention is as without special rule
Fixed, it is mass volume ratio.
Integral Thought and the Cleaning Principle of the present invention are as follows:
The present invention utilizes coliform can produce acid, biogas production characteristics with lactose fermenters at 37 DEG C, based on self-made medium, its
Middle interpolation indicator bromocresol purple and the colourless TTC of TTC() solution, using bromocresol purple and TTC as indicator,
Bromocresol purple and TTC are added in lactose fermentation culture medium, is configured to mixed-culture medium, utilize coliform lactose fermenters to produce acid
So that bromocresol purple turns yellow, simultaneously as the effect of hydrogenlyase, formic acid can be made to decompose and to produce CO2And H2, this
During the hydrogen taken off and TTC reaction produce red precipitation TTF(triphen first), therefore, in test tubes, culture fluid can become
Huang also generates with red precipitate, after reaction terminates, and centrifuging and taking supernatant, survey in specific nanometers with spectrophotometer
Amount, after gathering data, the culture fluid color intensity of variation Criterion curve caused according to variable concentrations coliform content, this
The measuring samples of rear unknown coliform content calculates coliform content according to standard curve.The inventive method is applicable to water quality
The quickly detection of coliform, analysis in monitoring, biology, health, food etc..
Being formulated as follows of lactose fermentation detection culture fluid used in the present invention:
50-90 μ L2%(mass concentration) bromocresol purple solution, 4g tryptone, 0.5g lactose, 0.5gD-mannitol,
0.75gNaCl, 0.02g sodium lauryl sulfate, 0.01g NaTDC, 100mL distilled water, pH6.8-7.2,115 DEG C, 15min
After sterilizing stand-by；
Being formulated as follows of TTC solution used in the present invention:
TTC adds sterilized water and makes 0.5-3%(mass concentration) solution, keeps in Dark Place, stand-by.
Embodiment 1
A kind of spectrophotography quickly detects the method for coliform, and step is as follows:
(1) solution preparation:
Lactose fermentation detection culture fluid: 50 μ L2%(mass concentrations) bromocresol purple solution, 4g tryptone, 0.5g lactose,
0.5gD-mannitol, 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 and makes 3%(mass concentration) solution, keeps in Dark Place, stand-by.
(2) detection process:
1. the preparation of the even liquid of sample:
Sterile working takes measuring samples 25g or 25mL, in the loading triangular flask containing 225mL physiological saline solution (if built-in
Dry bead), 8000r/min～10000r/min homogenizing 1min～2min, make volume ratio 1:10 the even liquid of sample (if liquid
During measuring samples detection limit relatively low (≤10cfu/mL), it is possible to direct stock solution detects)；
2. filter:
With the middling speed even liquid of quantitative filter paper filtered sample, repeatedly rinse once, take 5mL filtrate and concentrate out 1mL with film filter
Bacterium solution, the liquid to be checked taking concentration adds in the test tubes containing 9mL lactose fermentation detection culture fluid, moves with pipettor meanwhile
Take 30 μ LTTC solution in lactose test tubes, be placed in 37 DEG C of cultivations, cultivate 360min；
3. the detection of measuring samples:
Sample making standard curve with known coliform content.During detection sample, by step 1.-method 2. carries out
Processing and cultivate measuring samples, culture fluid has variable color and deposited phenomenon, takes the culture fluid in detection pipe by abovementioned steps, by centrifugation
Processing fermentation liquid, 7000r/min is centrifuged, centrifugal 5min, takes supernatant and measures light absorption value at 360nm 500nm, records number
According to, compare with standard curve, determine contained coliform group count in unknown sample according to comparing result, finally draw unknown sample
Contained coliform numerical value in product.
Embodiment 2
A kind of spectrophotography quickly detects the method for coliform, and step is as follows:
(1) solution preparation:
Lactose fermentation detection culture fluid: 90 μ L2%(mass concentrations) bromocresol purple solution, 4g tryptone, 0.5g lactose,
0.5gD-mannitol, 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 and makes 1.5%(mass concentration) solution, keeps in Dark Place, stand-by.
(2) detection process:
1. the preparation of the even liquid of sample:
Sterile working takes measuring samples 25g or 25mL, in the loading triangular flask containing 225mL physiological saline solution (if built-in
Dry bead), 8000r/min～10000r/min homogenizing 1min～2min, make volume ratio 1:10 the even liquid of sample (if liquid
Measuring samples detection limit is relatively low≤10cfu/mL time, it is possible to direct stock solution detects)；
2. filter:
With the middling speed even liquid of quantitative filter paper filtered sample, repeatedly rinse once, take 9mL filtrate and concentrate out 1mL with film filter
Bacterium solution, the liquid to be checked taking concentration adds in the test tubes containing 9mL lactose fermentation detection culture fluid, moves with pipettor meanwhile
Take 80 μ LTTC solution in lactose test tubes, be placed in 37 DEG C of cultivations, cultivate 300min；
3. the detection of measuring samples:
Sample making standard curve with known coliform content.During detection sample, by step 1.-method 2. carries out
Processing and cultivate measuring samples, culture fluid has variable color and deposited phenomenon, takes the culture fluid in detection pipe by abovementioned steps, by centrifugation
Processing fermentation liquid, 7000r/min is centrifuged, centrifugal 5min, takes supernatant and measures light absorption value at 360nm 500nm, records number
According to, compare with standard curve, determine contained coliform group count in unknown sample according to comparing result, finally draw unknown sample
Contained coliform numerical value in product.
Embodiment 3
A kind of spectrophotography quickly detects the method for coliform, and step is as follows:
(1) solution preparation:
Lactose fermentation detection culture fluid: 60 μ L2%(mass concentrations) bromocresol purple solution, 4g tryptone, 0.5g lactose,
0.5gD-mannitol, 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 and makes 2%(mass concentration) solution, keeps in Dark Place, stand-by.
(3) detection process:
1. the preparation of the even liquid of sample:
Sterile working takes measuring samples 25g or 25mL, in the loading triangular flask containing 225mL physiological saline solution (if built-in
Dry bead), 8000r/min～10000r/min homogenizing 1min～2min, make volume ratio 1:10 the even liquid of sample (if liquid
Measuring samples detection limit is relatively low≤10cfu/mL time, it is possible to direct stock solution detects)；
2. filter:
With the middling speed even liquid of quantitative filter paper filtered sample, repeatedly rinse once, take 10mL filtrate and concentrate out 1mL with film filter
Bacterium solution, the liquid to be checked taking concentration adds in the test tubes containing 9mL lactose fermentation detection culture fluid, moves with pipettor meanwhile
Take 50 μ LTTC solution in lactose test tubes, be placed in 37 DEG C of cultivations, cultivate 300min；
3. the detection of measuring samples:
Sample making standard curve with known coliform content.During detection sample, by step 1.-method 2. carries out
Processing and cultivate measuring samples, culture fluid has variable color and deposited phenomenon, takes the culture fluid in detection pipe by abovementioned steps, by centrifugation
Processing fermentation liquid, 7000r/min is centrifuged, centrifugal 5min, takes supernatant and measures light absorption value at 360nm 500nm, records number
According to, compare with standard curve, determine contained coliform group count in unknown sample according to comparing result, finally draw unknown sample
Contained coliform numerical value in product.
The manufacture method step of standard curve of the present invention is as follows:
The drafting of standard curve:
Add with the sample of known coliform content in the test tubes of lactose fermentation detection culture fluid, cultivate 60-
After 400min, detection culture medium in have color change and with redness sink to the bottom material produce, take detection pipe in culture fluid, through from
The heart processes fermentation liquid, and 5000-7000r/min is centrifuged, centrifugal 2min-5min, takes supernatant and is placed in quartz colorimetric utensil and uses light splitting
Photometer detects, and measures the light absorption value (i.e. OD value) of the special nanometers of 360nm 500nm, sets up different content coliform
Homologous pair sexual relationship between group's mixed-culture medium color and coliform content, draws standard curve.
The testing result of three embodiments of the present invention average after draw standard curve, result as it is shown in figure 1, from
In figure it can be seen that
The regression equation of standard spectral photometric value is:
lgY=14.89X-1.33
R2=0.991
In formula: Y: the predictive content of coliform in unknown sample
OD value at X:360nm 500nm
R: correlation coefficient.
Spectrophotography of the present invention quickly detects the related test results of the method for coliform:
Use detection method to the coliform in non-polluted egg, the coliform in nonfermenters gram negative bacilli
Detecting with the coliform in cookies, testing result is shown in Table 1, table 2, table 3.
Coliform testing result in table 1 non-polluted egg
Coliform testing result in table 2 nonfermenters gram negative bacilli
Coliform testing result in table 3 cookies
The present invention, with bromocresol purple and TTC as indicator, is changed by solution colour and produces red precipitate two more
Significantly phenomenon is as judging that coliform produces the standard of acid aerogenesis, meanwhile, predicts the unknown by solution colour intensity of variation
Coliform content in sample.Embodiment adds in 9mL test tubes with concentrating sample 1mL bacterium solution, cultivates 60min-for 37 DEG C
After 400min, at 360nm 500nm wavelength, carry out light absorption value measurement, draw large intestine contained by detection sample according to standard curve
After Microflora, contrast National Standard Method testing result, find testing result of the present invention all in the credible scope of National Standard Method testing result,
Illustrate that detection method of the present invention has certain credibility compared with National Standard Method, can be as effective detection method of coliform detection.Due to
National Standard Method detecting step is loaded down with trivial details, the detection time is longer, and the inventive method is the most easy and simple to handle, the detection time has and significantly reduces, and
And scope on probation is relatively wide, significantly meet the various demands of commercial production and small-size laboratory, it is contemplated that quickly detect neck in future
Territory has preferable application prospect.
Claims (3)
1. the method that a spectrophotography quickly detects coliform, it is characterised in that: step is as follows:
(1) solution preparation:
Lactose fermentation detection culture fluid: 50-90 μ L 2% (mass concentration) bromocresol purple solution, 4g tryptone, 0.5g lactose,
0.5g PEARLITOL 25C, 0.75g NaCl, 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 and makes 0.5-3% (mass concentration) solution, keeps in Dark Place, stand-by；
(2) detect process:
1. the preparation of the even liquid of sample:
Sterile working takes measuring samples, 8000r/min～10000r/min homogenizing 1min～2min, and making volume ratio is 1:10's
The even liquid of sample, or quality: volume (g:L) is the even liquid of sample of 1:10；
2. filter:
With the middling speed even liquid of quantitative filter paper filtered sample, obtain filtrate；By filtrate: the volume ratio of the liquid to be checked of concentration is the ratio of 5-10:1
Example film filter filter liquor, obtains the liquid to be checked of concentration；Take the liquid to be checked of concentration by volume for the ratio addition of 1:5-10
Containing in lactose fermentation detection culture fluid, meanwhile, by the liquid to be checked of concentration: volume ratio mL of TTC solution: μ L is 1:30-80's
Ratio takes TTC solution and detects in culture fluid to lactose fermentation, is placed in 37 DEG C of cultivations；
3. the drafting of standard curve:
After cultivating 60-400min, detection culture medium have color change and sinks to the bottom material production with redness, taking in detection pipe
Culture fluid, processes fermentation liquid by centrifugation, and 5000-7000r/min is centrifuged, centrifugal 2min-5min, takes supernatant and is placed in quartz cuvette
Ware detects with spectrophotometer, measures the light absorption value of the special nanometers of 360nm 500nm, set up different content large intestine
Homologous pair sexual relationship between flora mixed-culture medium color and coliform content, draws standard curve；
4. the detection of measuring samples:
During detection sample, by step 1.-method 3. carries out processing and cultivate measuring samples, culture fluid has variable color and precipitation existing
As, and 3. by abovementioned steps by after medium centrifugal, take supernatant at 360nm 500nm, measure light absorption value, record data,
Standard curve is compared, and determines contained coliform group count in unknown sample according to comparing result, finally draws measuring samples
Coliform numerical value contained by.
Spectrophotography the most according to claim 1 quickly detects the method for coliform, it is characterised in that: described step
The most 3. the regression equation of standard curve is:
LgY=14.89X-1.33
R2=0.991
In formula: Y: the predictive content of coliform in unknown sample
OD value at X:360nm 500nm
R: correlation coefficient.
3. spectrophotography as claimed in claim 1 or 2 quickly detects the method for coliform at water quality monitoring, biology, food
Application in the quick context of detection of the coliform in terms of product, health.
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| CN101613738A (en) * | 2009-07-15 | 2009-12-30 | 集美大学 | Coliform single-tube quantitative rapid detection method |
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| CN101914609A (en) * | 2010-08-27 | 2010-12-15 | 殷涌光 | Rapid detection method of coliform groups |
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| 硒对幽门螺杆菌空泡毒素及其重组影响;段秀杰等;《中国公共卫生》;20080731;第24卷(第07期);全文 * |
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