CN101497914A - Test chip for fast detecting microorganism, preparation and use thereof - Google Patents
Test chip for fast detecting microorganism, preparation and use thereof Download PDFInfo
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- CN101497914A CN101497914A CNA2009100079173A CN200910007917A CN101497914A CN 101497914 A CN101497914 A CN 101497914A CN A2009100079173 A CNA2009100079173 A CN A2009100079173A CN 200910007917 A CN200910007917 A CN 200910007917A CN 101497914 A CN101497914 A CN 101497914A
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Abstract
The invention discloses a testing piece for quickly testing microorganisms, as well as a preparation method and application thereof. The testing piece comprises a soleplate, a film with a hollow part in the middle and a plastic film. The bottom surface of the film with the hollow part in the middle is adhered to the soleplate; a groove culture area is formed at the hollow part in the middle of the film; a color development culture medium is arranged in the groove culture area; a cold water condensable gellant is adhered to the bottom surface of the plastic film; and the bottom surface of the plastic film and the upper surface of the film are adhered to the edge at one side. After the upper and the lower layers of the testing piece are adhered, irradiation and sterilization packages are used for standby. The testing piece can be applied to the detection of total colony count, coliform bacteria, coliform group, salmonella, staphylococcus aureus, subsidiary haemolytic vibrio, pseudomonas aeruginosa and other food-borne causative organisms and has the advantages of simple operation, convenient carrying, short testing time, accurate result and double-sided bacteria count.
Description
Technical field
The present invention relates to food microbiology safety verification detection range, specifically, relate to testing plate of a kind of rapid detection microorganism and its production and application.
Background technology
The complex operation step of existing state food sanitary microorganism examination criteria method in detection microorganism front and back, needs configuration substratum, cleaning cultivation vessel, sterilizes, increases bacterium, incubation time length etc., simultaneously to laboratory technician and demand of laboratory height.Under the current commerce and trade paces of accelerating day by day, traditional detection method can not satisfy food production or marketing enterprise, import and export the detection needs of commodity inspection, government administration section.
Testing plate can gel with the scraps of paper, cold water and non-woven fabrics etc. measure the content of microorganism in the food as culture medium carrier, with national standard method relatively, great advantage is to have save heavy preparation work; Sample does not need to increase bacterium, and direct inoculation testing plate, optimal temperature are cultivated the back counting, uses after sterilization is just disposable, simple and convenient, shortens detection time.
Yet the culture medium carrier of testing plate has material impact to detecting effect, and filter paper, gel and non-woven fabrics are three kinds of main flow carriers at present.The testing plate manufacture craft that with filter paper is carrier is simple, and material is cheap, but filter opening is excessive, and two-sided easily have colony growth and can't accurate counting; Detection is had fatty foods, and filter paper absorbs slower, is difficult to diffusion sample liquid is evenly distributed; With the non-woven fabrics is the testing plate of carrier, has the sample of making liquid and promptly evenly spreads advantage, but opaque and nonwoven layer is thicker because of it, carrier lower floor bacterium colony can not be counted and cause the rate of recovery low slightly.The gel testing plate of the petrifilm of Minnesota Mining and Manufacturing Company is because of the sheet upper strata covers thin film, when bacterium colony diffusion, the phenomenon that merges, influence counting can appear in bacterium aerogenesis, product mucus when too much; Slower because of the gel absorption speed in addition, sample liquid easily overflows outside the substratum scope, influences the rate of recovery.Testing plate in the market is all opaque, can not count bacterium in order to avoid omit by tow sides, and difficult by counting bacterium device counting automatically; In addition, except that the petrifilm testing plate of 3M company, various product all can not carry out next step and choose the dientification of bacteria.
Therefore, research and development fast, and are accurately easy, can carry out the microorganism testing slice of isolation identification, significant to the rapid detection food microorganisms safety of China.
Summary of the invention
The object of the present invention is to provide a kind ofly be convenient for carrying, easy to use, the test duration short, the result accurately, can two-sided several bacterium, be used for the disposable test sheet of microorganism detection, and the preparation method and the application of described testing plate be provided.
For achieving the above object, the present invention has adopted following technical scheme:
The testing plate of a kind of rapid detection microorganism, this testing plate comprises the film and the plastic film of base plate, middle hollow out, the bottom surface and the base plate of the film of middle hollow out are bonding, and openwork part forms the groove cultivation region in the middle of the film, and described groove cultivation region is built-in with color developing culture medium; The bottom surface of described plastic film is bonded with the gelifying agent that cold water can coagulate, and the bottom surface of described plastic film and the upper surface of film are bonding at a lateral edges.
Preferably, described base plate is consistent with the film length and width; Preferably, described plastic film is consistent with the width of described film, and the length of described plastic film is greater than the length of described film.
In order to differentiate different check samples, be preferably in a bonding label on the described plastic film clear and definite proving time, leave the hurdle of filling in of sample name and proving time on the label.
Described plastic film and base plate are prepared from by waterproof and breathable, transparent, nontoxic sealed polyethylene plastic.Plastic film thickness is 0.03～0.1mm, and length is 9～13cm, and width is 7.0～11.0cm.Base plate thickness is 0.03～0.25mm, and length is 8.0～12.0cm, and width is 7.0～11.0cm.
Described film is prepared from by poly terephthalic acid class plastics transparent, impermeable, light weight.Thickness is 0.3～0.5mm, and length is 8.0～12.0cm, and width is 7.0～11.0cm, and the intermediate groove cultivation region is that diameter is the circle of 4～7cm.
Described color developing culture medium is the corresponding commercial product culture medium of bacterium to be measured, is made up of nutritive substance, inductor and developer, and consumption is 1.5～4.0 * 10
-3G/cm
2
The gelifying agent that described cold water can coagulate is a polyose, and consumption is 1.0～4.0 * 10
-3G/cm
2
It is bonding that the gelifying agent that testing plate base plate of the present invention and film, film and plastic film, color developing culture medium and cold water can coagulate all adopts the polyacrylic resin pressure sensitive adhesive to carry out.
A kind of method for preparing the testing plate of rapid detection microorganism may further comprise the steps:
1) cuts out the film and the plastic film of base plate, middle hollow out on request;
2) bottom surface of bonding base plate and film obtains the groove cultivation region, and color developing culture medium is bonded in the groove cultivation region;
3) gelifying agent that cold water can be coagulated is bonded in the bottom surface of plastic film;
4) bottom surface of the upper surface of film and plastic film is bonding at a lateral edges;
5) irradiation sterilization, encapsulation.
Described irradiation adopts Co-60 gamma-rays, radiation dose 3K～5KGy.
The application of testing plate of the present invention in detecting the food-borne pathogenic microorganism comprises and measures total number of bacterial colony, detection intestinal bacteria, coliform, Salmonellas, streptococcus aureus, Vibrio parahemolyticus, Pseudomonas aeruginosa etc.
The use step of testing plate of the present invention is:
1) opens the upper strata plastic film;
2) drip the 1ml sample liquid in the centre circular hollow out groove cultivation region;
3) cover plastic film; Fail homodisperse as sample liquid, available handgrip sample liquid pushes away flat;
4) cultivate through suitable temp,, choose with colony colour bigger tally assembly is done according to the bacterium colony Show Color
Be background, place arbitrary counting of testing plate, or directly place automated colony counter to count.
Wherein the tally assembly is the enamelled paper material, and Huang, white, black three kinds of colors are arranged.Sizing grid is 1 * 1cm, and thickness is 0.03～0.25mm, and length is 9～13cm, and width is 7.0～11.0cm.
The testing plate of rapid detection microorganism of the present invention has following beneficial effect:
Simple to operate, be suitable for the unit that lacks the professional inspection personnel; Easy to carry, not limited by envrionment conditions; It is little to take up space during cultivation; Full sheet is transparent, can avoid bacterium colony to omit by two-sided several bacterium; Can be used for counting automatically bacterium device counting, time-saving and efficiency.
Description of drawings
Fig. 1 is the synoptic diagram of the testing plate of rapid detection microorganism of the present invention;
Fig. 2 is the side-view of Fig. 1 testing plate;
Fig. 3 is the synoptic diagram of the supporting tally of testing plate of rapid detection microorganism of the present invention;
Fig. 4 is the side-view of Fig. 3 tally;
Reference numeral: 1: label; 2: plastic film; 3 films; 4: base plate.
Embodiment
Below by specific embodiment the present invention is described.
Embodiment 1: the preparation of rapid determination microbe colony sum testing plate of the present invention
May further comprise the steps:
1) cut out each parts, the plastic film length and width are 10.5 * 7.5cm; Base plate and film length and width are 9.5 * 7.5cm; The film bottom surface of middle hollow out and the circular groove cultivation region that the bonding formation diameter of base plate is 6cm;
2) nutrient agar medium powder 6g and the 0.2gTTC that microorganism growth is needed mixes, and the 0.06g mixed powder is combined in the groove cultivation region by pressure-sensitive adhesive;
3) the polyacrylic resin pressure sensitive adhesive is heated into liquid, evenly is sprayed on the bottom surface of plastic film, the gelifying agent powder-stuck that 0.3g cold water can be coagulated is in the bottom surface of plastic film;
4) upper surface of the bottom surface of plastic film and film is bonding by pressure sensitive adhesive at a lateral edges, bond area is 7.5 * 1.0cm;
5) Co-60 gamma-rays, irradiation dose are the 5KGy sterilization;
6) package spare.
Embodiment 2: utilize embodiment 1 testing plate to measure total number of bacterial colony, and and international dull and stereotyped tilt-pour process comparison 1) preparation mixed bacteria liquid: picking intestinal bacteria (Escherichia coli 8099), Salmonella typhimurium (Salmonella typhiCMCC 50115), streptococcus aureus (Staphylococcus aureus ATCC 6538), streptococcus aureus (Staphylococcus aureus CMCC 26003), Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 9027) lawn is inoculated into respectively in the 5ml nutrient broth, behind 37 ℃ of cultivation 18-24h, respectively get 1ml and in a sterile test tube, obtain mixed bacteria liquid, adjust concentration and be about 10
5Cell/ml also carries out 10 times of gradient dilutions to 10 with physiological saline
4, 10
3, 10
2, 10
1, 10
0With 10
-1Cell/ml, each concentration gradient is provided with three repetitions, and is standby.
2) open testing plate upper strata plastic film, drip each concentration gradient mixed bacteria liquid 1ml in the groove cultivation region, cover plastic film, observe sample liquid and whether be dispersed in testing plate central authorities, leave standstill 1-2min, treat gel solidification after, cultivate 24h for 37 ℃, use the dull and stereotyped tilt-pour process of GB to contrast simultaneously;
3) observe red point and be bacterium colony, choose with the bigger tally assembly of bacterium colony aberration (yellow, white, black) and as a setting, place arbitrary counting of testing plate, or directly place automated colony counter to count.The total number of bacterial colony of testing plate and tilt-pour process is added up and average t check, and the result shows, both do not have significant difference (t=0.0397, p=0.8516〉0.05) at the colony counts of growing.
The total number of bacterial colony of two kinds of methods mensuration of table 1 mixed bacteria liquid (xS, n=3)
Embodiment 3: utilize embodiment 1 testing plate range estimation different strain colony number, and compare with the automated colony counter measurement result
1) preparation bacterium liquid: get each lawn intestinal bacteria (Escherichia coli 8099), Salmonella typhimurium (Salmonella typhiCMCC 50115), streptococcus aureus (Staphylococcus aureus ATCC 6538), streptococcus aureus (Staphylococcus aureus CMCC 26003), Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 9027) and be inoculated in the 5ml nutrient broth, behind 37 ℃ of cultivation 18-24h, respectively getting 1ml in sterile test tube, is 10 with 10 times of gradient dilutions of physiological saline to each bacterial concentration
1Cell/ml, every strain bacterium liquid is provided with two repetitions, and is standby.
2) get five testing plate, respectively determination step 1) five kinds of bacterium liquid.Open testing plate upper strata plastic film, drip concentration 10
1The bacterium liquid 1ml of cell/ml covers plastic film in lower floor's hollow out groove location, observes sample liquid and whether has been dispersed in testing plate central authorities, leaves standstill 1-2min, treat gel solidification after, cultivate 24h for 37 ℃;
3) observe red point and be bacterium colony, choose as a setting, place arbitrary appearance of testing plate to survey counting, directly place automated colony counter to count afterwards with the bigger tally assembly of bacterium colony aberration (yellow, white, black).The colony number of testing plate range estimation and automated colony counter added up with average t check, the result shows, both do not have significant difference (t=0.0739, p=0.7897〉0.05).
The colony number of each bacterial strain testing plate range estimation of table 2 and automated colony counter (x ±-S, n=2)
| Test strain | Intestinal bacteria 8099 | Salmonella typhimurium CMCC 50115 | The gold bacterium ATCC6538 of Portugal | The gold bacterium CMCC26003 of Portugal | Pseudomonas aeruginosa ATCC9027 |
| The range estimation colony number | 7 | 15 | 57 | 59 | 55 |
| Automatically count bacterium device colony number | 7 | 16 | 56 | 60 | 58 |
Embodiment 4: utilize testing plate of the present invention and tilt-pour process that the determination of recovery rates of staphylococcus aureus strains is compared
1) the testing plate preparation method by embodiment 1 prepares the streptococcus aureus testing plate, and different is that adherent is the 0.06g streptococcus aureus color developing culture medium of buying in the groove cultivation region;
2) preparation bacterium liquid: picking streptococcus aureus (Staphylococcus aureus ATCC 6538), streptococcus aureus (Staphylococcus aureus CMCC 26003) lawn are inoculated into respectively in the 5ml nutrient broth, behind 37 ℃ of cultivation 18-24h, respectively getting 1ml is 10 with 10 times of gradient dilutions of physiological saline to streptococcus aureus bacterial concentration
2Cell/ml, every strain bacterium is provided with three repetitions, and is standby.
3) open testing plate upper strata plastic film, drip step 2) bacterium liquid 1ml is in lower floor hollow out groove cultivation region position, covers plastic film, observes sample liquid and whether has been dispersed in testing plate central authorities, leaves standstill 1-2min, treat gel solidification after, cultivate 24h for 37 ℃, counting; Pour into streptococcus aureus colour developing flat board with 1ml bacterium liquid, cultivate 24h, in contrast, carry out operation count for 37 ℃ according to GB tilt-pour process specification step.
4) with purple and the positive bacterium colony of peach bacterium colony, calculate recovery rate.The rate of recovery of contrast in testing plate and tilt-pour process, streptococcus aureus ATCC6538, the CMCC26003 rate of recovery is 100%.
The rate of recovery=(testing plate colony number/tilt-pour process colony number) * 100% is if numerical value counts 100% greater than 100%.
The rate of recovery that each bacterial strain of table 3 is measured with testing plate and tilt-pour process
Annotate: the colony number mean value (cfu/ sheet or cfu/ ware) that data are measured with testing plate and tilt-pour process for each test strain in the table is the rate of recovery (%) in the bracket.
Embodiment 5: utilize testing plate of the present invention and tilt-pour process that the determination of recovery rates of Salmonellas bacterial strain is compared
1) the testing plate preparation method by embodiment 1 prepares the Salmonellas testing plate, and different is that adherent is the 0.06g salmonella color culture medium of buying in the groove cultivation region;
2) preparation bacterium liquid: picking Salmonella typhimurium (Salmonella typhi CMCC 50115) lawn is inoculated in the 5ml nutrient broth, and behind 37 ℃ of cultivation 18-24h, getting 1ml is 10 with 10 times of gradient dilutions of physiological saline to Salmonellas bacterial concentration
2Cell/ml is provided with three repetitions, and is standby.
3) open testing plate upper strata plastic film, drip step 2) bacterium liquid 1ml is in lower floor hollow out groove cultivation region position, covers plastic film, observes sample liquid and whether has been dispersed in testing plate central authorities, leaves standstill 1-2min, treat gel solidification after, cultivate 24h for 37 ℃, counting; Pour into the salmonella color flat board with 1ml bacterium liquid, cultivate 24h, in contrast, carry out operation count for 37 ℃ according to GB tilt-pour process specification step.
4) with purple and the positive bacterium colony of peach bacterium colony, calculate recovery rate.The rate of recovery of contrast in testing plate and tilt-pour process, the salmonella typhi CMCC50115 rate of recovery is 88.5%.
The rate of recovery=(testing plate colony number/tilt-pour process colony number) * 100% is if numerical value counts 100% greater than 100%.
The rate of recovery that table 4 Salmonellas is measured with testing plate and tilt-pour process
Annotate: the colony number mean value (cfu/ sheet or cfu/ ware) that data are measured with testing plate and tilt-pour process for each test strain in the table is the rate of recovery (%) in the bracket.
Embodiment 6: utilize testing plate of the present invention and tilt-pour process that the determination of recovery rates of coli strain is compared
1) the testing plate preparation method by embodiment 1 prepares the coliform testing plate, and different is that adherent is the 0.06g coliform chromogenic medium of buying in the groove cultivation region;
2) preparation bacterium liquid: picking intestinal bacteria (Escherichia coli 8099), intestinal bacteria (Escherichia coli ATCC 25922) lawn are inoculated into respectively in the 5ml nutrient broth, behind 37 ℃ of cultivation 18-24h, respectively getting 1ml is 10 with 10 times of gradient dilutions of physiological saline to Escherichia coli bacteria liquid concentration
2Cell/ml, every strain bacterium is provided with three repetitions, and is standby.
3) open testing plate upper strata plastic film, drip step 2) bacterium liquid 1ml is in lower floor hollow out groove cultivation region position, covers plastic film, observes sample liquid and whether has been dispersed in testing plate central authorities, leaves standstill 1-2min, treat gel solidification after, cultivate 24h for 37 ℃, counting; Pour into coliform colour developing flat board with 1ml bacterium liquid, cultivate 24h, in contrast, carry out operation count for 37 ℃ according to GB tilt-pour process specification step.
4) with the blue and green positive bacterium colony of bacterium colony, calculate recovery rate.The rate of recovery of contrast in testing plate and tilt-pour process, intestinal bacteria 8099 rate of recovery are 93.8%, and intestinal bacteria ATCC 25922 rate of recovery are 98.1%, and tilt-pour process colony number average recovery rate is 96% relatively.
The rate of recovery=(testing plate colony number/tilt-pour process colony number) * 100% is if numerical value counts 100% greater than 100%.
The rate of recovery that table 5 intestinal bacteria are measured with testing plate and tilt-pour process
Annotate: the colony number mean value (cfu/ sheet or cfu/ ware) that data are measured with testing plate and tilt-pour process for each test strain in the table is the rate of recovery (%) in the bracket.
Claims (11)
1. the testing plate of a rapid detection microorganism, it is characterized in that: this testing plate comprises the film and the plastic film of base plate, middle hollow out, the bottom surface and the base plate of the film of middle hollow out are bonding, and openwork part forms the groove cultivation region in the middle of the film, and described groove cultivation region is built-in with color developing culture medium; The bottom surface of described plastic film is bonded with the gelifying agent that cold water can coagulate, and the bottom surface of described plastic film and the upper surface of film are bonding at a lateral edges.
2. testing plate according to claim 1 is characterized in that: described base plate is consistent with the film length and width.
3. testing plate according to claim 2 is characterized in that: described plastic film is consistent with the width of described film, and the length of described plastic film is greater than the length of described film.
4. according to each described testing plate of claim 1-3, it is characterized in that: the upper surface of described plastic film is provided with label layer.
5. testing plate according to claim 1 is characterized in that: described base plate, plastic film are prepared from by sealed polyethylene plastic, and described film is prepared from by poly terephthalic acid class plastics, and described film thickness is 0.3～0.5mm.
6. testing plate according to claim 1 is characterized in that: openwork part is the circle of diameter 4～7cm in the middle of the described film.
7. testing plate according to claim 1 is characterized in that: described color developing culture medium is made up of the corresponding nutritive substance of bacterium to be measured, inductor and developer, and consumption is 1.5～4.0 * 10
-3G/cm
2The gelifying agent that described cold water can coagulate is a polysaccharide, and consumption is 1.0～4.0 * 10
-3G/cm
2
8. method for preparing the testing plate of rapid detection microorganism as claimed in claim 1 is characterized in that: may further comprise the steps:
1) cuts out the film and the plastic film of base plate, middle hollow out on request;
2) bottom surface of bonding base plate and film obtains the groove cultivation region, and color developing culture medium is bonded in the groove cultivation region;
3) gelifying agent that cold water can be coagulated is bonded in the bottom surface of plastic film;
4) bottom surface of the upper surface of film and plastic film is bonding at a lateral edges;
5) irradiation sterilization, encapsulation.
9. preparation method according to claim 8 is characterized in that: described irradiation adopts Co-60 gamma-rays, radiation dose 3K～5KGy.
10. the application of the described testing plate of claim 1 in detecting the food-borne pathogenic microorganism.
11. application according to claim 10 is characterized in that: described food-borne pathogenic microorganism is one or more in intestinal bacteria, coliform, Salmonellas, streptococcus aureus, Vibrio parahemolyticus, the Pseudomonas aeruginosa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100079173A CN101497914A (en) | 2008-12-30 | 2009-02-26 | Test chip for fast detecting microorganism, preparation and use thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810220569 | 2008-12-30 | ||
| CN200810220569.3 | 2008-12-30 | ||
| CNA2009100079173A CN101497914A (en) | 2008-12-30 | 2009-02-26 | Test chip for fast detecting microorganism, preparation and use thereof |
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| Publication Number | Publication Date |
|---|---|
| CN101497914A true CN101497914A (en) | 2009-08-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2009100079173A Pending CN101497914A (en) | 2008-12-30 | 2009-02-26 | Test chip for fast detecting microorganism, preparation and use thereof |
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| CN103343158A (en) * | 2013-07-20 | 2013-10-09 | 吉林农业大学 | Detection paper piece for clostridium perfringens, preparation method and application for same |
| CN104099244A (en) * | 2014-06-26 | 2014-10-15 | 上海市园林设计院有限公司 | Plate culture dish capable of quickly counting bacterial colonies |
| CN104611403A (en) * | 2014-12-18 | 2015-05-13 | 湖北工业大学 | Food and tableware coliform bacteria rapid detection scrip preparation and application |
| CN104673876A (en) * | 2015-02-09 | 2015-06-03 | 广州绿洲生化科技股份有限公司 | Transparent water-absorbing gel for microbial detection and detection plate |
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| CN105899653A (en) * | 2014-01-09 | 2016-08-24 | 生物梅里埃公司 | Method for detecting, identifying and enumerating micro-organisms in a porous support dry-impregnated with a dehydrated reaction medium |
| CN104099244A (en) * | 2014-06-26 | 2014-10-15 | 上海市园林设计院有限公司 | Plate culture dish capable of quickly counting bacterial colonies |
| CN104611403A (en) * | 2014-12-18 | 2015-05-13 | 湖北工业大学 | Food and tableware coliform bacteria rapid detection scrip preparation and application |
| CN104673876B (en) * | 2015-02-09 | 2017-08-15 | 广州绿洲生化科技股份有限公司 | A kind of transparent water absorbent gel and detection plate for microorganism detection |
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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 |
| CN107513493A (en) * | 2016-06-16 | 2017-12-26 | 孙百莉 | A kind of system for microorganism testing and preparation method thereof |
| CN105950459A (en) * | 2016-07-14 | 2016-09-21 | 孙百莉 | Microbial test strip and application thereof |
| CN106811404A (en) * | 2017-01-22 | 2017-06-09 | 贵州勤邦食品安全科学技术有限公司 | A kind of test piece of quick detection coliform and preparation method thereof, detection method |
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| CN108456711A (en) * | 2018-01-31 | 2018-08-28 | 广东环凯微生物科技有限公司 | It is a kind of to be resistant to the cold water soluble gel-type vehicle and its application that mesophilic bacterium is decomposed |
| CN109251957A (en) * | 2018-10-17 | 2019-01-22 | 长春理工大学 | Staphylococcus aureus selective coloration culture medium testing piece |
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Application publication date: 20090805 |