CN105754849A - Total bacterial count testing slip and preparation method and application thereof - Google Patents
Total bacterial count testing slip and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a total bacterial count testing slip and a preparation method and application thereof in the aspect of detecting the total bacterial count in food, and belongs to the technical field of food detection.The testing slip is composed of a transparent plastic upper-layer film with the sticky inner side, a plastic middle-layer plate with a hollow area and a bottom plate with printed squares.The inner side of the upper-layer film is coated with a composite cold water soluble gel, and the hollow area of the middle-layer plate is filled with a total bacterial count detection culture medium.After total bacterial count testing is carried out, a bacterial clump is red on the testing slip, and the test slip with the moderate bacterial count is selected for counting.The total bacterial count testing slip has the advantages that after sample liquid to be detected is added into the testing slip, a composite cold water gel and culture medium components form a compact net gel, a growth promoter in the culture medium has the effects of promoting and repairing bacteria growth, and a color developing agent, a bulking agent and a surfactant are beneficial to even distribution of sample liquid, bacterial clump forming and visual counting.When the total bacterial count testing slip is used for testing the total bacterial count in food, the program is easy and convenient to implement, the bacterial clump has the clear color, and counting is accurate.
Description
Technical field
The invention belongs to technical field of food detection, be specifically related to a kind of total plate count test sheet, preparation method and application in total plate count in detection food thereof.
Background technology
Total plate count is the important indicator of reflection food hygiene quality, is used for judging the degree that food is contaminated by bacterial.Total plate count mensuration is the Testing index in many agricultural products such as China's cooked meat product, aquatic product, cereal product, instant fruit and vegetable goods, instant seasoning product, beverage and food inspection standard, is also the important monitoring project of food risk profile and hazard evaluation.Current GB4789.2 " national food safety standard food microbiological examination total plate count mensuration " adopts colony counting method, and the method needs to carry out the work such as culture medium preparation, sterilizing, glass drying oven cleaning, and program is loaded down with trivial details, and workload is big.Microorganism testing slice class detection product eliminates a large amount of auxiliary works such as the cleaning treatment of preparation culture medium, sterilization and culture vessel, it is convenient to count, use and have only to 3 simple steps: sample preparation, enrichment culture and counting, be carry out microorganism both at home and abroad to pollute the detection product that counting mensuration is best.
In existing commercialization microorganism testing slice detection product, the Petrifilm that Minnesota Mining and Manufacturing Company is carrier with cold water solubles gelTMTest sheet is taken its place in the front ranks of the world always, the comparison used all over the world is many, domestic test flake products, mainly by the biochemical test flake products being carrier with non-woven fabrics researched and developed with Beijing overpass Science and Technology Ltd. etc. in oasis, Guangzhou, is in research and development and little scope operational phase in addition with some test sheets.Although these test sheets have a convenient and practical outstanding advantage, but test sheet still suffers from some shortcomings as the new technique of microorganism detection, and the technical bottleneck in bacterial growth, the diffusion of bacterium colony colour generation and accurate counting etc. is still needed and effectively solved.
Summary of the invention
An object of the present invention is in that to provide a kind of simple to operate, cheap, detect the sheet of total plate count cold water gel test accurately, this test sheet is (as high in transparency by the sticking transparent plastic upper layer film in inner side successively, nontoxic have no irritating odor, the PET pellosil of waterproof and breathable, thickness is 0.05mm～0.10mm), plastics la m (polypropylene plastics flitch as good in hardness or PET plate with void region, thickness is 0.15mm～0.30mm), it is printed with the base plate of grid (such as waterproof, impermeable white art paper, thickness is 0.10mm～0.20mm) composition；The resistant to elevated temperatures temperature of above material is 130 DEG C～150 DEG C；The void region of plastics la m is circular, oval or square structure, the proportion of void region area and la m area is 1:1.6～2.7, being filled with total plate count color developing detection culture medium in void region, the inner side of transparent plastic upper layer film is coated with composite cold water soluble gel.
Total plate count colour developing inspection side culture medium is except basal medium tryptone, yeast leaching powder, sodium chloride, glucose, separately add developer TTC (2,3,5-benzyltriphenylphosphonium chloride tetrazoles), somatomedin Sodium Pyruvate, surface active agent tween 80, mannitol and raising agent bean dregs；Wherein prepared by raising agent bean dregs oneself: after fresh bean dregs are placed in 50～60 DEG C of thermostatic drying chambers drying, grind, crosses 100～120 mesh sieves.
Composite cold water soluble gel is made up of guar gum, sodium polyacrylate and sodium carboxymethyl cellulose.
Total plate count colour developing inspection side culture medium composition by weight is: tryptone 15～30g, yeast leaching powder 5～15g, sodium chloride 5～10g, glucose 1～5g, Sodium Pyruvate 1～3g, mannitol 1～3g, Tween 80 1～3mL, TTC0.2～0.5g, bean dregs 3～10g.
Composite cold water soluble gel weight component is: guar gum 8g, sodium polyacrylate 1～2g and sodium carboxymethyl cellulose 1～2g.
The preparation method that another object of the present invention is to provide above-mentioned total plate count test sheet, its step is as follows:
(1) prepared by culture medium dry powder
Tryptone 15～30g, yeast leaching powder 5～15g, sodium chloride 5～10g, glucose 1～5g, bean dregs 3～10g are joined in 90mL distilled water, after heating, stirring and dissolving, add Sodium Pyruvate 1～3g, mannitol 1～3g, Tween 80 1～3mL stirring and dissolving, regulate pH to 6.8～7.2,121 DEG C of autoclaving 15～20min, are cooled to 45～55 DEG C；After TTC0.2～0.5g is dissolved with 10mL distilled water, join after 0.22 μm of condition filter is degerming in aforesaid culture medium, stirring and evenly mixing.
Above-mentioned culture medium solution is put in 45～50 DEG C of aseptic drying baker and is dried, grind into powder, crosses 100～120 order molecular sieves, standby.
(2) prepared by composite cold water gel
By cold water gel guar gum, sodium polyacrylate and sodium carboxymethyl cellulose grind into powder respectively, cross 100～120 order molecular sieves, fully mix by 8:1～2:1～2 part by weight.
(3) preparation of total plate count test sheet
Upper strata, middle level and bottom panel material are cut into onesize, adopt polyacrylic resin pressure sensitive adhesive (transparent, nontoxic, tasteless, aseptic) by one side closed bonding；
By above-mentioned composite cold water solubles gel powder, being coated in the sticking one side in inner side of transparent plastic upper layer film equably, thickness is 0.10mm～0.20mm, then after autoclaving 15～20min, is taken out and is cooled to room temperature under 121 DEG C of conditions by above-mentioned test sheet.
First uniform polyacrylic acid coating resin pressure-sensitive glue on the base plate that la m void region is corresponding, thickness is 0.05mm～0.10mm, then is filled in the void region of la m by total plate count colour developing inspection side culture medium powder, and thickness is 0.10mm～0.20mm；
Finally will test sheet sterile vacuum packaging.
(4) test sheet is adopted to measure the operating procedure of total plate count
Solid or semi-solid sample: weigh 25g food samples to be checked (food to be checked can be solid or the semi-solid sample such as ready-to-eat food, ripe cereal product, instant fruit and vegetable goods, instant bean product) and be placed in the aseptic homogenizing cup filling 225mL normal saline, 8000r/min～10000r/min homogenizing 1min～2min, makes the even liquid of sample of mass ratio 1:10.
Fluid sample: draw 25mL food samples to be checked (food to be checked can be the fluid sample such as beverage, instant seasoning product) with aseptic straw and be placed in the aseptic conical flask filling 225mL normal saline, fully mixing, makes the even liquid of sample of volume ratio 1:10.
Draw the even liquid 1mL of sample after 1:10 with 1mL pipettor, slowly note in the sterile test tube filling 9mL normal saline dilution liquid along tube wall, mix homogeneously, make the even liquid of sample of volume ratio 1:100.Further 10 times of gradient dilutions, the even liquid of sample of preparation 10 times of dilutions of series, standby.
According to the estimation to sample contamination situation, select the even liquid of sample of 2～3 acceptable diluent degree, raise the upper layer film of total plate count test sheet prepared by the present invention, take the even liquid 1mL of above-mentioned sample and add in the void region that la m contains culture medium, make the even liquid of sample be uniformly distributed in whole cultivation region.Upper layer film is slowly fallen, it is to avoid extruding, stand 30s and treat composite cold water soluble gel and water cure, put into incubator 37 DEG C and cultivate 48h, count according to the method for counting of total plate count in the assay method of state food safety criterion total plate count.Each dilution factor do two test sheets, meanwhile, respectively draw 1mL normal saline dilution liquid (without add sample) add two test sheets make blank.
nullWhat the present invention developed is based on the total plate count test sheet of cold water soluble gel，Compared with similar products，Have the prominent advantages that this test sheet total plate count culture medium is by basal medium、Somatomedin、Developer、Surfactant and raising agent composition，The total plate count test sheet that the present invention is prepared based on said components，(food to be checked can be ready-to-eat food to add food samples diluent to be checked、Ripe cereal product、Instant fruit and vegetable goods、Instant bean product、Beverage、Many based foods such as instant seasoning product) after la m cultivates region，Raising agent bean dregs and other component of culture medium in la m culture medium mix with the composite cold water soluble gel on upper strata，Gel water absorption course is formed stable、The moderate gel colloidal again with fine and close mesh space of hole，The bacterial growth being especially suitable under test sheet environment is formed with bacterium colony；Raising agent and surfactant are additionally favorable for that sample liquid to be checked is uniformly distributed, permeates, bacterium colony is formed and visual counting, and color developing effect can be made after mix with developer better, and bacterium colony is of moderate size, it is easier to recognize；In culture medium except carry out the screening of basal nutrient component also to somatomedin, surfactant screens, the surface active agent tween 80 with infiltration and emulsification is conducive to sample liquid to be checked to be uniformly distributed after adding cultivation region with mannitol component, plus having the somatomedin Sodium Pyruvate promoted with repairing damage bacterial growth effect, antibacterial well breeds in incubation；The present invention has also selected conventional total plate count to measure culture medium developer TTC, according to visual effect and scope of restraining fungi, its content is optimized, reach good color developing effect, after carrying out total plate count mensuration, bacterial clump presents red colonies on test sheet, and the test sheet selecting clump count moderate counts.The total plate count manufactured paper disc utilizing this patent detects total plate count in many based foods with current GB4789.2 " national food safety standard food microbiological examination total plate count mensuration " requirement, and program is easy, bacterium colony colour generation is clear, counting is accurate.This test sheet will have comparatively wide application prospect at agricultural product and the food enterprises exfactory inspection, health and epidemic prevention, market surveillance, commodity inspection and import and export quarantine field.
Accompanying drawing explanation
Fig. 1: total plate count test chip architecture schematic diagram.
As it is shown in figure 1, each branch is: label 1 (sticks with this name of product, sample title, the label 1 of detection time in test sheet side), upper strata overlay 2, with the plastics la m 3 of void region, cultivation region 4 (void region is cultivation region), base plate 5.
Detailed description of the invention
Embodiment 1 total plate count measures the preparation of culture medium
On the basis of basal medium screening, further relatively different somatomedins, surfactant, raising agent, developer on test sheet colony growth, colour developing, counting impact, content is optimized.
We determined that the original weight component of total plate count colour developing inspection side culture medium: tryptone 20g, glucose 2.5g, yeast leaching powder 5g, sodium chloride 5g, bean dregs 6g, mannitol 1g, Sodium Pyruvate 1g, Tween 80 1mL, TTC0.3g.
The preparation method of total plate count colour developing inspection side culture medium is:
20g tryptone, 5g yeast leaching powder, 5g sodium chloride, 2.5g glucose, 6g bean dregs are joined in 90mL distilled water, after heating, stirring and dissolving, add 1g Sodium Pyruvate, 1g mannitol, 1mL Tween 80 stirring and dissolving, with 1mol/L sodium hydroxide or 1mol/L salt acid for adjusting pH to 7.0,121 DEG C of autoclaving 15min, are cooled to room temperature；After separately the TTC of 0.3g being dissolved with 10mL distilled water, join in aforesaid culture medium with under 0.22 μm of condition of disposable filter after filtration sterilization, stirring and evenly mixing.Above-mentioned solution puts into 50 DEG C of sterile oven dries, and grind into powder crosses 120 order molecular sieves, standby.
The preparation method of composite cold water soluble gel is:
By cold water soluble gel guar gum, sodium polyacrylate and sodium carboxymethyl cellulose grind into powder respectively, cross 120 order molecular sieves, fully mix by 8g:1g:1g part by weight, standby.
The preparation of embodiment 2 total plate count test sheet
1) test sheet is by up of three layers, respectively upper strata PET pellosil, middle level is with the polypropylene plastics flitch of void region, base plate white boxes art paper.Three parts are all cut into 11cm × 9cm, and middle void region (7cm × 7cm) is bonded into cultivation region with base plate, and cardboard is printed on the grid of light orange 1cm × 1cm, convenient counting.By upper, middle, and lower part polyacrylic resin pressure sensitive adhesive by bonded to each other for a lateral edges, bonding width is 5mm.
2) by composite cold water solubles gel powder, being coated in upper layer film inboard equably, thickness is 0.15mm, and every built-in testing sheet cold water gel quality is 0.2g；Above-mentioned test sheet is cooled to room temperature after putting into 121 DEG C of autoclaving 15min of autoclave.
3) on the base plate that void region, middle level is corresponding, first it is uniformly coated with sterile transparent polyacrylic resin pressure sensitive adhesive 0.2g, then takes culture medium dry powder 0.2g and be added into cultivation region.
4) in the process surveying specimen preparation, each link to ensure aseptic.
5) asepsis vacuum packing is standby.
Embodiment 3 utilizes test sheet to measure total plate count method in food
1) weigh food samples 25g to be checked (measuring samples is the solid food such as cooked meat product, cookies) homogenizing with sterile working or food samples to be checked (measuring samples is the liquid foods such as beverage) 25mL joins in the aseptic conical flask of 225mL normal saline, fully mixing, makes the even liquid of sample of 1:10.
2) draw the even liquid 1mL of 1:10 sample with 1mL micropipettor, slowly note in the sterile test tube filling 9mL normal saline dilution liquid along tube wall, mix homogeneously, make the even liquid of sample of 1:100.Further 10 times of gradient dilutions, prepare the serial dilutions of 1:1000,1:10000,1:100000.Standby.
3) food samples diluent to be checked (step 1, the 2 five kinds of diluents obtained are taken, the fluid sample such as beverage that bacteria containing amount is few can directly use stock solution) 1mL, raise the upper layer film of test sheet, add in culture medium region, make sample liquid be uniformly distributed in whole cultivation region, upper film is slowly fallen, it is to avoid finger extrudes, stand 30s and treat gel solidification, put into incubator 37 DEG C and cultivate 48h.Each dilution factor does two test sheets, meanwhile, draws two test sheets of 1mL normal saline addition respectively and makes blank.
4) the test sheet cultivated puts into aseptic operating platform, observes it with or without red colonies, and red colonies is counted.Method of counting counts according to standard GB/T 4789.2.
If clump count is all higher than 300CFU on all dilution test sheets, then the test sheet that dilution factor is the highest being counted, other test sheets are recordable for how counting, and result is multiplied by most highly diluted multiple by average colony number and calculates.
If all dilution test sheet clump counts are respectively less than 30CFU, then should be multiplied by extension rate by the average colony number that dilution factor is minimum and calculate.
If all dilution factors (include fluid sample stock solution) tests sheet all without colony growth, then to be multiplied by the calculating of minimum extension rate less than 1.
If all dilution test sheet clump counts are not all between 30CFU～300CFU, when a portion is less than 30CFU or more than 300CFU, then it is multiplied by extension rate with the average colony number closest to 30CFU or 300CFU and calculates.
Embodiment 4: total plate count test sheet measures effect assessment
1) mensuration of test sheet method detection different experiments bacterial strain total plate count
Total plate count test sheet is prepared by embodiment 2, standby.
Take escherichia coli respectively, Salmonella, bacillus cereus, staphylococcus aureus, Pseudomonas aeruginosa are inoculated in LB fluid medium (the yeast leaching powder 0.5g of 100mL, peptone 1.0g, sodium chloride 0.5g, 100mL distilled water, pH value is 7.0) in, put into 37 DEG C of cultivation 12h in constant incubator, take 1mL bacterium solution and add the mixing of 9mL normal saline, making diluent 1, bacterium solution is diluted to the suitable dilution factor counted by operation more than repeating, and (concentration is 100～102CFU/mL), each strain takes 1mL and uses test sheet mensuration total plate count according to the method in embodiment 3.Carry out nutrient agar panel colony counting, secondary repeated trials according to standard GB/T 4789.2 " national food safety standard food microbiological examination total plate count mensuration " simultaneously.The results averaged of detection, in Table 1.
Table 1: the total plate count of test sheet method detection different experiments bacterial strain
2) mensuration of total plate count in food samples
Weigh 25g or draw 25mL food samples, adding 225mL physiological saline solution, patting mixing with slap type homogenizer, make the even liquid of 1:10 sample.The diluent 1mL taking 1:10 joins in 9mL normal saline, fully mixes, and makes 1:100 diluent, and sample liquid is diluted to 3 suitable dilution factors counted by operation more than repeating, and (concentration is 100～102CFU/mL), carry out total plate count mensuration according to the method for embodiment 3, compare test sheet and measure the goodness of fit with national standard method measurement result, carry out statistical analysis.Test sheet measures interpretation of result such as table 2 that in different food products sample, total plate count measures with national standard method.
Table 2: the experimental result of total plate count in test sheet detection food
By table 1, table 2 it is shown that use the test sheet mensuration total plate count that the present invention develops good with GB nutrient agar panel count results coincidence rate, two groups of data carry out after data analysis without significant difference through SPSS software.
Claims (8)
1. total plate count test sheet, it is characterised in that: from top to bottom successively by the sticking transparent plastic upper layer film in inner side, form with the plastics la m of void region, the base plate that is printed with grid；Being filled with total plate count color developing detection culture medium in void region, the inner side of transparent plastic upper layer film is coated with composite cold water soluble gel；The composition by weight of total plate count colour developing inspection side culture medium is tryptone 15～30g, yeast leaching powder 5～15g, sodium chloride 5～10g, glucose 1～5g, Sodium Pyruvate 1～3g, mannitol 1～3g, Tween 80 1～3mL, TTC0.2～0.5g, bean dregs 3～10g composition；The composition by weight of composite cold water soluble gel is for be made up of guar gum 8g, sodium polyacrylate 1～2g and sodium carboxymethyl cellulose 1～2g.
2. total plate count test sheet as claimed in claim 1 a kind of, it is characterised in that: transparent plastic upper layer film be transparency high, nontoxic have no irritating odor, the PET pellosil of waterproof and breathable, thickness is 0.05mm～0.10mm.
3. a kind of total plate count test sheet as claimed in claim 1, it is characterised in that: plastics la m is the good polypropylene plastics flitch of hardness or PET plate, and thickness is 0.15mm～0.30mm.
4. a kind of total plate count test sheet as claimed in claim 1, it is characterised in that: base plate is waterproof, impermeable white art paper, and thickness is 0.10mm～0.20mm.
5. a kind of total plate count test sheet as claimed in claim 1, it is characterised in that: the resistant to elevated temperatures temperature of upper layer film, la m and base plate is 130 DEG C～150 DEG C.
6. a kind of total plate count tests sheet as claimed in claim 1, it is characterised in that: the void region of plastics la m is circular, oval or square structure, void region area and la m area proportion is 1:1.6～2.7.
7. the preparation method of a kind of total plate count test sheet described in claim 1, its step is as follows:
(1) prepared by culture medium dry powder
Tryptone 15～30g, yeast leaching powder 5～15g, sodium chloride 5～10g, glucose 1～5g, bean dregs 3～10g are joined in 90mL distilled water, after heating, stirring and dissolving, add Sodium Pyruvate 1～3g, mannitol 1～3g, Tween 80 1～3mL stirring and dissolving, regulate pH to 6.8～7.2,121 DEG C of autoclaving 15～20min, are cooled to 45～55 DEG C；After TTC0.2～0.5g is dissolved with 10mL distilled water, join after 0.22 μm of condition filter is degerming in aforesaid culture medium, stirring and evenly mixing；
Above-mentioned culture medium solution is put in 45～50 DEG C of aseptic drying baker and is dried, and grind into powder crosses 100～120 order molecular sieves；
(2) prepared by composite cold water gel
By cold water gel guar gum, sodium polyacrylate and sodium carboxymethyl cellulose grind into powder respectively, cross 100～120 order molecular sieves, fully mix by 8:1～2:1～2 part by weight；
(3) preparation of total plate count test sheet
Upper strata, middle level and bottom panel material are cut into onesize, adopt polyacrylic resin pressure sensitive adhesive by one side closed bonding；
By above-mentioned composite cold water solubles gel powder, being coated in the sticking one side in inner side of transparent plastic upper layer film equably, thickness is 0.10mm～0.20mm, then after autoclaving 15～20min, is taken out and is cooled to room temperature under 121 DEG C of conditions by above-mentioned test sheet；
First uniform polyacrylic acid coating resin pressure-sensitive glue on the base plate that la m void region is corresponding, thickness is 0.05mm～0.10mm, then is filled in the void region of la m by total plate count colour developing inspection side culture medium powder, and thickness is 0.10mm～0.20mm；
(4) finally will test sheet sterile vacuum packaging.
8. the application in total plate count in detection food of a kind of total plate count test sheet described in claim 1.
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