CN105483204A - Detection vessel for rapidly detecting moulds and yeasts and preparation method - Google Patents
Detection vessel for rapidly detecting moulds and yeasts and preparation method Download PDFInfo
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- CN105483204A CN105483204A CN201510987947.0A CN201510987947A CN105483204A CN 105483204 A CN105483204 A CN 105483204A CN 201510987947 A CN201510987947 A CN 201510987947A CN 105483204 A CN105483204 A CN 105483204A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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Abstract
The invention provides a detection vessel capable of rapidly culturing and detecting moulds and yeasts at the same time. A biochemical reaction characteristic of microorganisms is utilized, types and reaction conditions of endoenzymes of the microorganisms are determined as main bases for microorganism classification and identification, a microorganism specific enzyme chromogenic substrate is added into an isolation medium or a selective medium, and when target bacteria grow on the medium, the chromogenic substrate can be degraded by specific enzymes and metabolites in special colors can be generated to realize specific colors or forms of colonies, thereby implementing culturing and detection of the microorganisms in a sample. The detection vessel has the advantages of convenience in operation, low cost, labor saving, simple judgment result, reliable detection result, short detection time and the like, and can play an important role in microorganism detection, and accuracy of a detection result reaches 90 percent or over 90 percent of a national standard detection method.
Description
Technical field
Technical field belonging to patent of the present invention is microbiologic inhibition tests, is specifically related to a kind of detection ware for rapid detection yeast and mold and preparation method.
Background technology
Yeast and mold is extensively present in air, water and soil, and the overwhelming majority is harmless, but some mould is harmful, mould can cause food to go mouldy, and can stimulate human body alimentary canal, stomach etc., and serious also can cause damage to liver, cause food poisoning, threat to life safety.Existing detection technique mainly contains GB4789.15-2010 national food safety standard, food microbiology detection-moulds and yeasts count, the method is flat band method, there is accurate, the reliable feature of detected result, classical microorganism detection and authentication method, right the method length consuming time, need now to configure substratum, complex operation step, be difficult to the actual requirement meeting fast inspection.
The appearance of paper disk method, compensate for the shortcoming of flat band method, progressively become main method and the standard of microorganism detection and qualification, 2007, Petrifilm testing plate is formally classified as Chinese inspection and quarantining for import/export industry standard, the method just gradually by increasing foodstuff production processing enterprise and all kinds of feeler mechanism approve.At present, more to the research of microorganism paper disk method both at home and abroad, Oasis Biochemistry Research Center discloses a utility model patent " the rapid detection inspection scraps of paper of yeast and mold " CN03274715.2, this patent adopts bottom-surface coated contain the upper epiphragma of enzyme developer water absorbent gel powder and adsorbed the cultivation sheet composition of liquid medium, the use of this product, need to carry out coating and the epiphragma operation of bacterium liquid to be measured, there is the improper epiphragma of manual operation and produce bubble and crawling and affect object bacteria and grow and the defect of experimental result interpretation difficulty.Jinan Institute of Product Quality Inspection and Shandong University disclose a patent of invention " detection method of foodstuff packaging material surface fungus and special filter paper " CN200110014458.2, and this invention is only applicable to packaging material for food, and only detects for mould.Hubei University Of Technology discloses the patent of invention preparation and application of coliform Rapid detecion paper " in food and the tableware " and filter paper is put into aseptic mold bag, assembling test sheet, there is object bacteria diffusion growth in assembling bag, colony edge is fuzzy, affects the shortcoming of detected result interpretation.3M Innovative Properties Company has applied for a patent of invention " for detecting method and the culture apparatus of yeast and mould " 201480007414.5, this culture apparatus comprises containing the main body from supporting substrate, base material has the first major surfaces and the second major surfaces, this culture apparatus needs testing sample to be coated with, be no more than the incubation time of 72 hours, can judge detected result, it is improper that its shortcoming is to there is manual operation, causes coating and epiphragma to produce the possibility of bubble.3M Innovative Properties Company has also applied for a patent of invention " device of rapid detection microorganism " 201180032280.9, the use of this device depends on coating and the epiphragma operation of testing sample, introduce because manual operation is improper, cause the possibility that sentence read result is wrong.
Summary of the invention
The problem to be solved in the present invention is to provide detection ware of a kind of yeast and mold fast culture and detection and preparation method thereof, to simplify and to accelerate the Testing and appraisal of yeast and mold, simplify laboratory operating procedures, improve the detection efficiency to microorganism, obtain reliable detected result.
The present invention is exactly to make up the deficiencies in the prior art and meeting the actual needs detected, on the basis of existing scraps of paper detection method, utilize the characteristic of microbial biochemical reaction, using the Main Basis that the kind of extracellular microbial endoenzyme and reaction conditions are identified as microorganism classification, by in isolation medium or selective medium, add microorganism specific enzymes chromogenic substrate, when object bacterium grows on substratum, its specific enzymes just can be degraded chromogenic substrate, and the metabolite produced with special color, bacterium colony is made to form specific color or form, thus carry out cultivation and the detection of microorganism in sample.The present invention adopts culture dish and filter paper to be carrier, exploitation yeast and mold is cultivated, is detected, incubation time is shortened to 48h by this detection ware, bacterium colony or bacterial plaque present three-dimensional growth, be easy to interpretation and analysis, can realize the detection of yeast and mold, and accuracy reaches GB more than 90% simultaneously, actual detection demand can be met.
Object of the present invention and solve its technical problem underlying and realize by the following technical solutions: a kind of detection ware for rapid detection yeast and mold, comprise sterile petri dish and testing plate, testing plate is laid in bottom sterile petri dish, testing plate is attached with yeast and mold feature colour developing liquid nutrient medium;
The composition of wherein said substratum, contain in every 100g distilled water: Tryptones 0.8g-2.0g, yeast powder 0.4g-0.8g, glucose 1g-4g, dipotassium hydrogen phosphate 0.05g-0.1g, sodium-chlor 0.05g-0.2g, potassium primary phosphate 0.2g-0.4g, magnesium sulfate 0.2g-0.4g, rose-bengal 0.04g-0.1g, paraxin 0.1g-0.3g, DMF 2g-4g, enzymolysis substrate mixture 0.015g-0.04g.
Described enzymolysis substrate mixture is dissolved in DMF by the chloro-3-indyl-phosphoric acid salt of the bromo-4-of 5-and NBT in 2:1-1:1 ratio and obtains.
The white Medium speed filter paper of described testing plate to be diameter be 70mm, every sheet filter paper uses 0.05Mol/LTriton-Tris buffer saline treatment solution to process, and treatment solution consumption is 1.0-1.5mL.Described sterile petri dish is the culture dish that single uses.
The preparation method of the detection ware of this rapid detection yeast and mold, follows these steps to preparation:
A. yeast and mold feature colour developing liquid nutrient medium is prepared: get 100g distilled water, add according to formula: after Tryptones 0.8g-2.0g, yeast powder 0.4g-0.8g, glucose 1g-4g, dipotassium hydrogen phosphate 0.05g-0.1g, sodium-chlor 0.05g-0.2g, potassium primary phosphate 0.2g-0.4g, magnesium sulfate 0.2g-0.4g, rose-bengal 0.04g-0.1g, heating for dissolving, adopt high-pressure sterilizing pot at 121 DEG C of sterilizing 15min, paraxin and the enzymolysis substrate mixture of sterilizing is after filtration added successively after being cooled to room temperature, obtain liquid nutrient medium, refrigerate for subsequent use at 0-4 DEG C.
B. testing plate is prepared:
Filter paper pre-treatment: absorbent filter paper is cut into the testing plate that diameter is 70mm, after adding the process of 1.0-1.5mL0.05Mol/LTriton-Tris buffer saline, put into 37 DEG C of thermostat containers or the dry 4h of loft drier, put into the valve bag containing siccative, room temperature preservation is for subsequent use.
Nutrient solution adsorbs: get above-mentioned process filter paper, 121 DEG C of sterilizing 15min, testing plate after sterilizing is immersed in the mould yeast feature colour developing liquid nutrient medium that steps A prepares, after soaking 5-10min, testing plate tweezers are pressed from both sides out, lies on clean Bechtop, after drying under aseptic condition, in the bacteria-free refrigerator of 0-4 DEG C, save backup;
C. assembling test sheet: by testing plate prepared in step B, be encased in sterile petri dish, after building culture dish lid, culture dish be placed in aluminum foil sack, vacuumize packaging, can product be obtained.
The present invention compared with prior art has obvious advantage and beneficial effect.From above technical scheme, concrete compared with prior art outstanding substantial advance has following 3 points:
The first, by medium optimization, a kind of substratum being simultaneously applicable to Molds and yeasts bacteria growing is obtained; Using good permeability, strong, the sizeable filter paper material of adsorptivity as the carrier of substratum, by the pre-treatment of filter paper, the absorption of substratum and oven dry, be attached to substratum on filter paper; By being directly inoculated on filter paper by testing sample, realize the fast culture to yeast and mold in testing sample and detection.
The second, the detection ware of rapid detection yeast and mold of the present invention, compared with existing 3MPetrifilm testing plate, avoid the phenomenon due to crawling and the improper generation bubble of epiphragma, the generation of bubble and existence, to considerable influence be produced for the count results detecting sample, avoid the improper error caused of manual operation.Meanwhile, take culture dish to be carrier, utilize distinctive culture medium prescription, yeast and mold three-dimensional growth can be realized, make it easy to observe and distinguish.
Three, the detection ware of rapid detection yeast and mold of the present invention, easy to use, can long-term storage, the feature of applying flexible, greatly can save the time of the process such as substratum configuration, culture dish sterilizing, cleaning, reduce labour intensity, shorten detection time.Meanwhile, it is simple, easy that the present invention detects ware preparation method, can realize automatic production, with low cost, easy to use.
Accompanying drawing explanation
The detection method detected result deck watch 1 that Fig. 1 is different.
Fig. 2 National Standard Method and this detection ware test result deck watch 2.
The process of Fig. 3 filter paper affects deck watch 3 to test-results.
Fig. 4 detects ware preparation flow figure.
Fig. 5 detects ware and uses schema.
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be understood that these embodiments are only for illustration of the present invention, instead of limit the scope of the invention.
Embodiment 1
For a detection ware for rapid detection yeast and mold, comprise sterile petri dish and testing plate, testing plate is laid in bottom sterile petri dish, testing plate is attached with yeast and mold feature colour developing liquid nutrient medium;
The wherein composition of substratum, contain in every 100g distilled water: Tryptones 0.8g, yeast powder 0.4g, glucose 1g, dipotassium hydrogen phosphate 0.05g, sodium-chlor 0.05g, potassium primary phosphate 0.2g, magnesium sulfate 0.2g, rose-bengal 0.04g, paraxin 0.1g, DMF 2g, enzymolysis substrate mixture 0.015g.Enzymolysis substrate mixture is dissolved in DMF by the chloro-3-indyl-phosphoric acid salt of the bromo-4-of 5-and NBT in 2:1 ratio and obtains.The white Medium speed filter paper of testing plate to be diameter be 70mm, every sheet filter paper uses 0.05Mol/LTriton-Tris buffer saline treatment solution to process, and treatment solution consumption is 1.0mL.Described sterile petri dish is the culture dish that single uses.
The preparation method of the detection ware of this rapid detection yeast and mold, follows these steps to preparation:
A. yeast and mold feature colour developing liquid nutrient medium is prepared: after recipe configuration substratum heating for dissolving, adopt high-pressure sterilizing pot at 121 DEG C of sterilizing 15min, paraxin and the enzymolysis substrate mixture of sterilizing is after filtration added successively after being cooled to room temperature, obtain liquid nutrient medium, refrigerate for subsequent use at 0-4 DEG C.
B. testing plate is prepared:
Filter paper pre-treatment: absorbent filter paper to be cut into area be diameter is the testing plate of 70mm, after adding the process of 1.0mL0.05Mol/LTriton-Tris buffer saline, put into 37 DEG C of thermostat containers or the dry 4h of loft drier, put into siccative with valve bag, room temperature preservation is for subsequent use.Nutrient solution adsorbs: get above-mentioned process filter paper, 121 DEG C of sterilizing 15min, testing plate after sterilizing is immersed in the mould yeast feature colour developing liquid nutrient medium that steps A prepares, after soaking 5min, testing plate tweezers are pressed from both sides out, lies on clean Bechtop, after drying under aseptic condition, in the bacteria-free refrigerator of 0-4 DEG C, save backup;
C. assembling test sheet: by testing plate prepared in step B, be encased in sterile petri dish, after building culture dish lid, culture dish be placed in aluminum foil sack, vacuumize packaging, can product be obtained.
When using the detection ware of this rapid detection yeast and mold, first, sample thief 25mL (g) puts into sampler or the homogeneous cup of 225mL sterile diluent (phosphate buffered saline buffer or physiological saline), make the even liquid of sample of 1:10, the even liquid 1mL of 1:10 sample is drawn with the sterilizing suction pipe of 1mL, inject containing 9mL diluent in vitro, obtain the sample diluting liquid of 1:100 after shaking up, down dilute successively.General sample selects 1 ~ 2 extent of dilution to detect, and the liquid sample (as drinking pure and mineral water etc.) that bacteria containing amount is few can directly be drawn stoste and detect.Next connects bacterium: testing plate is placed in smooth experiment table top, opens culture dish upper cover, draws the even liquid of 1mL sample be added in testing plate with sterilizing rifle head or dropper, and adjustment testing plate angle makes moistening whole the testing plate of sample liquid, closes the lid and can cultivate.Each extent of dilution inoculation two panels, gets 1mL diluent simultaneously and inoculates another testing plate and make blank.Then cultivate: the testing plate inoculated is positioned in 28 ± 1 DEG C of incubators and cultivates 48-72h.Ultimate analysis detected result: mould presents the bacterium colonies such as canescence, black, blue-greenish colour in testing plate, and bacterium colony presents more greatly the form of himself; Yeast presents green bacterium colony in testing plate, and bacterium colony is less more level and smooth.
Embodiment 2
For a detection ware for rapid detection yeast and mold, comprise sterile petri dish and testing plate, testing plate is laid in bottom sterile petri dish, testing plate is attached with yeast and mold feature colour developing liquid nutrient medium;
The wherein composition of substratum, contain in every 100g distilled water: Tryptones 1.2g, yeast powder 0.6g, glucose 3g, dipotassium hydrogen phosphate 0.08g, sodium-chlor 0.1g, potassium primary phosphate 0.3g, magnesium sulfate 0.3g, rose-bengal 0.06g, paraxin 0.2g, DMF 3g, enzymolysis substrate mixture 0.02g.Enzymolysis substrate mixture is dissolved in DMF by the chloro-3-indyl-phosphoric acid salt of the bromo-4-of 5-and NBT in 1:1 ratio and obtains.The white Medium speed filter paper of testing plate to be diameter be 70mm, every sheet filter paper uses 0.05Mol/LTriton-Tris buffer saline treatment solution to process, and treatment solution consumption is 1.2mL.Described sterile petri dish is the culture dish that single uses.
The preparation method of the detection ware of this rapid detection yeast and mold, follows these steps to preparation:
A. yeast and mold feature colour developing liquid nutrient medium is prepared: after recipe configuration substratum heating for dissolving, adopt high-pressure sterilizing pot at 121 DEG C of sterilizing 15min, paraxin and the enzymolysis substrate mixture of sterilizing is after filtration added successively after being cooled to room temperature, obtain liquid nutrient medium, refrigerate for subsequent use at 3 DEG C.
B. testing plate is prepared:
Filter paper pre-treatment: absorbent filter paper to be cut into area be diameter is the testing plate of 70mm, after adding the process of 1.2mL0.05Mol/LTriton-Tris buffer saline, put into 37 DEG C of thermostat containers or the dry 4h of loft drier, put into siccative with valve bag, room temperature preservation is for subsequent use.Nutrient solution adsorbs: get above-mentioned process filter paper, 121 DEG C of sterilizing 15min, testing plate after sterilizing is immersed in the mould yeast feature colour developing liquid nutrient medium that steps A prepares, after soaking 5min, testing plate tweezers are pressed from both sides out, lies on clean Bechtop, after drying under aseptic condition, in the bacteria-free refrigerator of 0-4 DEG C, save backup;
C. assembling test sheet: by testing plate prepared in step B, be encased in sterile petri dish, after building culture dish lid, culture dish be placed in aluminum foil sack, vacuumize packaging, can product be obtained.
When using the detection ware of this rapid detection yeast and mold, first, sample thief 25mL (g) puts into sampler or the homogeneous cup of 225mL sterile diluent (phosphate buffered saline buffer or physiological saline), make the even liquid of sample of 1:10, the even liquid 1mL of 1:10 sample is drawn with the sterilizing suction pipe of 1mL, inject containing 9mL diluent in vitro, obtain the sample diluting liquid of 1:100 after shaking up, down dilute successively.General sample selects 1 ~ 2 extent of dilution to detect, and the liquid sample (as drinking pure and mineral water etc.) that bacteria containing amount is few can directly be drawn stoste and detect.Next connects bacterium: testing plate is placed in smooth experiment table top, opens culture dish upper cover, draws the even liquid of 1mL sample be added in testing plate with sterilizing rifle head or dropper, and adjustment testing plate angle makes moistening whole the testing plate of sample liquid, closes the lid and can cultivate.Each extent of dilution inoculation two panels, gets 1mL diluent simultaneously and inoculates another testing plate and make blank.Then cultivate: the testing plate inoculated is positioned in 28 ± 1 DEG C of incubators and cultivates 48-72h.Ultimate analysis detected result: mould presents the bacterium colonies such as canescence, black, blue-greenish colour in testing plate, and bacterium colony presents more greatly the form of himself; Yeast presents green bacterium colony in testing plate, and bacterium colony is less more level and smooth.
Embodiment 3
For a detection ware for rapid detection yeast and mold, comprise sterile petri dish and testing plate, testing plate is laid in bottom sterile petri dish, testing plate is attached with yeast and mold feature colour developing liquid nutrient medium;
The wherein composition of substratum, contain in every 100g distilled water: Tryptones 2.0g, yeast powder 0.8g, glucose 4g, dipotassium hydrogen phosphate 0.1g, sodium-chlor 0.2g, potassium primary phosphate 0.4g, magnesium sulfate 0.4g, rose-bengal 0.1g, paraxin 0.3g, DMF 4g, enzymolysis substrate mixture 0.04g.Enzymolysis substrate mixture is dissolved in DMF by the chloro-3-indyl-phosphoric acid salt of the bromo-4-of 5-and NBT in 1:2 ratio and obtains.The white Medium speed filter paper of testing plate to be diameter be 70mm, every sheet filter paper uses 0.05Mol/LTriton-Tris buffer saline treatment solution to process, and treatment solution consumption is 1.5mL.Described sterile petri dish is the culture dish that single uses.
The preparation method of the detection ware of this rapid detection yeast and mold, follows these steps to preparation:
A. yeast and mold feature colour developing liquid nutrient medium is prepared: after recipe configuration substratum heating for dissolving, adopt high-pressure sterilizing pot at 121 DEG C of sterilizing 15min, paraxin and the enzymolysis substrate mixture of sterilizing is after filtration added successively after being cooled to room temperature, obtain liquid nutrient medium, refrigerate for subsequent use at 0-4 DEG C.
B. testing plate is prepared:
Filter paper pre-treatment: absorbent filter paper to be cut into area be diameter is the testing plate of 70mm, after adding the process of 1.5mL0.05Mol/LTriton-Tris buffer saline, put into 37 DEG C of thermostat containers or the dry 4h of loft drier, put into siccative with valve bag, room temperature preservation is for subsequent use.Nutrient solution adsorbs: get above-mentioned process filter paper, 121 DEG C of sterilizing 15min, testing plate after sterilizing is immersed in the mould yeast feature colour developing liquid nutrient medium that steps A prepares, after soaking 5min, testing plate tweezers are pressed from both sides out, lies on clean Bechtop, after drying under aseptic condition, in the bacteria-free refrigerator of 0-4 DEG C, save backup;
C. assembling test sheet: by testing plate prepared in step B, be encased in sterile petri dish, after building culture dish lid, culture dish be placed in aluminum foil sack, vacuumize packaging, can product be obtained.
When using the detection ware of this rapid detection yeast and mold, first, sample thief 25mL (g) puts into sampler or the homogeneous cup of 225mL sterile diluent (phosphate buffered saline buffer or physiological saline), make the even liquid of sample of 1:10, the even liquid 1mL of 1:10 sample is drawn with the sterilizing suction pipe of 1mL, inject containing 9mL diluent in vitro, obtain the sample diluting liquid of 1:100 after shaking up, down dilute successively.General sample selects 1 ~ 2 extent of dilution to detect, and the liquid sample (as drinking pure and mineral water etc.) that bacteria containing amount is few can directly be drawn stoste and detect.Next connects bacterium: testing plate is placed in smooth experiment table top, opens culture dish upper cover, draws the even liquid of 1mL sample be added in testing plate with sterilizing rifle head or dropper, and adjustment testing plate angle makes moistening whole the testing plate of sample liquid, closes the lid and can cultivate.Each extent of dilution inoculation two panels, gets 1mL diluent simultaneously and inoculates another testing plate and make blank.Then cultivate: the testing plate inoculated is positioned in 28 ± 1 DEG C of incubators and cultivates 48-72h.Ultimate analysis detected result: mould presents the bacterium colonies such as canescence, black, blue-greenish colour in testing plate, and bacterium colony presents more greatly the form of himself; Yeast presents green bacterium colony in testing plate, and bacterium colony is less more level and smooth.
In order to better embody beneficial effect of the present invention, contriver is by the present invention and national standard detection method, and it is as follows that 3M test-paper method compares detection analytical results:
By cultured yeast liquid, be diluted to 10 with sterile saline or phosphate buffered saline buffer
-6, 10
-7two gradients, using these two gradient yeast liquid of having diluted as mother liquor, are diluted to 10 by mould
-1, 10
-2, after dilution mixing, simultaneously containing yeast and mold in mother liquor, this mixed solution is the sample to be tested bacterium liquid handled well.Draw in the yeast and mold testing plate and detection ware of the present invention that 1mL sample to be tested bacterium liquid is uniformly coated on the flat board of National Standard Method recommendation, 3M produces with sterilizing rifle head or dropper, using 1mL sterile saline as blank, under 28 ± 1 DEG C of conditions, after cultivating 48h, record observations is as following table.
The detection method detected result deck watch (incubation time 48 hours, culture temperature 28 ± 1 DEG C) that table 1 is different
By cultured yeast liquid, be diluted to 10 with sterile saline or phosphate buffered saline buffer
-6, 10
-7two gradients, using these two gradient yeast liquid of having diluted as mother liquor, are diluted to 10 by mould
-1, 10
-2, after dilution mixing, simultaneously containing yeast and mold in mother liquor, this mixed solution is the sample to be tested bacterium liquid handled well.Drawing 1mL sample to be tested bacterium liquid with sterilizing rifle head or dropper is uniformly coated on flat board that National Standard Method recommends and on detection ware of the present invention respectively, using 1mL sterile saline as blank, under 28 ± 1 DEG C of conditions, cultivate after 66 hours, record observations is as following table 2.
Table 2 National Standard Method and this detection ware test result comparison (incubation time 66 hours, culture temperature 28 ± 1 DEG C)
After the present invention and National Standard Method and the comparison of 3M mould yeast detected result, experimental result shows that detected result of the present invention reaches GB standard, rate of accuracy reached more than 90%, shows with 3M mould yeast test result, and detected result accuracy of the present invention reaches 3M mould yeast testing plate.Simultaneously in order to verify that 0.05Mol/LTriton-Tris buffer saline treatment solution is to effect of the present invention and effect, devise following contrast experiment, same filter paper material is divided into two groups at random, one group of filter paper carries out pre-treatment, another group does not carry out any process, other process are consistent with culture condition, detect, using physiological saline as blank cultured mould yeast liquid.Laboratory test results is as table 3.
The process of table 3 filter paper affects deck watch to test-results
Simultaneous test shows that the filter paper detected result after processing is more accurate.Treated filter paper, more can be effective to the detection of yeast and mold in testing sample.
Claims (5)
1. the detection ware for rapid detection yeast and mold, comprise sterile petri dish and testing plate, testing plate is laid in bottom sterile petri dish, testing plate is attached with yeast and mold feature colour developing liquid nutrient medium, it is characterized in that: the composition of substratum, contain in every 100g distilled water: Tryptones 0.8g-2.0g, yeast powder 0.4g-0.8g, glucose 1g-4g, dipotassium hydrogen phosphate 0.05g-0.1g, sodium-chlor 0.05g-0.2g, potassium primary phosphate 0.2g-0.4g, magnesium sulfate 0.2g-0.4g, rose-bengal 0.04g-0.1g, paraxin 0.1g-0.3g, N, dinethylformamide 2g-4g, enzymolysis substrate mixture 0.015g-0.04g.
2. a kind of detection ware for rapid detection yeast and mold according to claim 1, it is characterized in that: described enzymolysis substrate mixture is dissolved in DMF by the chloro-3-indyl-phosphoric acid salt of the bromo-4-of 5-and NBT in 2:1-1:1 ratio and obtains.
3. a kind of detection ware for rapid detection yeast and mold according to claim 1, it is characterized in that: the white Medium speed filter paper of described testing plate to be diameter be 70mm, every sheet filter paper uses 0.05Mol/LTriton-Tris buffer saline treatment solution to process, and treatment solution consumption is 1.0-1.5mL.
4. a kind of detection ware for rapid detection yeast and mold according to claim 1, is characterized in that: described sterile petri dish is the culture dish that single uses.
5. prepare a preparation method for the detection ware of the rapid detection yeast and mold of claim 1-4 described in any one, it is characterized in that, follow these steps to preparation:
A. yeast and mold feature colour developing liquid nutrient medium is prepared: get 100g distilled water, add according to formula: after Tryptones 0.8g-2.0g, yeast powder 0.4g-0.8g, glucose 1g-4g, dipotassium hydrogen phosphate 0.05g-0.1g, sodium-chlor 0.05g-0.2g, potassium primary phosphate 0.2g-0.4g, magnesium sulfate 0.2g-0.4g, rose-bengal 0.04g-0.1g, heating for dissolving, adopt high-pressure sterilizing pot at 121 DEG C of sterilizing 15min, paraxin and the enzymolysis substrate mixture of sterilizing is after filtration added successively after being cooled to room temperature, obtain liquid nutrient medium, refrigerate for subsequent use at 0-4 DEG C;
B. testing plate is prepared:
Filter paper pre-treatment: absorbent filter paper to be cut into area be diameter is the testing plate of 70mm, after adding the process of 1.0-1.5mL0.05Mol/LTriton-Tris buffer saline, take out after putting into 37 DEG C of thermostat containers or the dry 4h of loft drier, put into the valve bag containing siccative, room temperature preservation is for subsequent use;
Nutrient solution adsorbs: get above-mentioned process filter paper, 121 DEG C of sterilizing 15min, testing plate after sterilizing is immersed in the mould yeast feature colour developing liquid nutrient medium that steps A prepares, after soaking 5-10min, testing plate tweezers are pressed from both sides out, lies on clean Bechtop, after drying under aseptic condition, in the bacteria-free refrigerator of 0-4 DEG C, save backup;
C. assembling test sheet: by testing plate prepared in step B, be encased in sterile petri dish, after building culture dish lid, culture dish be placed in aluminum foil sack, vacuumize packaging, can product be obtained.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510987947.0A CN105483204A (en) | 2015-12-25 | 2015-12-25 | Detection vessel for rapidly detecting moulds and yeasts and preparation method |
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| CN201510987947.0A CN105483204A (en) | 2015-12-25 | 2015-12-25 | Detection vessel for rapidly detecting moulds and yeasts and preparation method |
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| CN105483204A true CN105483204A (en) | 2016-04-13 |
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| CN106811403A (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 bacillus cereus and preparation method thereof, detection method |
| 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 |
| CN106834104A (en) * | 2017-01-22 | 2017-06-13 | 贵州勤邦食品安全科学技术有限公司 | A kind of test ware of quick detection total number of bacterial colonies and preparation method thereof |
| CN107043803A (en) * | 2017-05-24 | 2017-08-15 | 中检科（北京）实验室能力评价有限公司 | Yeast and mold sum numerical ability verification sample and preparation method thereof in medicine |
| CN109182445A (en) * | 2018-09-20 | 2019-01-11 | 吉林农业大学 | A kind of mould and yeast counts testing piece, preparation method and applications |
| WO2019064307A1 (en) * | 2017-09-27 | 2019-04-04 | Himedia Laboratories Pvt Ltd | Process for preparation of a chromogen based tool |
| CN109929903A (en) * | 2019-03-04 | 2019-06-25 | 内蒙古蒙牛乳业（集团）股份有限公司 | A kind of yeast and mold detection culture medium and its preparation method and application |
| CN112322692A (en) * | 2020-11-09 | 2021-02-05 | 安徽屾远材料技术有限公司 | Mould rapid detection patch and preparation method and use method thereof |
| CN112481350A (en) * | 2020-11-18 | 2021-03-12 | 中科健康产业集团股份有限公司 | Composite culture medium for rapidly detecting total number of moulds and yeasts and preparation method thereof |
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| CN106811403A (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 bacillus cereus and preparation method thereof, detection method |
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| CN106834104A (en) * | 2017-01-22 | 2017-06-13 | 贵州勤邦食品安全科学技术有限公司 | A kind of test ware of quick detection total number of bacterial colonies and preparation method thereof |
| CN107043803A (en) * | 2017-05-24 | 2017-08-15 | 中检科（北京）实验室能力评价有限公司 | Yeast and mold sum numerical ability verification sample and preparation method thereof in medicine |
| WO2019064307A1 (en) * | 2017-09-27 | 2019-04-04 | Himedia Laboratories Pvt Ltd | Process for preparation of a chromogen based tool |
| CN109182445A (en) * | 2018-09-20 | 2019-01-11 | 吉林农业大学 | A kind of mould and yeast counts testing piece, preparation method and applications |
| CN109182445B (en) * | 2018-09-20 | 2021-10-22 | 吉林农业大学 | Mould and yeast counting test piece, preparation method and application thereof |
| CN109929903A (en) * | 2019-03-04 | 2019-06-25 | 内蒙古蒙牛乳业（集团）股份有限公司 | A kind of yeast and mold detection culture medium and its preparation method and application |
| CN109929903B (en) * | 2019-03-04 | 2023-08-01 | 内蒙古蒙牛乳业（集团）股份有限公司 | Culture medium for detecting mold and saccharomycetes as well as preparation method and application thereof |
| CN112322692A (en) * | 2020-11-09 | 2021-02-05 | 安徽屾远材料技术有限公司 | Mould rapid detection patch and preparation method and use method thereof |
| CN112481350A (en) * | 2020-11-18 | 2021-03-12 | 中科健康产业集团股份有限公司 | Composite culture medium for rapidly detecting total number of moulds and yeasts and preparation method thereof |
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