CN109576338A - A kind of microorganism color developing agent and preparation method thereof and viable count detection device and preparation method thereof and detection method - Google Patents
A kind of microorganism color developing agent and preparation method thereof and viable count detection device and preparation method thereof and detection method Download PDFInfo
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- CN109576338A CN109576338A CN201811388389.6A CN201811388389A CN109576338A CN 109576338 A CN109576338 A CN 109576338A CN 201811388389 A CN201811388389 A CN 201811388389A CN 109576338 A CN109576338 A CN 109576338A
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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
- C12Q1/06—Quantitative determination
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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
- C12Q2304/00—Chemical means of detecting microorganisms
- C12Q2304/20—Redox indicators
- C12Q2304/22—Resazurin; Resorufin
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Abstract
The present invention relates to microorganism detection fields, in particular to a kind of microorganism color developing agent and preparation method thereof and viable count detection device and preparation method thereof and detection method, a kind of microorganism color developing agent, including following component and its mass ratio: carbon source and oxidation-reduction indicator, their mass ratioes are 100 ~ 1000:1 ~ 5.Nutriment needed for the present invention matches microbial metabolism and the oxidation-reduction indicator for indicating metabolic response, reflect the metabolic activity of bacterium to test sample viable count by the discoloration of oxidation-reduction indicator, rather than bacterium colony is directly counted, easy to operate and detection time is short.
Description
Technical field
The present invention relates to microorganism detection field, in particular to a kind of microorganism color developing agent and preparation method thereof and work
Fungal counting device and preparation method thereof and detection method.
Background technique
At present, the links such as the common detection methods of viable count, including sample preparation, culture, counting;Concrete operations are as follows: sample
After being prepared as the bacterium solution of debita spissitudo, then with suitable solid medium single Bacteria Culture at macroscopic single colonie,
These processes be unable to do without laboratory operation, and incubation time needs 48 hours.
Petrifilm total plate count testing piece (and the bacterium colony of other brands of domestic production of Minnesota Mining and Manufacturing Company's production
Total testing piece), it can be used for test sample viable count, it is solid medium is prefabricated and be applied on the scraps of paper or film, when use
The bacterium solution prepared is added dropwise in testing piece, is then flattened, and in 36 DEG C of constant temperature stationary cultures；Due to testing piece background color and bacterium
It is larger to fall color color difference, needing to cultivate 24 hours can count, and simplify the experimental implementation of culture link to a certain extent.
The use of above-mentioned Petrifilm total plate count testing piece operates, and there are still following shortcomings: (1) sample preparation link
For two class sample of body surface and liquid, different sampling consumptive materials is bought respectively --- sampling dropper or sampling rod, sample preparation
Trouble；(2) incubation time still needs to 24 hours, and test period is too long for family consumer；(3) testing piece is easy by microorganism
Pollution, causes the shelf-life short.
Summary of the invention
The first object of the present invention is to provide a kind of metabolic activity for reflecting bacterium by color change, to detect sample
The microorganism color developing agent of product viable count.
The second object of the present invention is to provide a kind of preparation method for operating simple, quick microorganism color developing agent.
The third object of the present invention is to provide a kind of detection viable count detection device simple, easy to use.
The fourth object of the present invention is to provide that a kind of detection is simple, and operation is easy, time-consuming short viable count detection device
Production method.
The fifth object of the present invention is that providing a kind of operate microorganism detection simplifies, and makes it possible to not depend on laboratory
Condition, consumer can be readily achieved detection at home, and the detection side of test period short viable count detection device
Method.
The first object of the present invention is achieved in that
A kind of microorganism color developing agent, including following component and its mass ratio: carbon source and oxidation-reduction indicator, their mass ratioes are
100 ~ 1000:1 ~ 5.
Above-mentioned first purpose of the invention can also be solved using following technical measures:
Further, further include having nitrogen source and thickener, the carbon source, oxidation-reduction indicator, nitrogen source and thickener quality
Than for 100 ~ 1000:1 ~ 5:0.1 ~ 5000:0.1 ~ 5000.
Further, the carbon source is glucose or beef extract, and the reductant-oxidant is the one of resazurin or methylenum careuleum
Kind or two kinds of combinations, the nitrogen source are peptone or yeast extract, and the thickener is agar or carragheen or starch or gathers
Sodium acrylate.
The second object of the present invention is achieved in that
A kind of preparation method of microorganism color developing agent, includes the following steps:
Step 1 prepares culture medium: weighing carbon source, distilled water is added and boils dissolution；
Step 2, cooling culture medium, controls the temperature of culture medium between 60-70 DEG C, oxidation-reduction indicator is then added, and makes
Obtain microorganism color developing agent；
Step 3 carries out aseptic packaging to microorganism color developing agent, is then cooled to room temperature.
Above-mentioned second purpose of the invention can also be solved using following technical measures:
Further, nitrogen source and thickener can also be added in the step 1.
Further, the microorganism color developing agent forms microorganism color developing agent powder after freeze-drying.
The third object of the present invention is achieved in that
A kind of viable count detection device, including microorganism color developing agent, test section and hand-held part, the test section and hand-held part connect
It connects, test section is adsorbed with microorganism color developing agent.
Above-mentioned third purpose of the invention can also be solved using following technical measures:
Further, the hand-held part is additionally provided with the liquid storage chamber for soaking test section, and the liquid storage chamber stores sterile slow
Fliud flushing.
The fourth object of the present invention is achieved in that
A kind of production method of viable count detection device, includes the following steps:
Step 1, the portion of will test are soaked into microorganism color developing agent, take out after wetting；
Test section after above-mentioned wetting is freeze-dried by step 2；
Viable count detection device is carried out aseptic sealed packages by step 3.
The fifth object of the present invention is achieved in that
A kind of detection method of viable count detection device, includes the following steps:
The test section of viable count detection device is immersed in tested solution wetting or by the inspection of viable count detection device by step 1
Survey portion wipes the surface of checking matter after soaking using sterile buffer；
Step 2 reacts viable count detection device 2-6 hours in the range of 25-37 DEG C；
Step 3, the color that will test portion are compared with the color of liquid viable count colorimetric card or object viable count colorimetric card,
Read the total plate count value of tested solution or checking matter.
Beneficial effects of the present invention are as follows:
(1) oxidation-reduction indicator of nutriment and instruction metabolic response needed for the microbial metabolism of present invention proportion, leads to
The discoloration of oxidization-reduction indicator reflects that the metabolic activity of bacterium to test sample viable count, rather than directly counts bacterium
It falls, easy to operate and detection time is short.
(2) microorganism color developing agent of the present invention, preparation method is simple, quick, and microorganism color developing agent can be prepared into powder
Last current state, convenient for storage.
(3) viable count detection device of the present invention, preparation method is simple, and viable count detection device passes through sterile sealing packet
It can be stored after dress, without remaking, user uses more convenient.
(4) viable count detection device of the present invention, including test section and hand-held part, above-mentioned test section are adsorbed with microorganism colour developing
Agent, in use, user holds hand-held part, the portion of will test, which is immersed in tested solution, to be soaked or the portion of will test is using sterile buffer
Checking matter surface is wiped after wetting, can collect microorganism, it is easy to operate, it is easy to use.
(5) production method of viable count detection device of the present invention, user collect microorganism, after reaction, cause to examine
Survey portion color generates variation, and user is compared according to the color of test section with the color of corresponding colorimetric card, thus know by
The total plate count value of object is examined, detection is simple, and operation is easy, and it is time-consuming short, greatly improve the practicability of viable count detection device.
(6) microorganism detection is operated and is simplified, makes it possible to not depend on by the detection method of viable count detection device of the present invention
Laboratory condition, consumer can be readily achieved detection at home, and test period is short, and entire detection only need to be 2 ~ 6 small
When can read as a result, detection cycle than conventional laboratory methods shorten 8 times.
Detailed description of the invention
Fig. 1 is the schematic diagram of the viable count detection device of the embodiment of the present invention one.
Fig. 2 is the schematic diagram of the viable count detection device of the embodiment of the present invention two.
Fig. 3 is fluid present invention viable count colorimetric card schematic diagram.
Fig. 4 is object viable count colorimetric card schematic diagram of the present invention.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and embodiments:
A kind of microorganism color developing agent, including following component and its mass ratio: carbon source, oxidation-reduction indicator, nitrogen source and thickener,
Mass ratio between them is 100 ~ 1000:1 ~ 5:0.1 ~ 5000:0.1 ~ 5000.
Wherein, the carbon source is glucose or beef extract, the reductant-oxidant be resazurin or methylenum careuleum one kind or
Two kinds of combinations, the nitrogen source are peptone or yeast extract, and the thickener is agar or carragheen or starch or polypropylene
Sour sodium.
A kind of preparation method of microorganism color developing agent:
Step 1: it prepares culture medium: weighing beef extract 0.3g, peptone 1g, sodium chloride 0.5g, agar 0.03g, add distillation boiling
Boil-off solution；
Step 2: cooling culture medium controls the temperature of culture medium at 65 degree, 0.05%(m/m is added) resazurin solution 2ml, it is made
Microorganism chromogenic reagent solution；
Step 3: aseptic packaging is carried out to microorganism color developing agent while hot, is then cooled to room temperature.
For the ease of saving, the microorganism chromogenic reagent solution can be formed into microorganism color developing agent after freeze-drying
Powder, to realize the purpose convenient for storage.
A kind of viable count detection device and preparation method thereof:
Embodiment one, as shown in connection with fig. 1, a kind of viable count detection device 3, including microorganism color developing agent, test section 1 and hand-held part
2, the test section 1 is hydrophilic and strong adsorption cotton swab, and hand-held part 2 is wooden stick, and the cotton swab is wrapped in wooden stick one end
Portion, test section 1 are adsorbed with microorganism color developing agent.
Embodiment two, as shown in connection with fig. 2, the hand-held part 1 of the viable count detection device are additionally provided with for soaking detection
The liquid storage chamber 11 in portion, the liquid storage chamber 11 store sterile buffer, and the sterile buffer is pure water.
A kind of production method of viable count detection device:
Step 1: it will test portion 1 and be soaked into microorganism chromogenic reagent solution, taken out after wetting；
Step 2: the test section 1 after wetting is freeze-dried；
Step 3: viable count detection device 3 is subjected to aseptic sealed packages.
Make liquid viable count colorimetric card and production body surface viable count colorimetric card:
As shown in connection with fig. 3, a kind of liquid viable count colorimetric card preparation method:
Step 1: nutrient broth 100ml is prepared in 250ml conical flask, is inoculated with 3 ring of Escherichia coli slant culture；
Step 2: cultivating 24 hours in 37 DEG C, and original bacteria liquid is made；
Step 3: original bacteria liquid is pressed into 10 times of gradient dilutions, respectively obtains 10-1、10-2、10-3、10-4、10-5Dilution gradient bacterium solution；
Step 4: detecting the viable count of original bacteria liquid and gradient dilution bacterium solution with pour plate method, completes normal concentration bacterium solution
Calibration, the viable count for measuring original bacteria liquid and each dilution gradient bacterium solution is respectively 10000CFU/ml, 1000CFU/ml, 100CFU/
ml,10CFU/ml,1CFU/ml,0.1CFU/ml；
Step 5: the test section 1 of viable count detection device 3 is separately immersed in the bacterium solution of different viable counts 1 second, is then taken
Out；
Step 6: viable count detection device 3 is reacted 3 hours within the scope of 25 DEG C, is then taken pictures and test section 1 and is extracted test section
1 color value；
Step 7: the color of the test section 1 in the bacterium solution for being immersed in different viable counts is mapped with its clump count, and production is not
With the colorimetric card of color.
As shown in connection with fig. 4, a kind of body surface viable count colorimetric card preparation method:
Step 1: inoculation staphylococcus aureus to inclined-plane nutrient agar slant medium；
Step 2: cultivating 24 hours in 37 DEG C, and slant culture is made；
Step 3: the starch salting liquid of starch-containing 0.5%, sodium chloride 0.5% is prepared；
Step 4: being washed down staphylococcus aureus slant culture with 2ml starch salting liquid, then supplements starch salting liquid extremely
100ml obtains original bacteria liquid；
Step 5: original bacteria liquid starch salting liquid is pressed into 10 times of gradient dilutions, respectively obtains 10-1、10-2、10-3、10-4、10-5Ladder
Degree dilution bacterium solution；
Step 6: cutting out 2.5cm × 4cm mute surface and PVC plastic sheet 36, and 6 one group, 6 groups of bacterium solutions for impregnating 6 concentration gradients respectively
In 1 minute, then take out and dry；
Step 7: every group of bacterium piece takes 3 to be wiped with the sterile cotton swab that sterile saline soaks respectively, then each cotton swab
It puts into 10ml sterile saline, vibrates 1 minute respectively, then take 1ml oscillation liquid pour plate method detection viable count, it is complete
At the calibration of standard bacteria piece: the viable count of 6 groups of bacterium pieces is respectively equal to 10000CFU/dm2、1000CFU/dm2、100CFU/dm2、
10CFU/dm2、1CFU/dm2、0.1CFU/dm2
Step 8: the test section of viable count detection device is soaked with pure water and uniformly wipes each standard bacteria piece, every group of bacterium piece
Test 3；
Step 9: viable count detection device 3 is reacted 3 hours in 25 DEG C of temperature ranges, test section 1 of then taking pictures, and extracts detection
Then test section 1 color of the wiping in the bacterium piece of different viable counts is mapped with its clump count, makes by the color value in portion 1
Make the colorimetric card of different colours.
A kind of method that microorganism color developing agent detects viable count:
Step 1: the test section 1 of viable count detection device 3 is immersed in tested solution and is soaked；
Step 2: viable count detection device 3 is reacted 2-6 hours in the range of 25-37 DEG C；
Step 3: the color that will test portion 1 is compared with the color of liquid viable count colorimetric card, reads the bacterium colony of tested solution
Total value.
For detecting the viable count of direct drinking fountain water sample, method is as follows:
Step 1: one glass of water is taken from direct drinking fountain with clean disposal plastic cup；
Step 2: the hand-held part 2 of hand-held viable count detection device 3 will test and soak in the immersion of portion 1 water flow, then takes out；
Step 3: by viable count detection device 3 30 DEG C at a temperature of react 3 hours；
Step 4: the color of the color and liquid viable count colorimetric card that will test portion carries out, if the color and work of discovery test section 1
Bacterium number is that the color of 10CFU/ml is identical, can judge that the viable count of direct drinking fountain water sample is 10CFU/ml.
The method of detection object surface viable count:
Step 1: by the test section 1 of viable count detection device 3 using the surface for wiping checking matter after sterile purified water wetting；
Step 2: viable count detection device 3 is reacted 2-3 hours in the range of 25-37 DEG C；
Step 3: the color that will test portion 1 is compared with the color of body surface viable count colorimetric card, reads the bacterium of checking matter
Fall total value.
Embodiment 4 detects the viable count of door knob watch face:
Step 1: the hand-held part 2 of hand-held viable count detection device 3 soaks test section 1 with sterile purified water；
Step 2: 10 dm on door handle is uniformly wiped with test section2Surface area；
Step 3: by viable count detection device 3 30 DEG C at a temperature of react 3 hours；
Step 4: the color of the color and object viable count colorimetric card that will test portion 1 carries out, and finds the color and work of test section 1
Bacterium number is 100CFU/dm2Color it is identical, can judge door knob watch face viable count be 100CFU/dm2。
Testing principle:
Nutriment needed for the present invention matches microbial metabolism and the oxidation-reduction indicator for indicating metabolic response, pass through oxygen
Change the discoloration of reduction indicator to reflect that the metabolic activity of bacterium to test sample viable count, rather than directly counts bacterium colony, behaviour
It is short to make simple and detection time.
Claims (10)
1. a kind of microorganism color developing agent, which is characterized in that including following component and its mass ratio: carbon source and redox instruction
Agent, their mass ratioes are 100 ~ 1000:1 ~ 5.
2. microorganism developer according to claim 1, which is characterized in that it further include having nitrogen source and thickener, the carbon source,
The mass ratio of oxidation-reduction indicator, nitrogen source and thickener is 100 ~ 1000:1 ~ 5:0.1 ~ 5000:0.1 ~ 5000.
3. microorganism developer according to claim 2, which is characterized in that the carbon source be glucose or beef extract, it is described
Reductant-oxidant is one or two kinds of combinations of resazurin or methylenum careuleum, and the nitrogen source is peptone or yeast extract, described
Thickener is agar or carragheen or starch or Sodium Polyacrylate.
4. a kind of preparation method of microorganism color developing agent as described in claim 1, which comprises the steps of:
Step 1 prepares culture medium: weighing carbon source, distilled water is added and boils dissolution；
Step 2, cooling culture medium, controls the temperature of culture medium between 60-70 DEG C, adds oxidation-reduction indicator, is made
Microorganism color developing agent；
Step 3 carries out aseptic packaging to microorganism color developing agent, is subsequently cooled to 4 ~ 35 DEG C.
5. the preparation method of microorganism color developing agent according to claim 4, which is characterized in that can also add in the step 1
Enter nitrogen source and thickener.
6. the preparation method of microorganism color developing agent according to claim 4, which is characterized in that the microorganism color developing agent passes through
Microorganism color developing agent powder is formed after freeze-drying.
7. a kind of viable count detection device containing microorganism color developing agent described in claim 1, which is characterized in that aobvious including microorganism
Toner, test section and hand-held part, the test section are connected with hand-held part, and test section is adsorbed with the microorganism color developing agent.
8. viable count detection device according to claim 7, which is characterized in that the hand-held part is additionally provided with for soaking inspection
The liquid storage chamber in survey portion, the liquid storage chamber store sterile buffer.
9. a kind of production method of viable count detection device as claimed in claim 7, which comprises the steps of:
Step 1, the portion of will test are soaked into microorganism color developing agent, take out after wetting；
Test section after above-mentioned wetting is freeze-dried by step 2,
Viable count detection device is carried out aseptic sealed packages by step 3.
10. a kind of detection method of viable count detection device as claimed in claim 7, which comprises the steps of:
The test section of viable count detection device is immersed in tested solution and soaks by step 1, or by viable count detection device
Test section wipes the surface of checking matter after soaking using sterile buffer；
Step 2 reacts viable count detection device 2-6 hours in the range of 25-37 DEG C；
Step 3, the color that will test portion are compared with the color of liquid viable count colorimetric card or object viable count colorimetric card,
Read the total plate count value of tested solution or checking matter.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811388389.6A CN109576338A (en) | 2018-11-21 | 2018-11-21 | A kind of microorganism color developing agent and preparation method thereof and viable count detection device and preparation method thereof and detection method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811388389.6A CN109576338A (en) | 2018-11-21 | 2018-11-21 | A kind of microorganism color developing agent and preparation method thereof and viable count detection device and preparation method thereof and detection method |
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