CN111206066A - Rapid detection method of microorganisms - Google Patents
Rapid detection method of microorganisms Download PDFInfo
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- CN111206066A CN111206066A CN202010218848.7A CN202010218848A CN111206066A CN 111206066 A CN111206066 A CN 111206066A CN 202010218848 A CN202010218848 A CN 202010218848A CN 111206066 A CN111206066 A CN 111206066A
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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
- C12Q1/10—Enterobacteria
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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
Abstract
The invention discloses a method for rapidly detecting microorganisms, which comprises the following steps: s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, and shaking to obtain 1:100 sample uniform solution; s2, inoculation: selecting 2-3 appropriate dilutions for food detection, placing the coliform test piece on a flat experiment table, and uncovering the upper layer film. The invention mainly adopts the coliform group test piece to carry out rapid detection, and the coliform group test piece contains a selective culture medium, a color indicator of coliform group specificity galactosidase and macromolecular absorbent gel, thereby not only greatly shortening the detection time, but also leading the detection result to be very close to the traditional method and the coincidence rate to reach about 90 percent, and solving the problems of long time consumption and low detection efficiency of the existing coliform group detection.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a rapid detection method of microorganisms.
Background
The coliform group mainly includes the genera Escherichia, Citrobacter, Redbergella and Enterobacter in the family Changium. Bacteria of these genera are both from the intestinal tract of humans and warm-blooded animals, aerobic and facultative anaerobic, do not form spores; can ferment lactose, lactic acid, lactic. The Escherichia coli group is mainly the Escherichia genus, which is commonly known as typical Escherichia coli. Coliform bacteria are faeces from humans and warm-blooded animals, either directly or indirectly. The genera in this group other than typical E.coli may arise from environmental variations after 7-30 days of excretion of typical E.coli, in addition to direct origin in feces. The detection of coliform groups in food products therefore indicates that the food product is contaminated, with typical coliform bacteria being recent contamination of faeces and other bacteria being old contamination of faeces
Coliform bacteria is one of important indexes for evaluating food hygiene quality, and is widely applied to food hygiene inspection work at present. Coliform bacteria are mostly present in excrement of warm-blooded animals, places where people frequently move and places where excrement is polluted, and the number of the coliform bacteria is high or low, which indicates the degree of pollution in food and food production processes. The existing coliform group bacteria detection takes long time and has low detection efficiency, so a method for rapidly detecting microorganisms is provided.
Disclosure of Invention
The present invention is directed to a method for rapidly detecting microorganisms, which solves the above problems of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme: a method for rapidly detecting microorganisms comprises the following steps:
s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, and shaking to obtain 1:100 sample uniform solution;
s2, inoculation: selecting 2-3 appropriate dilutions for food detection, placing a coliform group test piece on a flat experiment table, uncovering an upper layer film, sucking 1mL of sample uniform solution by using a sterile straw, slowly and uniformly dropping the sample uniform solution on a paper sheet, slowly covering the upper layer film, standing for about 10s to solidify a culture medium, inoculating two sheets at each dilution, and simultaneously making a blank negative control;
s3, culturing: placing the test pieces together into the original self-sealing bag, sealing, placing the bag in a constant temperature incubator with the transparent surface facing upwards horizontally, stacking the test pieces not more than 12, culturing at 35-37 deg.C for 15-24 h;
s4, counting: blue colonies on the cultured paper sheets are coliform colonies, and the paper sheets with the colony number between 15 and 150CFU are selected for counting.
Preferably, in step S1, the pH value of the sample homogenizing solution is controlled to be 6.5-7.5, and 1mol/L sodium hydroxide or 1mol/L hydrochloric acid is used to adjust the pH value of the sample homogenizing solution.
Preferably, the liquid sample with low bacteria content in step S2 can be directly absorbed to detect the original liquid, and the liquid sample with low bacteria content includes drinking pure water and mineral water.
Preferably, when the number of colonies on the test strip with two dilutions in the step S4 is between 15 and 150CFU, the average number of colonies is multiplied by the dilution factor to obtain the detection result, i.e. the number of coliform colonies per ml of sample.
Preferably, when the number of colonies on the test strip at all dilutions is less than 15CFU in step S4, the average colony on the test strip at the lowest dilution is counted and multiplied by the dilution factor to obtain the detection result.
Preferably, when all the test pieces with dilution in step S4 have been grown aseptically, the result is determined as less than 1 times the lowest dilution factor.
Preferably, when the number of colonies on the test strip with the highest dilution is greater than 150CFU in step S4, the average number of colonies on the test strip with the highest dilution is counted and multiplied by the dilution factor to obtain the detection result.
Preferably, when the number of colonies on the test strip with the highest dilution in the step S4 is greater than 150CFU, the number of colonies in one or two representative squares is counted, the counted number of colonies is converted into the number of colonies in a single square, and then the number of colonies is multiplied by the growth area of the test strip, so that the estimated number of colonies on the test strip is obtained, and the growth area of the paper surface is 20cm2。
Compared with the prior art, the invention has the beneficial effects that: the invention mainly adopts the coliform group test piece to carry out rapid detection, and the coliform group test piece contains a selective culture medium, a color indicator of coliform group specificity galactosidase and macromolecular absorbent gel, thereby not only greatly shortening the detection time, but also leading the detection result to be very close to the traditional method and the coincidence rate to reach about 90 percent, and solving the problems of long time consumption and low detection efficiency of the existing coliform group detection.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a method for rapidly detecting microorganisms comprises the following steps:
s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, and shaking to obtain 1:100 sample uniform solution;
s2, inoculation: selecting 2-3 appropriate dilutions for food detection, placing a coliform group test piece on a flat experiment table, uncovering an upper layer film, sucking 1mL of sample uniform solution by using a sterile straw, slowly and uniformly dropping the sample uniform solution on a paper sheet, slowly covering the upper layer film, standing for about 10s to solidify a culture medium, inoculating two sheets at each dilution, and simultaneously making a blank negative control;
s3, culturing: placing the test pieces together into the original self-sealing bag, sealing, placing the bag in a constant temperature incubator with the transparent surface facing upwards horizontally, stacking the test pieces not more than 12, culturing at 35-37 deg.C for 15-24 h;
s4, counting: blue colonies on the cultured paper sheets are coliform colonies, and the paper sheets with the colony number between 15 and 150CFU are selected for counting.
The first embodiment is as follows:
s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, and shaking to obtain 1:100 sample uniform solution;
s2, inoculation: selecting 2-3 appropriate dilutions for food detection, placing a coliform group test piece on a flat experiment table, uncovering an upper layer film, sucking 1mL of sample uniform solution by using a sterile straw, slowly and uniformly dropping the sample uniform solution on a paper sheet, slowly covering the upper layer film, standing for about 10s to solidify a culture medium, inoculating two sheets at each dilution, and simultaneously making a blank negative control;
s3, culturing: placing the test pieces together into the original self-sealing bag, sealing, placing the bag in a constant temperature incubator with the transparent surface facing upwards horizontally, stacking the test pieces not more than 12, culturing at 35-37 deg.C for 15-24 h;
s4, counting: blue colonies on the cultured paper sheets are coliform colonies, and the paper sheets with the colony number between 15 and 150CFU are selected for counting.
Example two:
s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, shaking to obtain 1:100 sample uniform solution, controlling the pH value of the sample uniform solution in the step S1 to be 6.5-7.5, and adjusting the pH value of the sample uniform solution by adopting 1mol/L sodium hydroxide or 1mol/L hydrochloric acid;
s2, inoculation: selecting 2-3 appropriate dilutions for food detection, placing a coliform group test piece on a flat experiment table, uncovering an upper layer film, sucking 1mL of sample uniform solution by using a sterile straw, slowly and uniformly dropping the sample uniform solution on a paper sheet, slowly covering the upper layer film, standing for about 10s to solidify a culture medium, inoculating two sheets at each dilution, and simultaneously making a blank negative control;
s3, culturing: placing the test pieces together into the original self-sealing bag, sealing, placing the bag in a constant temperature incubator with the transparent surface facing upwards horizontally, stacking the test pieces not more than 12, culturing at 35-37 deg.C for 15-24 h;
s4, counting: blue colonies on the cultured paper sheets are coliform colonies, and the paper sheets with the colony number between 15 and 150CFU are selected for counting.
Example three:
s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, shaking to obtain 1:100 sample uniform solution, controlling the pH value of the sample uniform solution in the step S1 to be 6.5-7.5, and adjusting the pH value of the sample uniform solution by adopting 1mol/L sodium hydroxide or 1mol/L hydrochloric acid;
s2, inoculation: selecting 2-3 appropriate dilutions for food detection, wherein a liquid sample with low bacteria content can directly absorb stock solution for detection, the liquid sample with low bacteria content comprises drinking pure water and mineral water, placing a coliform group test piece on a flat experiment table, uncovering an upper layer film, absorbing 1mL of sample uniform solution by using a sterile straw, slowly and uniformly dripping the sample uniform solution on a paper sheet, slowly covering the upper layer film, standing for about 10s to solidify a culture medium, inoculating two sheets of the culture medium at each dilution, and simultaneously making a blank negative control;
s3, culturing: placing the test pieces together into the original self-sealing bag, sealing, placing the bag in a constant temperature incubator with the transparent surface facing upwards horizontally, stacking the test pieces not more than 12, culturing at 35-37 deg.C for 15-24 h;
s4, counting: blue colonies on the cultured paper sheets are coliform colonies, and the paper sheets with the colony number between 15 and 150CFU are selected for counting.
Example four:
s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, shaking to obtain 1:100 sample uniform solution, controlling the pH value of the sample uniform solution in the step S1 to be 6.5-7.5, and adjusting the pH value of the sample uniform solution by adopting 1mol/L sodium hydroxide or 1mol/L hydrochloric acid;
s2, inoculation: selecting 2-3 appropriate dilutions for food detection, wherein a liquid sample with low bacteria content can directly absorb stock solution for detection, the liquid sample with low bacteria content comprises drinking pure water and mineral water, placing a coliform group test piece on a flat experiment table, uncovering an upper layer film, absorbing 1mL of sample uniform solution by using a sterile straw, slowly and uniformly dripping the sample uniform solution on a paper sheet, slowly covering the upper layer film, standing for about 10s to solidify a culture medium, inoculating two sheets of the culture medium at each dilution, and simultaneously making a blank negative control;
s3, culturing: placing the test pieces together into the original self-sealing bag, sealing, placing the bag in a constant temperature incubator with the transparent surface facing upwards horizontally, stacking the test pieces not more than 12, culturing at 35-37 deg.C for 15-24 h;
s4, counting: the blue bacterial colony on the cultured paper sheet is coliform bacterial colony, the paper sheet with bacterial colony number between 15-150CFU is selected for counting, when the bacterial colony number on the test sheet with two dilutions is between 15-150CFU, the average bacterial colony number is multiplied by the dilution multiple to be used as a detection result, namely the coliform bacterial colony number in each milliliter of sample, when the bacterial colony number on the test sheet with all dilutions is less than 15CFU, the average bacterial colony number on the test sheet with the lowest dilution is multiplied by the dilution multiple to be used as a detection result, when no bacterial colony grows on the test sheet with all dilutions, the average bacterial colony number on the test sheet with the highest dilution is multiplied by the dilution multiple to be used as a detection result, and when the bacterial colony number on the test sheet with the highest dilution is more than 150CFU, the average bacterial colony number on the test sheet with the highest dilution is multiplied by the dilution multiple to be used as a detection result.
Example five:
s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, shaking to obtain 1:100 sample uniform solution, controlling the pH value of the sample uniform solution in the step S1 to be 6.5-7.5, and adjusting the pH value of the sample uniform solution by adopting 1mol/L sodium hydroxide or 1mol/L hydrochloric acid;
s2, inoculation: selecting 2-3 appropriate dilutions for food detection, wherein a liquid sample with low bacteria content can directly absorb stock solution for detection, the liquid sample with low bacteria content comprises drinking pure water and mineral water, placing a coliform group test piece on a flat experiment table, uncovering an upper layer film, absorbing 1mL of sample uniform solution by using a sterile straw, slowly and uniformly dripping the sample uniform solution on a paper sheet, slowly covering the upper layer film, standing for about 10s to solidify a culture medium, inoculating two sheets of the culture medium at each dilution, and simultaneously making a blank negative control;
s3, culturing: placing the test pieces together into the original self-sealing bag, sealing, placing the bag in a constant temperature incubator with the transparent surface facing upwards horizontally, stacking the test pieces not more than 12, culturing at 35-37 deg.C for 15-24 h;
s4, counting: the blue bacterial colony on the cultured paper sheet is coliform bacterial colony, the paper sheet with bacterial colony number between 15-150CFU is selected for counting, when the bacterial colony number on the test sheet with two dilutions is between 15-150CFU, the average bacterial colony number is multiplied by the dilution multiple to be used as a detection result, namely the coliform bacterial colony number in each milliliter of sample, when the bacterial colony number on the test sheet with all dilutions is less than 15CFU, the average bacterial colony number on the test sheet with the lowest dilution is multiplied by the dilution multiple to be used as a detection result, when the bacterial colony number on the test sheet with all dilutions is grown, the bacterial colony number in one or two representative squares is counted, when the bacterial colony number on the test sheet with the highest dilution is more than 150CFU, the bacterial colony number in one square is multiplied by the paper sheet growth area to be used as the estimated bacterial colony number on the test sheet, the growth area of the paper surface is 20cm2。
The invention mainly adopts the coliform group test piece to carry out rapid detection, and the coliform group test piece contains a selective culture medium, a color indicator of coliform group specificity galactosidase and macromolecular absorbent gel, thereby not only greatly shortening the detection time, but also leading the detection result to be very close to the traditional method and the coincidence rate to reach about 90 percent, and solving the problems of long time consumption and low detection efficiency of the existing coliform group detection.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A method for rapidly detecting microorganisms, which is characterized in that: the method comprises the following steps:
s1, sample treatment: putting 25mL of a sample into a sampling tank or a homogenizing cup containing 225mL of sterilized phosphate buffer solution diluent or physiological saline to prepare 1:10 sample uniform solution, sucking 1:10 sample uniform solution 1mL by using a 1mL sterilizing suction pipe, injecting into a test tube containing 9mL of the diluent, and shaking to obtain 1:100 sample uniform solution;
s2, inoculation: selecting 2-3 appropriate dilutions for food detection, placing a coliform group test piece on a flat experiment table, uncovering an upper layer film, sucking 1mL of sample uniform solution by using a sterile straw, slowly and uniformly dropping the sample uniform solution on a paper sheet, slowly covering the upper layer film, standing for about 10s to solidify a culture medium, inoculating two sheets at each dilution, and simultaneously making a blank negative control;
s3, culturing: placing the test pieces together into the original self-sealing bag, sealing, placing the bag in a constant temperature incubator with the transparent surface facing upwards horizontally, stacking the test pieces not more than 12, culturing at 35-37 deg.C for 15-24 h;
s4, counting: blue colonies on the cultured paper sheets are coliform colonies, and the paper sheets with the colony number between 15 and 150CFU are selected for counting.
2. The method for rapid detection of microorganisms according to claim 1, wherein: in the step S1, the pH value of the sample homogenizing solution is controlled to be 6.5-7.5, and 1mol/L sodium hydroxide or 1mol/L hydrochloric acid is adopted to adjust the pH value of the sample homogenizing solution.
3. The method for rapid detection of microorganisms according to claim 1, wherein: in step S2, the liquid sample with low bacteria content can be directly absorbed to detect, and the liquid sample with low bacteria content includes drinking pure water and mineral water.
4. The method for rapid detection of microorganisms according to claim 1, wherein: and when the colony numbers of the test pieces with the two dilutions in the step S4 are both between 15 and 150CFU, taking the average colony number multiplied by the dilution times as a detection result, and obtaining the colony number of the coliform group in each milliliter of sample.
5. The method for rapid detection of microorganisms according to claim 1, wherein: and when the number of colonies on the test strip of all dilutions is less than 15CFU in the step S4, multiplying the average colony on the test strip with the lowest counting dilution by the dilution factor to obtain the detection result.
6. The method for rapid detection of microorganisms according to claim 1, wherein: when the sterile colonies grow on the test pieces with all the dilutions in the step S4, the result of the detection is obtained by multiplying the minimum dilution times by less than 1.
7. The method for rapid detection of microorganisms according to claim 1, wherein: and when the number of the colonies on the test strip with the highest dilution in the step S4 is more than 150CFU, counting the average number of the colonies on the test strip with the highest dilution multiplied by the dilution factor to obtain a detection result.
8. The method for rapid detection of microorganisms according to claim 1, wherein: when the number of the bacterial colonies on the test piece with the highest dilution degree in the step S4 is more than 150CFU, counting the bacterial colonies in one or two representative squares, converting the bacterial colonies into the bacterial colonies in a single square, and multiplying the bacterial colonies by the growth area of the paper sheet to obtain the estimated bacterial colonies on the test piece, wherein the growth area of the paper sheet is 20cm2。
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Application publication date: 20200529 |