阅读说明：本技术 一种检测粽子中微生物的方法 (Method for detecting microorganisms in rice dumplings ) 是由 张国强 李江华 陈坚 堵国成 于 2020-05-28 设计创作，主要内容包括：本发明涉及一种检测粽子中微生物的方法,属于微生物检测领域。本发明的方法包括以下步骤：(1)菌落总数的测定；(2)霉菌和酵母计数；(3)大肠菌群计数。本发明的检测方法,具备一定操作经验的技术人员、或具备实验室均可进行操作。可将固体中不同的菌株快速分离并检测出来,操作简便、快速、误差小,可用于多种场景的菌株分离检测及数量检测,具有良好的应用前景。(The invention relates to a method for detecting microorganisms in rice dumplings, and belongs to the field of microorganism detection. The method of the invention comprises the following steps: (1) measuring the total number of colonies; (2) mold and yeast counts; (3) coliform counts. The detection method of the present invention can be performed by a technician having a certain operation experience or a laboratory. The method can quickly separate and detect different strains in the solid, is simple and convenient to operate, is quick, has small error, can be used for strain separation detection and quantity detection in various scenes, and has a good application prospect.)

1. A method for detecting microorganisms in rice dumplings is characterized by comprising the following steps:

A. measuring the total number of colonies; B. mold and yeast counts; C. counting coliform bacteria;

the step A is specifically as follows:

a1: taking a certain amount of samples from different parts of the samples, and respectively adding physiological saline for homogenization to prepare sample homogeneous solution;

a2: gradually diluting according to a proper multiple;

a3, selecting 2-3 sample homogeneous solutions with proper dilution, respectively adding 1m L into sterile culture dishes, adding 15-20m L into each dish, melting, keeping the temperature to 46 ℃ and counting agar culture medium on a flat plate, uniformly mixing, simultaneously, respectively sucking 1m L blank dilution solution and adding the blank dilution solution into two sterile dishes as blank control;

a4: after the agar had solidified, the plates were inverted and brought to 36 deg.C+1 cultivation of 48+2h；

A5: counting colonies on each plate, calculating the average value of the colonies on the parallel plates, and multiplying the average value by the corresponding dilution times to obtain the result of the total number of the colonies in each g of samples;

the step B is specifically as follows:

b1: taking a certain amount of samples from different parts of the samples, and respectively adding physiological saline for homogenization to prepare sample homogeneous solution;

b2: gradually diluting according to a proper multiple;

b3, selecting 2-3 sample homogeneous solutions with proper dilution, respectively adding 1m L into sterile culture dishes, adding 20-25m L into each dish, melting, keeping the temperature to 46 ℃ and adding potato glucose agar culture medium, mixing uniformly, simultaneously, respectively sucking 1m L blank dilution solution and adding the blank dilution solution into two sterile plates as blank control;

b4: after the agar had solidified, the plates were inverted and brought to 28 deg.C+1, culturing for 5 d;

b5: counting colonies on each plate, calculating the average value of the colonies on the parallel plates, and multiplying the average value by the corresponding dilution times to obtain the result of the total number of the colonies in each g of samples;

the step C is specifically as follows:

c1: taking a certain amount of samples from different parts of the samples, and respectively adding physiological saline for homogenization to prepare sample homogeneous solution;

c2: gradually diluting according to a proper multiple;

c3, selecting 2-3 sample homogeneous solutions with proper dilution, respectively adding 1m L into a sterile culture dish, adding 15-20m L into each dish, melting, keeping the temperature to 46 ℃, uniformly mixing, simultaneously, respectively absorbing 1m L blank dilution solution, and adding the blank dilution solution into two sterile plates as blank control;

c4: after the agar had solidified, the plates were inverted and brought to 36 deg.C+1, culturing for 18-24 h;

c5: typical and suspect coliform colonies present on each plate were counted.

2. The method of claim 1, wherein different types of canonical and suspect colonies are picked from VRBA plates and separated in step C5 of step CInoculating to Brilliant green lactose, bile salt and broth tube at 36 deg.C+1, culturing for 24-48h, and observing gas production conditions;

the specific operation steps are as follows:

(a) checking whether the plate has typical bacterial colony which is mauve and has red bile salt precipitation ring around;

(b) if typical colonies were present, 10 different types of typical and suspect colonies were picked from each plate, less than 10 colonies were picked and all typical and suspect colonies were individually plated in BG L B broth tubes at 36 deg.C+1, culturing for 24-48 h;

(c) observing the gas production condition, and reporting that the coliform group is positive when the BG L B broth tube produces gas;

(d) and finally, multiplying the ratio of the test tubes which are proved to be positive by the coliform group by the average value of the plate colony number, and then multiplying by the dilution times to obtain the coliform group number in each gram of sample.

3. The method according to claim 1, wherein the colonies are counted by visual observation, optionally with a magnifying glass or a colony counter, and the dilution factor and the corresponding number of colonies are recorded.

4. The method according to claim 3, wherein the total number of colonies is counted by selecting a plate with the colony count of between 30CFU and 300CFU and no spread colony growth; plates below 30CFU record specific colony numbers, more than 300CFU can be recorded as more than unmeasurable; the number of colonies per dilution should be taken as the average of two plates.

5. The method of claim 4, wherein when one of the plates has a large plate-like colony growth, it is not preferable to use the plate having no plate-like colony growth as the number of colonies at the dilution; if the plate-shaped colonies are less than half of the plate and the colonies in the other half are uniformly distributed, the number of the colonies on one plate can be represented by multiplying half of the plate by 2.

6. The method of claim 4, wherein each single strand is counted as a colony when chain growth occurs on the plate without a distinct boundary between colonies.

7. The method of claim 4, wherein the colony count is less than 100CFU, rounded off and reported as an integer.

8. The method of claim 4, wherein when the colony count is greater than or equal to 100CFU, the 3 rd digit is reduced by the "rounding off" rule, the first 2 digits are taken, and the digits are replaced by 0; it can also be expressed in the form of an index of 10, reduced by the "rounding" principle, followed by two significant digits.

9. The method of claim 4, wherein if colonies grow on the blank, the assay result is invalid.

10. The method of any one of claims 1 to 9, wherein the weight samples are reported in CFU/g and the volume samples are reported in CFU/m L.

Technical Field

The invention relates to a method for detecting microorganisms in rice dumplings, and belongs to the field of microorganism detection.

Background

In the production of food and beverages, the presence of certain microorganisms is concomitantly detected to ensure the quality of the final product. The detection of total number of colonies, moulds and yeasts and coliform groups is an important evidence of food quality. Microbial flora, including mold and yeast, is regulated in many countries and regions to allow for the content of certain food products, such as dairy, in the coliform.

The current detection methods usually require very skilled technicians to observe and interpret the detection results, the detection steps are complicated and tedious, and errors caused by operation are large. There is thus a need for a simple, accurate method for determining the number of microorganisms in a solid sample.

Disclosure of Invention

In order to solve the problems, the invention provides a detection method capable of detecting microbial flora, mould and yeast flora and coliform flora contained in the rice dumplings.

The invention provides a detection method of microorganisms in rice dumplings, which comprises the following steps:

A. measuring the total number of colonies; B. mold and yeast counts; C. coliform counts.

In one embodiment of the present invention, the step a comprises the steps of:

a1: taking a certain amount of samples from different parts of the samples, and respectively adding physiological saline for homogenization to prepare sample homogeneous solution;

a2: gradually diluting according to a proper multiple;

a3, selecting 2-3 sample homogeneous solutions with proper dilution, respectively adding 1m L into sterile culture dishes, adding 15-20m L into each dish, melting, keeping the temperature to 46 ℃ and counting agar culture medium (PCA) on a plate, uniformly mixing, simultaneously, respectively sucking 1m L blank dilution solution and adding the blank dilution solution into two sterile plates as blank control;

a4: after the agar is solidified, the plate is turned over and cultured for 48 +/-2 hours at 36 +/-1 ℃;

a5: the number of colonies on each plate was counted, and the mean of the number of colonies on the parallel plates was calculated and multiplied by the corresponding dilution factor as a result of the total number of colonies per g of sample.

In one embodiment of the present invention, the step B comprises the steps of:

b1: taking a certain amount of samples from different parts of the samples, and respectively adding physiological saline for homogenization to prepare sample homogeneous solution;

b2: gradually diluting according to a proper multiple;

b3, selecting 2-3 sample homogeneous solutions with proper dilution, respectively adding 1m L into sterile culture dishes, adding 20-25m L into each dish, melting, keeping the temperature to 46 ℃ and adding potato glucose agar culture medium, mixing uniformly, simultaneously, respectively sucking 1m L blank dilution solution and adding the blank dilution solution into two sterile plates as blank control;

b4: after the agar is solidified, turning over the plate, and culturing for 5d at 28 +/-1 ℃;

b5: the number of colonies on each plate was counted, and the mean of the number of colonies on the parallel plates was calculated and multiplied by the corresponding dilution factor as a result of the total number of colonies per g of sample.

In one embodiment of the present invention, the step C comprises the steps of:

c1: taking a certain amount of samples from different parts of the samples, and respectively adding physiological saline for homogenization to prepare sample homogeneous solution;

c2: gradually diluting according to a proper multiple;

c3, selecting 2-3 sample homogeneous solutions with proper dilution, respectively adding 1m L into a sterile culture dish, adding 15-20m L into each dish, melting, keeping the temperature to 46 ℃, uniformly mixing crystal violet neutral red bile salt agar (VRBA), and simultaneously respectively sucking 1m L blank diluent and adding the blank diluent into two sterile plates as blank control;

c4: after the agar is solidified, turning over the plate, and culturing for 18-24h at 36 +/-1 ℃;

c5: enumerating representative and suspect coliform colonies (e.g., colonies of smaller diameter than representative colonies) present on each plate; typical colonies are purple red, have red bile salt precipitation rings around the colony, and have a diameter of 0.5mm or greater.

In one embodiment of the present invention, different types of typical and suspicious colonies are picked from VRBA plates in the C5 step, and inoculated into Brilliant Green lactose bile salt (BG L B) broth tube, cultured at 36 ℃ +/-1 for 24-48h, and the gas production is observed, and the specific operation steps are as follows:

(a) checking whether the plate has typical bacterial colony which is mauve and has red bile salt precipitation ring around;

(b) if typical colonies exist, 10 different types of typical and suspicious colonies are picked from each plate, less than 10 colonies are picked, all typical and suspicious colonies are respectively transplanted into a BG L B broth tube, and the typical and suspicious colonies are cultured for 24-48h at 36 ℃ +/-1;

(c) observing the gas production condition, and reporting that the coliform group is positive when the BG L B broth tube produces gas;

(d) and finally, multiplying the ratio of the test tubes which are proved to be positive by the coliform group by the average value of the plate colony number, and then multiplying by the dilution times to obtain the coliform group number in each g of samples.

The invention has the beneficial effects that: the detection method of the present invention can be performed by a technician having a certain operation experience or a laboratory. The method can quickly separate and detect different strains in the solid, is simple and convenient to operate, is quick, has small error, can be used for strain separation detection and quantity detection in various scenes, and has a good application prospect.

Drawings

FIG. 1 is a flowchart of the test of the total number of colonies.

FIG. 2 is a flow chart of the assay for mold and yeast plate counts.

FIG. 3 is a flowchart of the test for coliform plate count.

Detailed Description