CN113999776A - Eurotium cristatum culture medium and eurotium cristatum plate counting method - Google Patents
Eurotium cristatum culture medium and eurotium cristatum plate counting method Download PDFInfo
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- CN113999776A CN113999776A CN202111406864.XA CN202111406864A CN113999776A CN 113999776 A CN113999776 A CN 113999776A CN 202111406864 A CN202111406864 A CN 202111406864A CN 113999776 A CN113999776 A CN 113999776A
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Abstract
The invention provides a eurotium cristatum culture medium and a eurotium cristatum plate counting method, wherein the eurotium cristatum culture medium comprises the following components in percentage by weight: 3-7g of peptone, 8-12g of glucose, 18-23g of sucrose, 0.7-1.3g of monopotassium phosphate, 0.2-0.7g of magnesium sulfate, 13-18g of agar, 0.01-0.05g of Bengal, 0.05-0.2g of chloramphenicol, 10% -15% of sodium chloride and 5-7g of lithium chloride, wherein after the components are mixed, the pH is adjusted to 7.2 +/-0.2. The invention discloses a new culture medium for eurotium cristatum, which can inhibit bacteria, control the growth of part of fungi and control the spread of colonies, wherein the eurotium cristatum has relatively unique colony characteristics and can be easily separated from other fungi, and the problem that the eurotium cristatum in the Fuzhuan tea does not have a special quantitative counting method can be solved through a subsequent corresponding eurotium cristatum plate counting method.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a eurotium cristatum culture medium and a eurotium cristatum plate counting method.
Background
The most distinctive characteristic of the dark tea is the Fuzhuan tea, and the probiotics Eurotium cristatum in the Fuzhuan tea has important influence on the tea quality of the Fuzhuan tea. Fuzhuan tea is a unique tea variety in China, and is specified in the current national standard GB/T9833.3-2013: the qualified standard of the Fuzhuan tea is that the number of the Eurotium cristatum is more than or equal to 200000CFU/g, but no detection standard for detecting the Eurotium cristatum exists at home and abroad at present, so GB/T9833.3 only can temporarily designate the standard GB4789.15 (the national food inspection standard, the count of mould and yeast) to detect the Eurotium cristatum; GB4789.15 uses potato glucose agar or a Bengal culture medium to culture the mold, the 2 mold culture media have no selectivity to the mold, although the Eurotium cristatum belongs to the mold, a plurality of pathogenic bacteria exist in the mold and the yeast, for example, the colony color of virulent Aspergillus flavus is yellow on a mold counting plate, so that the confusion is very easy, and the detection of the Eurotium cristatum by using the mold and yeast counting method is not only inaccurate, but also has the possibility of serious safety accidents. Therefore, the research of a rapid and convenient quantitative detection method of eurotium cristatum is an urgent need in the industry, and for this reason, the invention provides a eurotium cristatum culture medium and a eurotium cristatum plate counting method.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a eurotium cristatum culture medium and a eurotium cristatum plate counting method, the eurotium cristatum culture medium and the eurotium cristatum plate counting method are reasonable in design, and the problem that the eurotium cristatum in the Fuzhuan tea has no special quantitative counting method can be solved, the invention discloses a novel eurotium cristatum culture medium which can inhibit bacteria, control the growth of part of fungi and control the spread of the bacterial colony simultaneously, the eurotium cristatum has relatively unique colony characteristics on the eurotium cristatum culture medium, can be easily separated from other fungi, the counting result of the eurotium cristatum can be obtained through simple primary screening identification, if the identification needs to be more accurate, because the eurotium cristatum can be obviously distinguished from other fungi in the eurotium cristatum on the culture medium of the invention, therefore, by identifying the ITS nucleic acid sequence of a typical colony and determining that the colony belongs to the genus Eurotium, the colony can be confirmed to be Eurotium cristatum.
In order to achieve the purpose, the invention is realized by the following technical scheme: a eurotium cristatum culture medium comprises the following components by weight: 3-7g of peptone, 8-12g of glucose, 18-23g of sucrose, 0.7-1.3g of monopotassium phosphate, 0.2-0.7g of magnesium sulfate, 13-18g of agar, 0.01-0.05g of Bengal, 0.05-0.2g of chloramphenicol, 10% -15% of sodium chloride and 5-7g of lithium chloride, wherein after the components are mixed, the pH is adjusted to 7.2 +/-0.2.
A method for counting eurotium cristatum plates comprises the following specific steps:
the method comprises the following steps: preparing a sample; selecting a proper amount of tea samples for later use, and taking a proper amount of eurotium cristatum culture medium raw materials for later use;
step two: homogenizing the sample; crushing a tea sample by using an aseptic crusher, and weighing a proper amount of crushed sample by using a quartering method for later use;
step three: diluting a sample; the method comprises the following specific steps:
the method comprises the following steps: sucking 25mL of the crushed sample by using a sterile suction pipe into a suitable container or a sterile homogenizing bag containing 225mL of sterile diluent, fully shaking or beating by using a beating type homogenizer for 1min-2min to prepare a product 1: 10, homogenizing the sample;
secondly, the step of: taking 1mL of 1: injecting 10 sample homogenized solution into a test tube containing 9mL of sterile diluent, and repeatedly blowing and sucking by replacing 1 sterile suction tube with 1mL, or uniformly mixing on a vortex mixer, wherein the solution is 1: 100 sample homogenizing liquid;
③: preparing 10 times of serial dilution sample homogenizing solution, and replacing 1 sterile pipette with 1mL each time of incremental dilution;
fourthly, the method comprises the following steps: selecting 2-3 sample homogeneous solutions with appropriate dilution, respectively sucking 1mL of the sample homogeneous solution into 2 sterile plates for each dilution while performing 10-fold incremental dilution, and simultaneously respectively adding 1mL of the sterile dilution solution into 2 sterile plates for blank comparison;
fifthly: pouring 20-25 mL of the Eurotium cristatum counting plate cooled to 46 ℃ into a plate in time, rotating the plate to uniformly mix the Eurotium cristatum counting plate and the Eurotium cristatum counting plate, and placing the plate on a horizontal table until the Eurotium cristatum culture medium is completely solidified;
step four: culturing; after the agar is solidified, placing the flat plate in the right direction, culturing the flat plate in an incubator at the temperature of 28 +/-1 ℃, and observing and recording the results from the culture to the culture at the 7 th day from the 2 nd day;
step five: counting typical colonies; the characteristic of the typical colony morphology of the Eurotium cristatum on a Eurotium cristatum counting plate is that the colony is a white flocculent circular colony when 2d-3d is carried out, the colony is a bright golden circular colony after 3d-4d is carried out, the colony is a round colony with bright golden edges and black center and always has water beads, the back of the colony is dark red to red brown, the colony is compact, the plate with the suspected Eurotium cristatum colonies of yellow, bright golden and the like is selected, the plate with the suspected Eurotium cristatum colony number of the plate with the same dilution degree between 10CFU and 150CFU is counted;
step six: calculating a primary screening result; calculating the average value of the colony numbers of two plates with the same dilution degree, and multiplying the average value by the corresponding dilution times, wherein the specific calculation steps are as follows:
the method comprises the following steps: if the colony number on two dilution plates is between 10 and 150CFU, calculating according to the corresponding regulation of GB 4789.2;
secondly, the step of: if the colony number on all the plates is more than 150CFU, counting the plate with the highest dilution, recording the number of other plates as the number of the plates which can not be counted, and calculating the result according to the average colony number multiplied by the highest dilution times;
③: if the colony number on all the plates is less than 10CFU, calculating according to the average colony number with the lowest dilution multiplied by the dilution times;
fourthly, the method comprises the following steps: if all dilution (including liquid sample stock) plates were grown aseptically, calculated as less than 1 times the lowest dilution;
fifthly: if the colony number of the plate at all dilutions is not between 10CFU and 150CFU, and a part of the colony number is less than 10CFU or more than 150CFU, the colony number is calculated by multiplying the average colony number closest to 10CFU or 150CFU by the dilution factor;
sixthly, the method comprises the following steps: when the determination is further confirmed, at least 5 suspicious colonies are selected from typical colonies for ITS identification, and an identification test is carried out, wherein the ITS identification result belongs to the Eurotium cristatum, and the colony culture characteristic is the typical characteristic of the Eurotium cristatum, so that the Eurotium cristatum is confirmed;
step seven: calculating a final result; the calculation formula is as follows:
calculating formula 1: t ═ AB/Cd
In the formula:
t-the number of colonies of eurotium cristatum in the sample;
a-total number of typical colonies at a certain dilution;
b-number of colonies identified as positive at a certain dilution;
c-one dilution used to identify the number of colonies tested;
d-dilution factor;
calculating formula 2:
T＝(A1B1/C1+A2B2/C2)/1.1d
in the formula:
t-the number of colonies of eurotium cristatum in the sample;
a1 — first dilution, i.e. total number of representative colonies at low dilution;
b1 — number of colonies identified as positive at the first dilution, i.e. low dilution;
c1 — first dilution, low dilution, is used to identify the number of colonies tested;
a2-second dilution-total number of representative colonies at high dilution;
b2-number of colonies positive for the second dilution, i.e.high dilution, identification;
c2 — second dilution, high dilution, is used to identify the number of colonies tested;
1.1-calculating the coefficient;
d-dilution factor of the first dilution;
and determining the typical colony ITS as Eurotium cristatum after being identified as Eurotium positive, and specifically calculating the following steps:
the method comprises the following steps: counting the typical colonies on the dilution plate if the typical colony number of only one dilution plate is between 10CFU and 150CFU, and calculating according to the formula 1;
secondly, the step of: if the number of typical colonies on the lowest dilution plate is less than 10CFU, counting the typical colonies on the dilution plate, and calculating according to the formula 1;
③: if the number of typical colonies on one dilution plate is more than 150CFU but no typical colony on the next dilution plate, counting the typical colonies on the dilution plate according to formula 1;
fourthly, the method comprises the following steps: if the number of typical colonies on one dilution plate is more than 150CFU, and the number of typical colonies on the next dilution plate is not in the range of 10CFU-150CFU, counting the typical colonies on the dilution plate, and calculating according to the formula 1;
fifthly: if the number of typical colonies on the plate at 2 serial dilutions is between 10CFU and 150CFU, it is calculated as formula 2.
As a preferred embodiment of the present invention, the sterile diluent in the (c) of the third step includes, but is not limited to, distilled water, physiological saline or phosphate buffer.
As a preferred embodiment of the present invention, in the sixth step, if there are less than 5 suspicious colonies, the total selection is performed.
In the third step (i), a proper amount of sterile glass beads are placed in the container.
In a preferred embodiment of the present invention, in the fifth step, the Eurotium cristatum counting plate is placed in a thermostatic water bath box at 46 ℃ +/-1 ℃ for heat preservation.
The invention has the beneficial effects that: the invention can solve the problem that the Eurotium cristatum in the Fuzhuan tea has no special quantitative counting method, the invention not only can inhibit bacteria, but also can control the growth of part of fungi and simultaneously control the spread of bacterial colonies by inventing a novel culture medium for the Eurotium cristatum, the Eurotium cristatum has relatively unique bacterial colony characteristics on the culture medium for the Eurotium cristatum, the Eurotium cristatum can be easily separated from other fungi, the counting result of the Eurotium cristatum can be obtained by simple primary screening identification, if the identification is more accurate, the Eurotium cristatum can be obviously distinguished from other fungi in the Eurotium cristatum on the culture medium, so that the bacterial colony can be confirmed to be the Eurotium cristatum by identifying the ITS nucleic acid sequence of a typical bacterial colony and determining that the bacterial colony belongs to the Eurotium cristatum.
Drawings
FIG. 1 is a schematic flow chart of a method for counting Eurotium cristatum plates according to the present invention;
FIG. 2 is a detailed flowchart of the method for counting Eurotium cristatum plates of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Referring to fig. 1 to 2, the present invention provides a technical solution: a eurotium cristatum culture medium comprises the following components by weight: 5g of peptone, 10g of glucose, 20g of sucrose, 1g of monopotassium phosphate, 0.5g of magnesium sulfate, 15g of agar, 0.03g of Bengal, 0.1g of chloramphenicol, 10% -15% of sodium chloride and 6g of lithium chloride, wherein after the components are mixed, the pH is adjusted to 7.2 +/-0.2.
A method for counting eurotium cristatum plates comprises the following specific steps:
the method comprises the following steps: preparing a sample; selecting a proper amount of tea samples for later use, and taking a proper amount of eurotium cristatum culture medium raw materials for later use;
step two: homogenizing the sample; crushing a tea sample by using an aseptic crusher, and weighing a proper amount of crushed sample by using a quartering method for later use;
step three: diluting a sample; the method comprises the following specific steps:
the method comprises the following steps: sucking 25mL of the crushed sample by using a sterile suction pipe into a suitable container or a sterile homogenizing bag containing 225mL of sterile diluent, fully shaking or beating by using a beating type homogenizer for 1min-2min to prepare a product 1: 10, homogenizing the sample;
secondly, the step of: taking 1mL of 1: injecting 10 sample homogenized solution into a test tube containing 9mL of sterile diluent, and repeatedly blowing and sucking by replacing 1 sterile suction tube with 1mL, or uniformly mixing on a vortex mixer, wherein the solution is 1: 100 sample homogenizing liquid;
③: preparing 10 times of serial dilution sample homogenizing solution, and replacing 1 sterile pipette with 1mL each time of incremental dilution;
fourthly, the method comprises the following steps: selecting 2-3 sample homogeneous solutions with appropriate dilution, respectively sucking 1mL of the sample homogeneous solution into 2 sterile plates for each dilution while performing 10-fold incremental dilution, and simultaneously respectively adding 1mL of the sterile dilution solution into 2 sterile plates for blank comparison;
fifthly: pouring 20-25 mL of the Eurotium cristatum counting plate cooled to 46 ℃ into a plate in time, rotating the plate to uniformly mix the Eurotium cristatum counting plate and the Eurotium cristatum counting plate, and placing the plate on a horizontal table until the Eurotium cristatum culture medium is completely solidified;
step four: culturing; after the agar is solidified, placing the flat plate in the right direction, culturing the flat plate in an incubator at the temperature of 28 +/-1 ℃, and observing and recording the results from the culture to the culture at the 7 th day from the 2 nd day;
step five: counting typical colonies; the characteristic of the typical colony morphology of the Eurotium cristatum on a Eurotium cristatum counting plate is that the colony is a white flocculent circular colony when 2d-3d is carried out, the colony is a bright golden circular colony after 3d-4d is carried out, the colony is a round colony with bright golden edges and black center and always has water beads, the back of the colony is dark red to red brown, the colony is compact, the plate with the suspected Eurotium cristatum colonies of yellow, bright golden and the like is selected, the plate with the suspected Eurotium cristatum colony number of the plate with the same dilution degree between 10CFU and 150CFU is counted;
step six: calculating a primary screening result; calculating the average value of the colony numbers of two plates with the same dilution degree, and multiplying the average value by the corresponding dilution times, wherein the specific calculation steps are as follows:
the method comprises the following steps: if the colony number on two dilution plates is between 10 and 150CFU, calculating according to the corresponding regulation of GB 4789.2;
secondly, the step of: if the colony number on all the plates is more than 150CFU, counting the plate with the highest dilution, recording the number of other plates as the number of the plates which can not be counted, and calculating the result according to the average colony number multiplied by the highest dilution times;
③: if the colony number on all the plates is less than 10CFU, calculating according to the average colony number with the lowest dilution multiplied by the dilution times;
fourthly, the method comprises the following steps: if all dilution (including liquid sample stock) plates were grown aseptically, calculated as less than 1 times the lowest dilution;
fifthly: if the colony number of the plate at all dilutions is not between 10CFU and 150CFU, and a part of the colony number is less than 10CFU or more than 150CFU, the colony number is calculated by multiplying the average colony number closest to 10CFU or 150CFU by the dilution factor;
sixthly, the method comprises the following steps: when the determination is further confirmed, at least 5 suspicious colonies are selected from typical colonies for ITS identification, and an identification test is carried out, wherein the ITS identification result belongs to the Eurotium cristatum, and the colony culture characteristic is the typical characteristic of the Eurotium cristatum, so that the Eurotium cristatum is confirmed;
step seven: calculating a final result; the calculation formula is as follows:
calculating formula 1: t ═ AB/Cd
In the formula:
t-the number of colonies of eurotium cristatum in the sample;
a-total number of typical colonies at a certain dilution;
b-number of colonies identified as positive at a certain dilution;
c-one dilution used to identify the number of colonies tested;
d-dilution factor;
calculating formula 2:
T＝(A1B1/C1+A2B2/C2)/1.1d
in the formula:
t-the number of colonies of eurotium cristatum in the sample;
a1 — first dilution, i.e. total number of representative colonies at low dilution;
b1 — number of colonies identified as positive at the first dilution, i.e. low dilution;
c1 — first dilution, low dilution, is used to identify the number of colonies tested;
a2-second dilution-total number of representative colonies at high dilution;
b2-number of colonies positive for the second dilution, i.e.high dilution, identification;
c2 — second dilution, high dilution, is used to identify the number of colonies tested;
1.1-calculating the coefficient;
d-dilution factor of the first dilution;
and determining the typical colony ITS as Eurotium cristatum after being identified as Eurotium positive, and specifically calculating the following steps:
the method comprises the following steps: counting the typical colonies on the dilution plate if the typical colony number of only one dilution plate is between 10CFU and 150CFU, and calculating according to the formula 1;
secondly, the step of: if the number of typical colonies on the lowest dilution plate is less than 10CFU, counting the typical colonies on the dilution plate, and calculating according to the formula 1;
③: if the number of typical colonies on one dilution plate is more than 150CFU but no typical colony on the next dilution plate, counting the typical colonies on the dilution plate according to formula 1;
fourthly, the method comprises the following steps: if the number of typical colonies on one dilution plate is more than 150CFU, and the number of typical colonies on the next dilution plate is not in the range of 10CFU-150CFU, counting the typical colonies on the dilution plate, and calculating according to the formula 1;
fifthly: if the number of typical colonies on the plate at 2 serial dilutions is between 10CFU and 150CFU, it is calculated as formula 2.
As a preferred embodiment of the present invention, the sterile diluent in the (c) of the third step includes, but is not limited to, distilled water, physiological saline or phosphate buffer.
As a preferred embodiment of the present invention, in the sixth step, if there are less than 5 suspicious colonies, the total selection is performed.
In the third step (i), a proper amount of sterile glass beads are placed in the container.
In a preferred embodiment of the present invention, in the fifth step, the Eurotium cristatum counting plate is placed in a thermostatic water bath box at 46 ℃ +/-1 ℃ for heat preservation.
As a preferred embodiment of the invention, the invention can solve the problem that the prior art has no special quantitative counting method for Eurotium cristatum in Fuzhuan tea, and the invention can inhibit bacteria, control the growth of part of fungi and simultaneously control the spread of bacterial colonies by inventing a novel culture medium for Eurotium cristatum, the Eurotium cristatum has relatively unique colony characteristics on the Eurotium cristatum culture medium and can be easily separated from other fungi, the count result of the eurotium cristatum can be obtained by simple preliminary screening and identification, if the identification is more accurate, since eurotium cristatum can be clearly distinguished from various other fungi within the genus eurotium on the medium of the present invention, therefore, by identifying the ITS nucleic acid sequence of a typical colony and determining that the colony belongs to the genus Eurotium, the colony can be confirmed to be Eurotium cristatum.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. A eurotium cristatum culture medium is characterized by comprising the following components in percentage by weight: 3-7g of peptone, 8-12g of glucose, 18-23g of sucrose, 0.7-1.3g of monopotassium phosphate, 0.2-0.7g of magnesium sulfate, 13-18g of agar, 0.01-0.05g of Bengal, 0.05-0.2g of chloramphenicol, 10% -15% of sodium chloride and 5-7g of lithium chloride, wherein after the components are mixed, the pH is adjusted to 7.2 +/-0.2.
2. The culture medium for Eurotium cristatum according to claim 1, wherein: the components and contents are preferably as follows: 5g of peptone, 10g of glucose, 20g of sucrose, 1g of monopotassium phosphate, 0.5g of magnesium sulfate, 15g of agar, 0.03g of Bengal, 0.1g of chloramphenicol, 10% -15% of sodium chloride and 6g of lithium chloride, wherein after the components are mixed, the pH is adjusted to 7.2 +/-0.2.
3. A eurotium cristatum flat plate counting method is characterized by comprising the following specific steps:
the method comprises the following steps: preparing a sample; selecting a proper amount of tea samples for later use, and taking a proper amount of eurotium cristatum culture medium raw materials for later use;
step two: homogenizing the sample; crushing a tea sample by using an aseptic crusher, and weighing a proper amount of crushed sample by using a quartering method for later use;
step three: diluting a sample; the method comprises the following specific steps:
the method comprises the following steps: sucking 25mL of the crushed sample by using a sterile suction pipe into a suitable container or a sterile homogenizing bag containing 225mL of sterile diluent, fully shaking or beating by using a beating type homogenizer for 1min-2min to prepare a product 1: 10, homogenizing the sample;
secondly, the step of: taking 1mL of 1: injecting 10 sample homogenized solution into a test tube containing 9mL of sterile diluent, and repeatedly blowing and sucking by replacing 1 sterile suction tube with 1mL, or uniformly mixing on a vortex mixer, wherein the solution is 1: 100 sample homogenizing liquid;
③: preparing 10 times of serial dilution sample homogenizing solution, and replacing 1 sterile pipette with 1mL each time of incremental dilution;
fourthly, the method comprises the following steps: selecting 2-3 sample homogeneous solutions with appropriate dilution, respectively sucking 1mL of the sample homogeneous solution into 2 sterile plates for each dilution while performing 10-fold incremental dilution, and simultaneously respectively adding 1mL of the sterile dilution solution into 2 sterile plates for blank comparison;
fifthly: pouring 20-25 mL of the Eurotium cristatum counting plate cooled to 46 ℃ into a plate in time, rotating the plate to uniformly mix the Eurotium cristatum counting plate and the Eurotium cristatum counting plate, and placing the plate on a horizontal table until the Eurotium cristatum culture medium is completely solidified;
step four: culturing; after the agar is solidified, placing the flat plate in the right direction, culturing the flat plate in an incubator at the temperature of 28 +/-1 ℃, and observing and recording the results from the culture to the culture at the 7 th day from the 2 nd day;
step five: counting typical colonies; the characteristic of the typical colony morphology of the Eurotium cristatum on a Eurotium cristatum counting plate is that the colony is a white flocculent circular colony when 2d-3d is carried out, the colony is a bright golden circular colony after 3d-4d is carried out, the colony is a round colony with bright golden edges and black center and always has water beads, the back of the colony is dark red to red brown, the colony is compact, the plate with the suspected Eurotium cristatum colonies of yellow, bright golden and the like is selected, the plate with the suspected Eurotium cristatum colony number of the plate with the same dilution degree between 10CFU and 150CFU is counted;
step six: calculating a primary screening result; calculating the average value of the colony numbers of two plates with the same dilution degree, and multiplying the average value by the corresponding dilution times, wherein the specific calculation steps are as follows:
the method comprises the following steps: if the colony number on two dilution plates is between 10 and 150CFU, calculating according to the corresponding regulation of GB 4789.2;
secondly, the step of: if the colony number on all the plates is more than 150CFU, counting the plate with the highest dilution, recording the number of other plates as the number of the plates which can not be counted, and calculating the result according to the average colony number multiplied by the highest dilution times;
③: if the colony number on all the plates is less than 10CFU, calculating according to the average colony number with the lowest dilution multiplied by the dilution times;
fourthly, the method comprises the following steps: if all dilution plates grow aseptically, calculating by multiplying the lowest dilution times by less than 1;
fifthly: if the colony number of the plate at all dilutions is not between 10CFU and 150CFU, and a part of the colony number is less than 10CFU or more than 150CFU, the colony number is calculated by multiplying the average colony number closest to 10CFU or 150CFU by the dilution factor;
sixthly, the method comprises the following steps: when the determination is further confirmed, at least 5 suspicious colonies are selected from typical colonies for ITS identification, and an identification test is carried out, wherein the ITS identification result belongs to the Eurotium cristatum, and the colony culture characteristic is the typical characteristic of the Eurotium cristatum, so that the Eurotium cristatum is confirmed;
step seven: calculating a final result; the calculation formula is as follows:
calculating formula 1: t ═ AB/Cd
In the formula:
t-the number of colonies of eurotium cristatum in the sample;
a-total number of typical colonies at a certain dilution;
b-number of colonies identified as positive at a certain dilution;
c-one dilution used to identify the number of colonies tested;
d-dilution factor;
calculating formula 2:
T＝(A1B1/C1+A2B2/C2)/1.1d
in the formula:
t-the number of colonies of eurotium cristatum in the sample;
a1 — first dilution, i.e. total number of representative colonies at low dilution;
b1 — number of colonies identified as positive at the first dilution, i.e. low dilution;
c1 — first dilution, low dilution, is used to identify the number of colonies tested;
a2-second dilution-total number of representative colonies at high dilution;
b2-number of colonies positive for the second dilution, i.e.high dilution, identification;
c2 — second dilution, high dilution, is used to identify the number of colonies tested;
1.1-calculating the coefficient;
d-dilution factor of the first dilution;
and determining the typical colony ITS as Eurotium cristatum after being identified as Eurotium positive, and specifically calculating the following steps:
the method comprises the following steps: counting the typical colonies on the dilution plate if the typical colony number of only one dilution plate is between 10CFU and 150CFU, and calculating according to the formula 1;
secondly, the step of: if the number of typical colonies on the lowest dilution plate is less than 10CFU, counting the typical colonies on the dilution plate, and calculating according to the formula 1;
③: if the number of typical colonies on one dilution plate is more than 150CFU but no typical colony on the next dilution plate, counting the typical colonies on the dilution plate according to formula 1;
fourthly, the method comprises the following steps: if the number of typical colonies on one dilution plate is more than 150CFU, and the number of typical colonies on the next dilution plate is not in the range of 10CFU-150CFU, counting the typical colonies on the dilution plate, and calculating according to the formula 1;
fifthly: if the number of typical colonies on the plate at 2 serial dilutions is between 10CFU and 150CFU, it is calculated as formula 2.
4. The method for counting Eurotium cristatum plates according to claim 3, wherein the method comprises the following steps: and the sterile diluent in the third step is distilled water, physiological saline or phosphate buffer solution.
5. The method for counting Eurotium cristatum plates according to claim 3, wherein the method comprises the following steps: in the sixth step, if the number of the suspicious colonies is less than 5, the suspicious colonies are selected completely.
6. The method for counting Eurotium cristatum plates according to claim 3, wherein the method comprises the following steps: in the third step, a proper amount of sterile glass beads are preset in the container.
7. The method for counting Eurotium cristatum plates according to claim 3, wherein the method comprises the following steps: and in the fifth step, placing the Eurotium cristatum counting plate in a thermostatic water bath box with the temperature of 46 +/-1 ℃ for heat preservation.
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