CN114107432B - Viable bacillus subtilis counting culture medium, diluent and viable bacillus counting method - Google Patents
Viable bacillus subtilis counting culture medium, diluent and viable bacillus counting method Download PDFInfo
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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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
Abstract
The invention belongs to the technical field of microbial strains, and particularly relates to a viable bacteria counting culture medium, a sterilization diluent and a viable bacteria counting method for bacillus subtilis. Wherein, the solid culture medium formula is as follows: 4-6g of peeled soybean meal (crushed and sieved by a 100-mesh sieve), 3-5g of corn flour (crushed and sieved by a 100-mesh sieve), 5-10g of sodium chloride, 3-5g of xanthan gum, 11-13g of agar and 1000mL of distilled water, wherein the pH value is 7.3 +/-0.2; sterilizing the diluent: 8-9g of sodium chloride, 1-2g of monopotassium phosphate, 0.3-0.5g of anhydrous glucose, 0.3-0.5g of peptone, 0.1-0.2g of defoaming agent and 1000mL of distilled water; and a culture mode of firstly heating and then cooling is adopted, so that secretion of bacteriostatic substances of the bacterial cells is reduced, the speed of colony expansion is reduced, and competitive inhibition of bacterial cell growth is reduced. The method for counting the viable bacillus subtilis improves the accuracy of dilution, reduces the death rate of bacterial cells in the process of oscillation dilution, reduces the aggregation of the bacterial cells, controls the spread of bacterial colonies, reduces the size of the bacterial colonies, and improves the counting accuracy and the repeatability of the viable bacillus subtilis.
Description
Technical Field
The invention belongs to the technical field of microbial strains, and particularly relates to a bacillus subtilis viable count solid culture medium, a sterilization diluent and a viable count method.
Background
The bacillus subtilis exists in the preparation in the form of endospore, and after the spores enter animal intestinal tracts, the spores can be rapidly reactivated at the upper parts of the intestinal tracts and secrete high-activity protease, lipase and amylase, so that the bacillus subtilis is beneficial to degrading complex carbohydrate, generates polypeptide substances with antagonistic intestinal pathogenic bacteria and the like, and plays a role in bacteriostasis and prevention. In addition, the bacillus subtilis is aerobic bacteria, can cause an anaerobic environment by consuming oxygen in the intestinal tract, promotes the propagation of dominant bacteria anaerobic bacteria in the intestinal tract, and maintains the ecological balance of the intestinal tract.
The application of the bacillus subtilis in livestock and poultry is mainly characterized in that the bacillus subtilis is made into an additive for use after liquid propagation, the viable count is an important index for measuring the quality of the bacillus subtilis viable preparation, and the optimal addition amount can be better adjusted only by knowing the accurate viable count, so that the beneficial health-care effect of the bacillus subtilis applied in livestock and poultry is ensured.
The viable count method is also called indirect count method, the direct count method measures the total number of dead cells and viable cells, and the indirect count method measures only the number of viable cells, and the value obtained by the method is often smaller than that measured by the direct count method. The viable bacteria counting method generally includes plate colony counting, liquid dilution maximum likelihood (MPN) counting, membrane filtration counting, and the like, wherein the plate colony counting method is the most common counting method. Plate colony counts are based on the ability of each discrete viable cell to grow and form a colony in a suitable medium, and thus the number of colonies is the number of viable bacteria contained in the sample to be tested. After the test solution of the single-cell microorganism is serially diluted by 10 times, the diluent with a certain concentration is quantitatively inoculated on an agar plate culture medium for culture, the number of grown colonies is the number of living cells contained in the diluent, and the number of the living cells in a sample to be tested can be calculated. However, it should be noted that, because individual colonies on a plate may not be formed by one bacterial cell but may be formed by several bacterial cells due to various bacterial species characteristics, dilution procedures, solid media, culture methods, and the like, the amount of viable bacteria counted by the colonies is lower than the actual amount of viable bacteria, and thus, when the number of bacteria contained in a unit sample is expressed, it is expressed in terms of colony forming units per unit sample, i.e., CFU/mL or CFU/g.
At present, there are two ways for counting viable bacillus subtilis, which are respectively counting total viable bacteria and counting spore individuals, wherein the total viable bacteria comprises trophosome and spore, and counting spore individuals requires high-temperature water bath treatment on a sample, so that thermolabile trophosome is killed, and heat-resistant spore is reserved.
Because the same strain can correspondingly generate different colonies on different nutrient media, the size and the shape of the colonies can be changed. Therefore, the bacillus subtilis has a plurality of problems in the aspect of flat viable count at present, for example, the viscosity of fermentation liquor for liquid propagation of the bacillus subtilis is high, and the number of adjacent gradient bacterial colonies is seriously deviated from a 10-fold relation due to insufficient oscillation dilution, so that the count error is large; the bacillus subtilis not only easily forms bacterial colonies formed by gathering a plurality of somatic cells, but also has larger actual bacterial colonies, the number of the bacterial colonies which can be contained in a single plate is limited, and larger errors are easily caused; secondly, the difference of the operation methods of the bacillus subtilis liquid or the bacillus powder in the process of long-time severe oscillation dilution can also cause death in different degrees, which also inevitably affects the accuracy of detection.
The existing culture medium commonly used for counting the bacillus subtilis, such as NA culture medium and LB culture medium, is suitable for culturing most of bacteria, but has weak pertinence; there are also tests for Bacillus subtilis in some microbial preparations (GB/T26428-2010) which prescribe the use of Nutrient Agar (NA) medium (peptone 10g, beef extract 3g, sodium chloride 5g, agar 16g, distilled water 1000mL, pH 7.3. + -. 0.2), diluted in 0.85% physiological saline, incubated at 37 ℃. + -. 1 ℃ in an incubator for 48. + -. 2 h. However, substances such as peptone and beef extract are adopted, so that the bacillus subtilis is easy to be over-nourished, and the problem that bacterial colonies are large and easy to spread finally occurs.
Liu baofeng etc. (2011) carry out the experimental study of viable count to bacterial colony height adhesion bacillus subtilis, carry out the comparative study to different diluents such as normal saline, sterilized water, various PBS blank culture mediums, the result shows that normal saline, sterilized water, various PBS can't disperse the fungus group, be difficult to form effectual viable count gradient, only can make the even dilution gradient of thalli dispersion obvious when the blank culture medium with this bacterial strain is as the diluent, make the count result accurate, good reproducibility. The Wangma and the like (2020) research optimizes a viable count culture medium of bacillus subtilis, changes beef powder into beef extract, and adjusts the agar powder from 1.5% to 3.0% so that the counted bacterial colony does not spread, adhere and grow independently, and the counting accuracy and repeatability are improved.
However, although the above methods can solve the problems of partial bacterial cell aggregation and colony spread in counting, the above methods cannot solve the problems of more foam, high bacterial cell death rate, overlarge colony of counting plate, and the like in the shaking dilution process. Based on the problems, the invention develops a viable bacteria counting culture medium, a sterilization diluent and a viable bacteria counting method for bacillus subtilis.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a bacillus subtilis viable count solid culture medium, a sterilization diluent and a viable count method. The method for counting the viable bacillus subtilis not only improves the dilution accuracy and reduces the death rate of bacterial cells in the dilution process, but also reduces the aggregation of the bacterial cells, effectively controls the spread of bacterial colonies, reduces the size of the bacterial colonies, and improves the accuracy and the repeatability of the counting of the bacillus subtilis.
The technical scheme of the invention is as follows:
a method for counting bacillus subtilis comprises the following operation steps:
s1: preparation of the initial suspension:
weighing 25g (mL) of a sample by aseptic operation, adding 225mL of sterilized diluent, adding 50 sterilized glass beads, and oscillating for 30min to prepare an initial suspension of 1: 10;
s2: gradient dilution:
placing 9mL of sterilized diluent and 10 sterilized glass beads into a penicillin bottle with the volume of 15mL, sucking 1mL of the initial suspension obtained in the step S1 into the penicillin bottle, oscillating for 20-30S by using a vortex oscillator to prepare a diluent I with the ratio of 1:100, and performing ten-fold serial incremental dilution according to the viable count of the sample until the diluent I has a proper dilution gradient of 10nObtaining a diluent; the n is the number of times of dilution, and the specific number of times of dilution is determined according to the quantity of the living bacteria.
S3: coating and plate culture:
the dilution obtained in step S2 was adjusted to 10n-2，10n-1，10nThe diluted solution is subjected to water bath for 10 min at the temperature of 80 +/-1 ℃, then 100 uL of diluted solution is sucked by a sterile gun head and coated on a solid culture medium, and a sterile coating rod is used for coating, wherein 3 gradient coatings are formed;
the culture conditions are as follows: culturing at 36-40 deg.C for 6 hr, and then lowering the culture temperature to 28-32 deg.C for further culturing for 24 hr;
s4: counting:
selecting a plate with the colony number of 30-300 after culture for counting, and if large flaky colonies grow in the plate, the plate is not suitable for use; 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 in the whole plate can be represented by multiplying the number of the colonies in the half plate by 2.
Further, the invention also provides a sterilization diluent in the step S2, which is specifically 8-9g of sodium chloride, 1-2g of monopotassium phosphate, 0.3-0.5g of anhydrous glucose, 0.3-0.5g of peptone, 0.1-0.2g of antifoaming agent and 1000mL of distilled water.
Further, the sterilized diluent in the step S2 is 9g of sodium chloride, 2g of potassium dihydrogen phosphate, 0.5g of anhydrous glucose, 0.5g of peptone, 0.2g of antifoaming agent, and 1000mL of distilled water.
Further, the defoaming agent in the sterilization diluent is a polyether defoaming agent.
Further, the polyether defoaming agent in the sterilization diluent is preferably polyoxypropylene ethylene oxide glyceryl ether. The polyether defoamer has the characteristics of long foam inhibition time, good effect, high defoaming speed, good thermal stability and the like, wherein the foam inhibition is the greatest advantage of the defoamer, and the defoamer can well inhibit the generation of foam in the oscillation process.
Further, the invention also provides a solid culture medium formula in the step S3, which specifically comprises 4-6g of peeled soybean meal, 3-5g of corn flour, 5-10g of sodium chloride, 3-5g of xanthan gum, 11-13g of agar and 1000mL of distilled water.
Further, the solid medium formula in step S3 is 4g of peeled soybean meal, 3g of corn flour, 5g of sodium chloride, 3g of xanthan gum, 13g of agar and 1000mL of distilled water.
Further, the pH value of the solid medium in the step S3 is 7.3 ± 0.2.
Further, the culture conditions in step S3 are that the culture is firstly carried out at 40 ℃ for 6h, and then the culture temperature is reduced to 32 ℃ for further culture for 24 h.
In the prior art, normal saline is usually used as a sterilization diluent for viable count of bacillus subtilis, but the mortality of the bacillus subtilis is high in the process of violent oscillation and dilution, bacterial cells are easy to gather and are not easy to dilute and scatter, and great interference is caused to subsequent culture and count. The sterilization diluent provided by the invention is supplemented with two nutrient substances of trace anhydrous glucose (carbon source) and peptone (nitrogen source), and the combined action of the two nutrient substances is found to improve the capability of bacterial cells to resist severe oscillation dilution stress and reduce the death rate of the bacterial cells in the severe oscillation dilution process; in addition, experiments prove that the bacterial cells can be uniformly dispersed by adding a trace amount of anhydrous glucose and peptone to the sterilized diluent. However, due to the addition of organic matters such as peptone and glucose, the system is easier to form foam in the oscillation process, the accuracy of gradient dilution is influenced, and the polyether defoamer, especially polyoxypropylene ethylene oxide glycerol ether, can well inhibit the formation of a large amount of foam in the bacterial cell dilution oscillation process, so that the dilution effect is ensured, and the counting accuracy is improved. In addition, sodium chloride and potassium dihydrogen phosphate are supplemented in the sterilization diluent, so that osmotic pressure balance can be effectively adjusted, and the death rate of bacterial cells can be effectively reduced; and the potassium dihydrogen phosphate solution is weakly acidic, and can acidify an alkaline bacillus subtilis sample, reduce the viscosity of the bacillus subtilis sample, enable bacterial cells to be more easily dispersed, and further improve the dilution accuracy.
The microbial culture medium is an artificially prepared nutrient for the growth and maintenance of microorganisms, and generally contains carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins, water and the like. Although it is known in the prior art that peeled soybean meal and corn flour can be used as culture medium raw materials for microbial growth, soluble fine raw materials such as peptone, beef extract, yeast powder and glucose and easily utilized by bacillus subtilis are always selected as nutrient components on a viable count culture medium, the culture medium is rich in nutrition and is suitable for culturing most of bacteria, however, the bacillus subtilis is not high in nutrition requirement, the existing bacillus subtilis count culture medium enables the bacillus subtilis to grow vigorously, bacterial colonies are large and easy to spread, and viable count results are affected, so the existing bacillus subtilis count culture medium does not have pertinence. Moreover, experimental researches show that when the peeled soybean meal and the corn meal are used as raw materials of the viable count culture medium, the bioavailability is low, and agglomeration and delamination phenomena are easy to occur, so that the peeled soybean meal and the corn meal are not used as raw material components of the viable count culture medium. The xanthan gum, the peeled soybean meal and the corn flour in a specific ratio are reasonably matched to solve the problems, and the solid culture medium which meets the nutritional characteristics of the bacillus subtilis is combined.
Experiments prove that the xanthan gum can not be degraded by a plurality of enzymes generated by the bacillus subtilis, such as protease, amylase, cellulase, hemicellulase and the like, so that the xanthan gum with a specific proportion can be used for effectively inhibiting the spread of bacillus subtilis colonies, and the counting accuracy is greatly improved. Moreover, the inventor unexpectedly finds that xanthan gum molecules at a certain concentration can form a super-bonded banded spiral copolymer to form a fragile gel-like net structure, can effectively maintain the shapes of peeled soybean meal powder and corn flour solid particles, shows strong emulsification stability and high suspension capacity, and prevents the precipitation and delamination of insoluble particle soybean meal powder and corn flour in the preparation process of a culture medium. In addition, the combination of xanthan gum and agar has a synergistic effect, so that the viscosity and hardness of the gel can be increased, and the utilization of the thalli to nutrient substances in a solid culture medium can be reasonably controlled.
In addition, in the culture method, the invention changes the conventional constant-temperature culture mode, selects the optimum temperature culture (which means the appropriate growth temperature,i.e. 36-40 °) Then, the temperature is reduced for cultivation (namely 28-32)°) Further overcomes the defects of the bacillus subtilis in counting. The early-stage proper-temperature culture can enable bacterial cells under oscillation stress to recover smoothly, and the later-stage cooling culture can effectively reduce the speed of bacterial colony expansion, properly reduce the activity of the bacterial cells and reduce the secretion of antibacterial substances, so that the growth of the bacterial cells with high activity and low inhibitory activity of the bacterial cells is avoided, and the accuracy of viable count of bacillus subtilis is improved.
Compared with the prior art, the method for counting the bacillus subtilis has the following advantages:
(1) according to the invention, peeled soybean meal and corn flour are used for replacing peptone and beef powder (extract) in the bacillus subtilis NA counting culture medium, so that the nutrition requirement is more targeted, the problem of large counting bacterial colony of the NA culture medium is solved, and the counting accuracy is improved; the xanthan gum can be used for cooperating with the agar to increase the viscosity and hardness of the colloid, and can also be used for reasonably controlling the utilization of thalli to nutrient substances in a solid culture medium and inhibiting the spread of bacterial colonies, thereby improving the counting accuracy.
(2) The dilution liquid is added with trace amounts of carbon source and nitrogen source, monopotassium phosphate, defoaming agent and the like, so that the death rate of bacterial cells in the process of violent oscillation dilution is effectively reduced, the formation of foam in the process of dilution oscillation is reduced, the oscillation effect is improved, and the dilution efficiency and accuracy are improved.
(3) According to the invention, a culture mode of firstly heating and then cooling is selected, so that secretion of bacteriostatic substances of bacterial cells is reduced, the speed of bacterial colony expansion is reduced, competitive inhibition of bacterial cell growth is reduced, the defect of bacillus subtilis in counting culture is further overcome, and the accuracy of viable count is improved.
Drawings
FIG. 1 is a graph of viable count colonies from example 3, wherein the dilution gradient is 1X 108；
FIG. 2 is a graph of viable count colonies from example 5, wherein the dilution gradient is 1X 108；
FIG. 3 is a graph of viable count colonies of comparative example 1, wherein the dilution gradient is 1X 108；
FIG. 4 is a graph of viable count colonies of comparative example 2, wherein the dilution gradient is 1X 108；
FIG. 5 is a graph of viable count colonies of comparative example 3, wherein the dilution gradient is 1X 108；
FIG. 6 is a graph of viable count colonies of comparative example 4, wherein the dilution gradient is 1X 108；
FIG. 7 is a graph of viable count colonies of comparative example 5, wherein the dilution gradient is 1X 108；
FIG. 8 is a graph of viable count colonies of comparative example 6, wherein the dilution gradient is 1X 108；
FIG. 9 is a graph of viable count colonies of comparative example 7, wherein the dilution gradient is 1X 108；
FIG. 10 is a graph of viable count colonies of comparative example 8, wherein the dilution gradient is 1X 108。
Detailed Description
The present invention is further illustrated by the following description of specific embodiments, which are not intended to limit the invention, and various modifications and improvements can be made by those skilled in the art based on the basic idea of the invention, but the invention is within the protection scope of the invention.
Wherein, the bacillus subtilis sample used by the invention can be purchased from Shandong blue biological science and technology limited company,the product has a trade name of 500 hundred million Yuankangbao, a product code of YKB-500 and a viable count of about 5.0 multiplied by 1010CFU/g; the other reagents are common reagents and can be purchased from conventional reagent production and sale companies.
Example 1 viable bacteria count solid Medium for Bacillus subtilis according to the invention
The solid culture medium (formula 1) of the invention: 4g of peeled soybean meal (crushed by a 100-mesh sieve), 3g of corn flour (crushed by a 100-mesh sieve), 5g of sodium chloride, 3g of xanthan gum, 13g of agar and 1000mL of distilled water, wherein the pH value is 7.3.
The solid culture medium of the invention (formula 2): 6g of peeled soybean meal (crushed by 100 meshes), 5g of corn flour (crushed by 100 meshes), 10g of sodium chloride, 5g of xanthan gum, 11g of agar and 1000mL of distilled water, wherein the pH value is 7.5.
Example 2 viable count sterilized diluent of Bacillus subtilis
The sterilization diluent (formula 1) of the invention: 8g of sodium chloride, 2g of monopotassium phosphate, 0.3g of anhydrous glucose, 0.3g of peptone, 0.1g of polyoxypropylene ethylene oxide glycerol ether defoamer and 1000mL of distilled water.
The sterilization diluent (formula 2) of the invention: 9g of sodium chloride, 2g of monopotassium phosphate, 0.5g of anhydrous glucose, 0.5g of peptone, 0.2g of polyoxypropylene ethylene oxide glycerol ether defoamer and 1000mL of distilled water.
Example 3 viable bacteria counting method of Bacillus subtilis
Sample preparation: the viable count of the Bacillus subtilis sample is about 5.0 multiplied by 1010 CFU/g。
S1: preparation of the initial suspension:
25g of the sample was weighed in a sterile manner, 225mL of sterile diluent was added, 50 sterilized glass beads were added, and the mixture was shaken for 30min to prepare an initial suspension of 1: 10.
S2: ten-fold gradient dilution:
preparing a penicillin bottle with the capacity of 15mL, adding 9mL of sterilized diluent and 10 sterilized glass beads into the penicillin bottle, sucking 1mL of the initial suspension obtained in the step S1 into the penicillin bottle, oscillating the solution for 20-30S by using a vortex oscillator to prepare a 1:100 diluent I, and further performing ten times of processing on the diluent IMultiple serial incremental dilution, and gradient dilution to dilution degree of 107，108，109；
S3: coating and plate culture:
the dilution obtained in step S2 was adjusted to 107，108，109The diluted solution was subjected to water bath at 80 ℃ for 10 min, and 100 uL of the diluted solution was then applied to a solid medium by pipetting with a sterile pipette tip, and 3 was applied to each gradient using a sterile applicator rod.
Wherein the culture conditions are as follows: culturing at 36 deg.C for 6h, and then lowering the culture temperature to 28 deg.C for further culturing for 24 h.
S4: counting:
and counting plates with the colony number of 30-300 after culture. If large flaky bacterial colonies grow in the flat plate, the method is not suitable for use; 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 in the whole plate can be represented by multiplying the number of the colonies in the half plate by 2.
The solid medium formulation adopts the formulation one in example 1: 4g of peeled soybean meal (crushed by a 100-mesh sieve), 3g of corn flour (crushed by a 100-mesh sieve), 5g of sodium chloride, 3g of xanthan gum, 13g of agar and 1000mL of distilled water, wherein the pH value is 7.3.
The sterile diluent used in example 2 was the first formulation: 8g of sodium chloride, 2g of monopotassium phosphate, 0.3g of anhydrous glucose, 0.3g of peptone, 0.1g of polyoxypropylene ethylene oxide glycerol ether defoamer and 1000mL of distilled water.
Example 4 viable bacteria counting method of Bacillus subtilis
The enumeration method of example 4 is similar to example 3, except that the solid medium formulation of example 4 employs formulation two of example 1: 6g of peeled soybean meal (crushed by 100 meshes), 5g of corn flour (crushed by 100 meshes), 10g of sodium chloride, 5g of xanthan gum, 11g of agar and 1000mL of distilled water, wherein the pH value is 7.5.
Example 5 viable bacteria counting method of Bacillus subtilis
The counting procedure of example 5 was similar to that of example 3, except that the sterile diluent of example 5 was the same as the second formulation of example 2: 9g of sodium chloride, 2g of monopotassium phosphate, 0.5g of anhydrous glucose, 0.5g of peptone, 0.2g of polyoxypropylene ethylene oxide glycerol ether defoamer and 1000mL of distilled water.
Example 6 viable bacteria counting method of Bacillus subtilis
The counting method of example 6 is similar to that of example 3, except for the culture conditions, the culture conditions of example 6: culturing at 40 deg.C for 6 hr, and then lowering the culture temperature to 32 deg.C for further culturing for 24 hr.
Comparative example 1 method for counting Bacillus subtilis
The counting method of comparative example 1 is similar to that of example 5, except that the sterilized diluent does not contain peptone and glucose, and the specific sterilized diluent formulation: 9g of sodium chloride, 2g of monopotassium phosphate, 0.2g of polyoxypropylene ethylene oxide glycerol ether antifoaming agent and 1000mL of distilled water.
Comparative example 2 method for counting Bacillus subtilis
The counting method of comparative example 2 is similar to that of example 5, except that the sterile diluent is a conventional sterile diluent, and the specific formulation of the sterile diluent is as follows: 8.5g of sodium chloride and 1000mL of distilled water.
Comparative example 3 method for counting Bacillus subtilis
The counting method of comparative example 3 is similar to that of example 5, except that the defoaming agent selected for the sterilizing diluent is an organic silicon defoaming agent (manufacturer: Guangbaowoff environmental protection technology Co., Ltd., model: BWF-WB 880), and the specific sterilizing diluent: 9g of sodium chloride, 2g of monopotassium phosphate, 0.5g of anhydrous glucose, 0.5g of peptone, 0.2g of organic silicon defoamer and 1000mL of distilled water.
Comparative example 4 method for counting Bacillus subtilis
The counting method of comparative example 4 is similar to that of example 5, except that a conventional bacillus subtilis counting medium (NA), specifically a Nutrient Agar (NA) medium, is selected: 10g of peptone, 3g of beef extract, 5g of sodium chloride, 16g of agar and 1000mL of distilled water, and the pH value is 7.3.
Comparative example 5 method for counting Bacillus subtilis
The counting method of comparative example 5 is similar to that of example 5 except that xanthan gum is not contained in the solid medium.
Comparative example 6 method for counting Bacillus subtilis
The counting method of comparative example 6 is similar to that of example 5 except that corn meal is not included in the solid medium.
Comparative example 7 method for counting Bacillus subtilis
The counting method of comparative example 7 is similar to that of example 5, except that the culture conditions: directly culturing for 32h under the condition of 37 ℃.
Comparative example 8 method for counting Bacillus subtilis
The counting method of comparative example 8 is similar to that of example 5, except that in the solid medium: the soybean meal is increased to 10g, the corn flour is increased to 6g, and the xanthan gum is reduced to 2 g.
Test example one, counting
1. Test subjects: the results of counting Bacillus subtilis obtained according to the counting methods of examples 3 to 6 and comparative examples 1 to 8.
2. And (3) test results:
the test results are shown in Table 1.
TABLE 1 results of Bacillus subtilis count obtained in examples 3 to 6 and comparative examples 1 to 8
As can be seen from Table 1: the colony count of the group of the embodiment 3, the group of the embodiment 4, the group of the embodiment 5 and the group of the embodiment 6 is smaller, the single plate can contain more bacteria, the colony spread quantity is small, the colony number among gradients is closer to 10 times, and the coating plates have good repeatability and higher accuracy. The test results of the comparative example group and the example group show that the solid culture medium and the sterilization diluent provided by the invention have few or no components, and the death rate of bacillus subtilis cells in the process of violent shaking dilution is greatly reduced, so that the dilution efficiency and the dilution accuracy are improved.
As shown in fig. 3: comparative example 1 group dilution stress resulted in low viability of bacterial cells and lower total viable count than actual values without peptone and glucose in the sterilization diluent.
As shown in fig. 4: in the comparative example 2, the traditional 0.85% sodium chloride is adopted to easily dilute the live bacteria to cause the stress death of the bacteria, and the activity of the stressed bacteria cells is low, so that the total number of the live bacteria is lower than the actual value.
As shown in fig. 5: in the case of adding peptone and glucose to the sterilization diluent of the group 3 of comparative examples without adding an antifoaming agent, more foams are caused, the gradient is far away from a 10-time relation, the number of bacterial colonies among parallel plates is large in fluctuation, errors are large, and the number of viable bacteria is seriously low.
As shown in fig. 6: comparative example 4 group selected conventional bacillus subtilis enumeration medium (NA), the colonies were large, resulting in a small number of colonies contained in the medium plate; in addition, the spread condition of more colonies affects the judgment of single colony individuals, and further affects the accuracy of final counting.
As shown in fig. 7: compared with the solid culture medium of the group 5, xanthan gum is not contained, and insoluble peeled soybean meal and corn flour in the culture medium sink on the bottom of the culture medium, so that more bacterial colonies are spread, the judgment of a single bacterial colony individual is influenced, and the accuracy of counting is finally influenced.
As shown in fig. 8: the absence of corn flour in the solid medium of the comparative example 6 group resulted in not only minimal colonies but also extremely low numbers of colonies, leading to severely low counts.
As shown in fig. 9: the group of comparative example 7 was cultured for 32 hours at 37 ℃ directly, resulting in continued competition between the cells. Under the condition of not reducing the temperature, the growth of partial bacteria cells with higher activity is over-vigorous to inhibit the growth of other bacteria, so that the number of colonies is lower than the actual number, and the accuracy of viable count can be improved by adopting a midway temperature reduction culture mode in the embodiment of the application.
As shown in fig. 10: the mass ratio of the peeled soybean meal, the corn flour and the xanthan gum in the culture medium of the comparative example 8 group was 5: 3: 1, under the condition that the specific gravity of the xanthan gum is lower, the nutrition distribution of the solid culture medium is uneven, the colony sizes are different, and the viable count result is lower.
In conclusion, the polyoxypropylene ethylene oxide glyceryl ether in the sterilization diluent provided by the invention can well inhibit a large amount of foams from being formed in the bacterial cell dilution oscillation process, so that the dilution effect is ensured, the bacterial cell survival rate is improved, and the counting accuracy is improved. The solid culture medium provided by the invention uses peeled soybean meal and corn flour to replace peptone and beef powder (paste) in the existing counting culture medium of bacillus subtilis, and is matched with a certain amount of rehmannia root crude gum for combined action, so that the culture medium not only can meet the nutritional requirement of the bacillus subtilis, but also solves the problems of large counting bacterial colony and easy spread of the solid culture medium, and improves the counting accuracy; and the combination of xanthan gum and agar has a synergistic effect, so that the viscosity and hardness of the gel are increased, and the utilization of the thalli to nutrient substances in a solid culture medium can be reasonably controlled.
Meanwhile, in the culture method, the traditional constant-temperature culture mode is changed, the proper-temperature culture is selected, and then the temperature reduction culture is performed, so that the defects in the counting of the bacillus subtilis are further overcome, and the accuracy of the counting of the viable bacillus subtilis is improved.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (6)
1. A viable bacillus subtilis counting method is characterized by comprising the following steps:
s1: preparation of the initial suspension:
weighing a sample by aseptic operation, adding a diluent, adding sterilized glass beads, and oscillating to prepare an initial suspension of 1: 10;
s2: gradient dilution:
putting the diluent and the sterilized glass beads into a penicillin bottle, sucking the initial suspension obtained in the step S1 into the penicillin bottle, oscillating the initial suspension by using a vortex oscillator to prepare a diluent I with the ratio of 1:100, and performing ten times of serial incremental dilution according to the number of viable bacteria of a sample to obtain a diluent II;
s3: coating and plate culture:
carrying out water bath on the diluent II obtained in the step S2 at the temperature of 80 +/-1 ℃, sucking by using a sterile gun head, coating on a solid culture medium, and coating by using a sterile coating rod, wherein 3 coatings are coated on each gradient;
the culture conditions are as follows: firstly, culturing for 6h at the temperature of 36-40 ℃, and then, reducing the culture temperature to 28-32 ℃ for continuous culture for 24 h;
s4: counting:
selecting a plate with the colony number of 30-300 for counting after culture, and if large flaky colonies grow in the plate, the plate is not suitable for use; if the flaky bacterial colony is less than one half of the flat plate and the bacterial colonies in the other half are uniformly distributed, calculating the number of the bacterial colonies in the half flat plate and multiplying the number by 2 to represent the number of the bacterial colonies in the whole plate;
the solid culture medium consists of 4-6g of peeled soybean meal, 3-5g of corn flour, 5-10g of sodium chloride, 3-5g of xanthan gum, 11-13g of agar and 1000mL of distilled water;
the diluent is a sterilization diluent and consists of 8-9g of sodium chloride, 1-2g of monopotassium phosphate, 0.3-0.5g of anhydrous glucose, 0.3-0.5g of peptone, 0.1-0.2g of antifoaming agent and 1000mL of distilled water;
the defoaming agent in the sterilization diluent is a polyether defoaming agent.
2. The viable count method of bacillus subtilis according to claim 1, wherein the sterile diluent is 9g of sodium chloride, 2g of monopotassium phosphate, 0.5g of anhydrous glucose, 0.5g of peptone, 0.2g of antifoaming agent, and 1000mL of distilled water.
3. The viable bacillus subtilis count method of claim 1 wherein the antifoaming agent is polyoxypropylene ethylene oxide glyceryl ether.
4. The viable count method of bacillus subtilis according to claim 1, wherein the formula of the solid culture medium comprises 4g of peeled soybean meal, 3g of corn meal, 5g of sodium chloride, 3g of xanthan gum, 13g of agar and 1000mL of distilled water.
5. The viable bacillus subtilis count method of claim 1, wherein the pH of the solid medium is 7.3 ± 0.2.
6. The viable count method of bacillus subtilis according to claim 1, wherein the culturing conditions in step S3 are that the culturing is first carried out at 40 ℃ for 6 hours, and then the culturing is continued for 24 hours by reducing the culturing temperature to 32 ℃.
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