CN111088315A - Flat plate counting method for anaerobic bacteria - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 241001148471 unidentified anaerobic bacterium Species 0.000 title claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 41
- 241001148470 aerobic bacillus Species 0.000 claims abstract description 33
- 239000010410 layer Substances 0.000 claims abstract description 28
- 239000002356 single layer Substances 0.000 claims abstract description 23
- 238000012258 culturing Methods 0.000 claims abstract description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 42
- 239000001963 growth medium Substances 0.000 claims description 35
- 241000894006 Bacteria Species 0.000 claims description 33
- 239000000725 suspension Substances 0.000 claims description 23
- 239000006916 nutrient agar Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000004321 preservation Methods 0.000 claims description 14
- 229920001817 Agar Polymers 0.000 claims description 12
- 244000063299 Bacillus subtilis Species 0.000 claims description 12
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 11
- 238000010790 dilution Methods 0.000 claims description 11
- 239000012895 dilution Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 8
- 241000194108 Bacillus licheniformis Species 0.000 claims description 7
- 238000009631 Broth culture Methods 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 7
- 238000000576 coating method Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 238000007711 solidification Methods 0.000 claims description 2
- 230000008023 solidification Effects 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 abstract description 9
- 239000001301 oxygen Substances 0.000 abstract description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 8
- 238000012136 culture method Methods 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 7
- 239000000843 powder Substances 0.000 description 5
- 241000193468 Clostridium perfringens Species 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 241000193171 Clostridium butyricum Species 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- LIRDHUDRLFDYAI-UHFFFAOYSA-H iron(3+);trisulfite Chemical compound [Fe+3].[Fe+3].[O-]S([O-])=O.[O-]S([O-])=O.[O-]S([O-])=O LIRDHUDRLFDYAI-UHFFFAOYSA-H 0.000 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
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- 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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- 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)
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Abstract
The invention relates to a plate counting method of anaerobic bacteria, which comprises the following steps: preparing aerobic bacteria liquid, preparing a single-layer plate, preparing a double-layer plate, culturing, observing and counting. The invention has the advantages that by using the double-layer flat plate method, the oxygen in the culture dish is utilized for the growth of the aerobic bacteria in the upper flat plate, the oxygen concentration in the culture dish is reduced, the anaerobic environment is created for the growth of the anaerobic bacteria, compared with the conventional anaerobic culture mode, the cost is lower, and the culture method is simple and easy to implement.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a flat plate counting method for anaerobic bacteria.
Background
The cultivation of microorganisms requires two basic conditions to be met: nutrients and culture environment required for growth; the nutrient substances mainly comprise a culture medium; the culture environment mainly comprises the culture temperature, the oxygen content in the culture process and the pH value of the culture medium. Anaerobic microorganisms do not have a complete metabolic enzyme system, so energy metabolism is performed in an anaerobic fermentation mode, and survival of the anaerobic microorganisms can be performed under the condition of lower oxidation-reduction potential.
Bacteria growing on the surface of a solid medium can be classified into obligate anaerobes, microaerophilic anaerobes, and facultative anaerobes according to their tolerance to oxygen. Anaerobic bacteria can only grow under the condition of low oxygen partial pressure, but cannot grow under the conditions of air (18% oxygen) and/or 10% carbon dioxide concentration.
Although many methods are currently used for culturing anaerobic bacteria, the methods have wide application and are effective: anaerobic bag method, anaerobic box method, candlesting method, anaerobic incubator method and intelligent anaerobic system method. The above detection methods have respective defects, or are complicated to operate, expensive in equipment, or time-consuming and consumable. Due to the defects, the anaerobic bacteria culture method which is low in price, simple and convenient in method and free of catalyst is necessary.
Disclosure of Invention
In view of the above, the present invention provides a plate counting method for anaerobic bacteria, which overcomes the problems of complicated structure, high price and the need of adding additional deoxidizer or catalyst in the anaerobic bacteria culture device in the prior art.
In order to achieve the above object, the present invention provides a plate counting method of anaerobic bacteria, comprising the steps of:
1) preparing an aerobic bacteria suspension: absorbing aerobic bacteria glycerol tube preservation bacteria liquid, inoculating the aerobic bacteria glycerol tube preservation bacteria liquid into 50ml of nutrient broth culture medium with the inoculation amount of 0.5-1.0% (v/v), culturing for 20-30 h at the culture temperature of 30-40 ℃ and the rotation speed of 100-200 rpm/min, adding 10ml of bacteria liquid into a sterile test tube, treating for 5-15 min under the water bath condition, absorbing 1ml of bacteria liquid under the sterile condition, diluting the bacteria liquid by 10-1000 times according to the volume by using sterile normal saline to obtain aerobic bacteria suspension, and preserving at 4 ℃ for later use;
2) preparation of a single-layer flat plate: under the aseptic condition, after a plurality of anaerobe solid samples or fermentation liquid samples to be detected are subjected to gradient dilution, 1.0ml of diluent is respectively sucked and added into a plurality of culture dishes, then 5-15 ml of semi-solid agar culture medium is respectively added, the culture dishes are placed on a horizontal desktop, and natural solidification is carried out at room temperature to obtain a plurality of single-layer flat plates;
3) preparation and culture of double-layer plates: under an aseptic condition, respectively adding 5-15 ml of semisolid agar culture medium into the plurality of single-layer plates prepared in the step 2), placing the single-layer plates on a horizontal desktop, naturally solidifying to obtain a plurality of double-layer plate nutrient agar culture media, sucking 0.1-1 ml of aerobic bacteria suspension obtained in the step 1), respectively adding the aerobic bacteria suspension into the surfaces of the plurality of double-layer plate nutrient agar culture media obtained previously, uniformly coating the aerobic bacteria suspension by using an aseptic coater, covering a dish cover when no obvious water stain exists on the surfaces, and inversely placing the culture dish cover in an incubator at 30-40 ℃ for culture;
4) and (3) observation and counting: and after macroscopic bacteria appear in the plurality of culture dishes, taking out the culture dishes for counting.
Further, in step 1), the aerobic bacteria comprise spore-forming aerobic bacteria.
Further, in step 1), the aerobic bacteria are selected from at least one of bacillus subtilis and bacillus licheniformis.
Further, in the step 1), the temperature of the water bath is 80-90 ℃.
Further, in step 1), the preparation of the aerobic bacterial suspension specifically comprises: absorbing aerobic bacteria glycerol tube preservation bacteria liquid, inoculating the aerobic bacteria glycerol tube preservation bacteria liquid into a 250ml shake flask filled with 50ml nutrient broth culture medium with the inoculation amount of 1.0% (v/v), culturing for 30h under the conditions that the culture temperature is 35 ℃ and the rotation speed is 180rpm/min, adding 10ml bacteria liquid into a sterile test tube, treating for 10min under the condition of 85 ℃ water bath, absorbing 1ml bacteria liquid, diluting the bacteria liquid by 100 times according to the volume by using sterile physiological saline under the sterile condition to obtain aerobic bacteria suspension, and preserving for later use at 4 ℃.
Further, in the step 2), the diameter of the bottom of the culture dish is 90mm, and the height of the bottom of the dish is 20 mm; the amount of the semi-solid agar medium added was 15 ml.
Further, in the step 2), three dilutions are selected for each anaerobic bacteria solid sample or fermentation liquid sample to be detected, and three parallel counting plates are arranged for each dilution.
Further, in step 3), the preparation of the double-layer flat plate specifically comprises: under the aseptic condition, adding 10ml of semisolid nutrient agar culture medium on the surface of the single-layer plate prepared in the step 2), naturally solidifying at room temperature to obtain a double-layer plate nutrient agar culture medium, sucking 0.1ml of aerobic bacteria liquid obtained in the step 1), adding the surface of the double-layer plate nutrient agar culture medium obtained before, uniformly coating by using an aseptic coater, covering a dish cover when no obvious water stain exists on the surface, and inversely placing the dish cover in an incubator at 37 ℃ for culture.
Further, in step 4), the observing and counting specifically includes: and after macroscopic single colonies appear in the lower-layer flat plates of the plurality of culture dishes, taking out the culture dishes to count the colonies.
Further, in the step 4), the colony number in the lower plate of each culture dish is within the range of 30-300, and all the colonies are statistical objects.
By the technical scheme, the invention at least has the following advantages:
the invention utilizes the characteristic that bacillus subtilis or bacillus licheniformis consumes a large amount of oxygen during germination, reduces the oxygen concentration in a sealed environment, and creates a low-oxygen or anaerobic condition for the culture of anaerobic bacteria.
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following description is given with reference to the preferred embodiments.
The following materials and reagents are commercially available unless otherwise specified.
Example 1
A sample to be tested: 100 hundred million Clostridium butyricum powder.
1) Preparing an aerobic bacteria suspension: absorbing bacillus subtilis glycerol tube preservation bacteria liquid, inoculating the bacillus subtilis glycerol tube preservation bacteria liquid into 50ml of nutrient broth culture medium with the inoculation amount of 1.0% (v/v), culturing for 30h at the culture temperature of 35 ℃ and the rotation speed of 180rpm/min, adding 10ml of the bacteria liquid into a sterile test tube, treating for 10min under the condition of 85 ℃ water bath, absorbing 1ml of the bacteria liquid under the sterile condition, diluting the bacteria liquid by 100 times according to the volume by using sterile normal saline, obtaining bacillus subtilis spore suspension, and preserving for later use at 4 ℃;
2) preparation of a single-layer flat plate: under aseptic condition, 10.0g of bacterial powder to be detected is added into 90ml of physiological saline and respectively diluted to 10 degrees in a gradient way-7，10-8，10-9Diluting, namely respectively sucking 1.0ml of diluent, adding the diluent into a plurality of culture dishes with the diameter of 90mm at the bottom of the dish and the height of 20mm at the bottom of the dish, and then respectively adding 15ml of melted ferric sulfite agar culture medium cooled to 45-55 DEG CPlacing the culture dishes on a horizontal desktop, and naturally solidifying at room temperature for later use to obtain a plurality of single-layer flat plates;
3) preparing a double-layer flat plate: under the aseptic condition, adding 10ml of semisolid nutrient agar culture medium into the plurality of single-layer plates prepared in the step 2), placing the single-layer plates on a horizontal desktop, naturally solidifying at room temperature to obtain a plurality of double-layer plate nutrient agar culture media, sucking 0.1ml of bacillus subtilis spore suspension obtained in the step 1), respectively adding the surfaces of the plurality of double-layer plate nutrient agar culture media obtained in the previous step, uniformly coating by using an aseptic coater, covering a dish cover when no obvious water stain exists on the surface, and inversely placing the dish cover in an incubator at 37 ℃ for culture.
Each dilution of the bacterial powder to be tested is provided with three parallels, and a control group is arranged at the same time: in the control group, ferric sulfate agar medium was used as the counting medium, the counting plate was inverted in an anaerobic bag, a commercial anaerobic gas-generating bag was added, the air in the bag was evacuated, and the cells were cultured at 37 ℃.
After the culture is finished, the number of colonies in the lower-layer plate of each culture dish of the experimental group and the control group is within the range of 30-300, and the colonies are all statistical objects.
The results of colony counting for the experimental group and the control group are shown in table 1 below.
TABLE 1
As can be seen from the data in table 1, the standard deviation and error of the method (experimental group) described in example 1 are smaller than those of the conventional detection method (control group), and the detection method is simple and easy to implement and has lower cost.
Example 2
A sample to be tested: the mixed strain microecological preparation contains 20 hundred million enterococcus faecalis and 90 hundred million Bacillus subtilis.
1) Preparing an aerobic bacteria suspension: sucking bacillus subtilis glycerol tube preservation bacteria liquid, inoculating the bacillus subtilis glycerol tube preservation bacteria liquid into 50ml of nutrient broth culture medium with the inoculation amount of 0.5% (v/v), culturing for 24 hours at the culture temperature of 37 ℃ and the rotation speed of 150rpm/min, adding 10ml of the bacteria liquid into a sterile test tube, treating for 15 minutes under the water bath condition of 80 ℃, sucking 1ml of the bacteria liquid under the sterile condition, diluting the bacteria liquid by 10 times according to the volume by using sterile normal saline to obtain bacillus subtilis spore suspension, and preserving for later use at 4 ℃;
2) preparation of a single-layer flat plate: under aseptic condition, 10.0g of bacterial powder to be detected is added into 90ml of physiological saline and respectively diluted to 10 degrees in a gradient way-6，10-7，10-8Sucking 1.0ml of diluent in each dilution, adding the diluent into a plurality of culture dishes with the diameter of 90mm at the bottom of the dish and the height of 20mm at the bottom of the dish, respectively adding 10ml of MRS agar culture medium which is melted and then cooled to 45-55 ℃, placing the culture dishes on a horizontal desktop, naturally solidifying at room temperature for later use to obtain a plurality of single-layer plates,
3) preparing a double-layer flat plate: under the aseptic condition, adding 10ml of nutrient agar culture medium which is melted and then cooled to 45-55 ℃ into the plurality of single-layer plates prepared in the step 2), placing the single-layer plates on a horizontal desktop, naturally solidifying at room temperature to obtain a plurality of double-layer plate nutrient agar culture media, sucking 0.1ml of bacillus subtilis spore suspension prepared in the step 1), respectively adding the surfaces of the plurality of double-layer plate nutrient agar culture media obtained in the previous step, uniformly coating by using an aseptic coater, covering a culture dish cover when no obvious water stain exists on the surfaces, and inversely placing the culture dish cover in an incubator at 37 ℃ for culture.
Each dilution of the bacterial powder to be tested is provided with three parallels, and a control group is arranged at the same time: control group MRS + CaCO3The agar medium was used as the counting medium, the counting plate was inverted in an anaerobic bag, a commercial anaerobic gas-generating bag was added, the air in the bag was evacuated, and the culture was carried out at 37 ℃.
After the culture is finished, the number of colonies in the lower-layer plate of each culture dish of the experimental group and the control group is within the range of 30-300, and the colonies are all statistical objects.
The results of colony counts for the experimental group and the control group are shown in Table 2 below.
TABLE 2
As can be seen from the data in table 2, the standard deviation and error of the method (experimental group) described in example 2 are smaller than those of the conventional detection method (control group), and the detection method is simple and easy to implement and has lower cost.
Example 3
A sample to be tested: a clostridium perfringens fermentation broth.
1) Preparing an aerobic bacteria suspension: absorbing bacillus licheniformis glycerol tube preservation bacterial liquid, inoculating the bacillus licheniformis glycerol tube preservation bacterial liquid into 50ml of nutrient broth culture medium with the inoculation amount of 0.5% (v/v), culturing for 24 hours at the culture temperature of 37 ℃ and the rotation speed of 150rpm/min, adding 10ml of bacterial liquid into a sterile test tube, treating for 5 minutes under the water bath condition of 90 ℃, absorbing 1ml of bacterial liquid under the aseptic condition, diluting the bacterial liquid by 10 times according to the volume by using sterile normal saline, obtaining bacillus licheniformis spore suspension, and preserving for later use at 4 ℃;
2) preparation of a single-layer flat plate: under the aseptic condition, 10ml of clostridium perfringens fermentation liquor to be detected is added into 90ml of physiological saline and is respectively diluted to 10 in a gradient way-5，10-6，10-7Respectively sucking 1.0ml of diluent at three dilution degrees, adding the diluent into a plurality of culture dishes with the diameter of 90mm and the height of 20mm at the bottom of the dish, respectively adding 15ml of melted RCM agar culture medium cooled to 45-55 ℃, placing the culture dishes on a horizontal desktop, and naturally solidifying at room temperature for later use to obtain a plurality of single-layer flat plates;
3) preparing a double-layer flat plate: adding 10ml of melted nutrient agar culture medium cooled to 45-55 ℃ into the plurality of single-layer plates prepared in the step 2) under an aseptic condition, placing the single-layer plates on a horizontal desktop, naturally solidifying at room temperature to obtain a plurality of double-layer plate nutrient agar culture media, sucking 0.1ml of bacillus licheniformis spore suspension obtained in the step 1), respectively adding the surfaces of the plurality of double-layer plate nutrient agar culture media obtained in the previous step, uniformly coating by using an aseptic coater, covering a culture dish cover when no obvious water stain exists on the surfaces, and inversely placing the culture dish cover in an incubator at 37 ℃ for culture.
Three parallels are set for each dilution of clostridium perfringens fermentation liquor to be detected, and a comparison is set at the same time: the control group used RCM agar medium as the counting medium, the counting plate was inverted in an anaerobic bag, a commercial anaerobic gas-generating bag was added, the air in the bag was evacuated, and the culture was carried out at 37 ℃.
After the culture is finished, the number of colonies in the lower-layer plate of each culture dish of the experimental group and the control group is within the range of 30-300, and the colonies are all statistical objects.
The results of colony counts for the experimental group and the control group are shown in Table 3 below.
TABLE 3
As can be seen from the data in table 3, the standard deviation of the method (experimental group) described in example 3 is smaller than that of the conventional detection method (control group), and the detection method is simple and easy to implement and has lower cost.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A plate counting method for anaerobic bacteria is characterized by comprising the following steps:
1) preparing an aerobic bacteria suspension: absorbing aerobic bacteria glycerol tube preservation bacteria liquid, inoculating the aerobic bacteria glycerol tube preservation bacteria liquid into 50ml of nutrient broth culture medium with the inoculation amount of 0.5-1.0% (v/v), culturing for 20-30 h at the culture temperature of 30-40 ℃ and the rotation speed of 100-200 rpm/min, adding 10ml of bacteria liquid into a sterile test tube, treating for 5-15 min under the water bath condition, absorbing 1ml of bacteria liquid under the sterile condition, diluting the bacteria liquid by 10-1000 times according to the volume by using sterile normal saline to obtain aerobic bacteria suspension, and preserving at 4 ℃ for later use;
2) preparation of a single-layer flat plate: under the aseptic condition, after a plurality of anaerobe solid samples or fermentation liquid samples to be detected are subjected to gradient dilution, 1.0ml of diluent is respectively sucked and added into a plurality of culture dishes, then 5-15 ml of semi-solid agar culture medium is respectively added, the culture dishes are placed on a horizontal desktop, and natural solidification is carried out at room temperature to obtain a plurality of single-layer flat plates;
3) preparation and culture of double-layer plates: under an aseptic condition, respectively adding 5-15 ml of semisolid agar culture medium into the plurality of single-layer plates prepared in the step 2), placing the single-layer plates on a horizontal desktop, naturally solidifying to obtain a plurality of double-layer plate nutrient agar culture media, sucking 0.1-1 ml of aerobic bacteria suspension obtained in the step 1), respectively adding the aerobic bacteria suspension into the surfaces of the plurality of double-layer plate nutrient agar culture media obtained previously, uniformly coating the aerobic bacteria suspension by using an aseptic coater, covering a dish cover when no obvious water stain exists on the surfaces, and inversely placing the culture dish cover in an incubator at 30-40 ℃ for culture;
4) and (3) observation and counting: and after macroscopic bacteria appear in the plurality of culture dishes, taking out the culture dishes for counting.
2. The plate counting method of claim 1, wherein in step 1), the aerobic bacteria comprise spore-forming aerobic bacteria.
3. The plate counting method according to claim 2, wherein in the step 1), the aerobic bacteria are selected from at least one of bacillus subtilis and bacillus licheniformis.
4. The plate counting method according to claim 3, wherein the temperature of the water bath in step 1) is 80 to 90 ℃.
5. The plate counting method of claim 4, wherein the preparing of the aerobic bacterial suspension in step 1) specifically comprises: absorbing aerobic bacteria glycerol tube preservation bacteria liquid, inoculating the aerobic bacteria glycerol tube preservation bacteria liquid into a 250ml shake flask filled with 50ml nutrient broth culture medium with the inoculation amount of 1.0% (v/v), culturing for 30h under the conditions that the culture temperature is 35 ℃ and the rotation speed is 180rpm/min, adding 10ml bacteria liquid into a sterile test tube, treating for 10min under the condition of 85 ℃ water bath, absorbing 1ml bacteria liquid, diluting the bacteria liquid by 100 times according to the volume by using sterile physiological saline under the sterile condition to obtain aerobic bacteria suspension, and preserving for later use at 4 ℃.
6. The plate counting method according to claim 1, wherein in step 2), the diameter of the bottom of the culture dish is 90mm, and the height of the bottom of the dish is 20 mm; the amount of the semi-solid agar medium added was 15 ml.
7. The plate counting method according to claim 1, wherein in step 2), three dilutions are selected for each of the anaerobe solid or fermentation broth samples to be tested, and three parallel counting plates are provided for each dilution.
8. The plate counting method of claim 1, wherein in step 3), the preparation of the bi-layer plate specifically comprises: under the aseptic condition, adding 10ml of semisolid nutrient agar culture medium on the surface of the single-layer plate prepared in the step 2), naturally solidifying at room temperature to obtain a double-layer plate nutrient agar culture medium, sucking 0.1ml of aerobic bacteria liquid obtained in the step 1), adding the surface of the double-layer plate nutrient agar culture medium obtained before, uniformly coating by using an aseptic coater, covering a dish cover when no obvious water stain exists on the surface, and inversely placing the dish cover in an incubator at 37 ℃ for culture.
9. The plate counting method of claim 1, wherein in step 4), said observing and counting specifically comprises: and after macroscopic single colonies appear in the lower-layer flat plates of the plurality of culture dishes, taking out the culture dishes to count the colonies.
10. The plate counting method according to claim 9, wherein the number of colonies in the lower plate of each dish in step 4) is in the range of 30 to 300, which is a statistical object.
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|---|---|---|---|---|
| CN1940085A (en) * | 2006-09-28 | 2007-04-04 | 东北林业大学 | Double-layer planar accounting method of bifidobacterium etc. strict anacerobe |
| US20150291994A1 (en) * | 2014-04-10 | 2015-10-15 | Mak Wood, Inc. | Microorganism enumeration methods |
| CN108641977A (en) * | 2018-04-16 | 2018-10-12 | 贵州理工学院 | A method of mixing high density fermentation improves bifidobacterium cells density and metabolite |
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