CN117165652A - Method for detecting bacillus subtilis in feed - Google Patents
Method for detecting bacillus subtilis in feed Download PDFInfo
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- CN117165652A CN117165652A CN202311372659.5A CN202311372659A CN117165652A CN 117165652 A CN117165652 A CN 117165652A CN 202311372659 A CN202311372659 A CN 202311372659A CN 117165652 A CN117165652 A CN 117165652A
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- bacillus subtilis
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- normal saline
- agar plates
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 39
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000006916 nutrient agar Substances 0.000 claims abstract description 32
- 238000011081 inoculation Methods 0.000 claims abstract description 31
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 238000012360 testing method Methods 0.000 claims abstract description 21
- 238000012790 confirmation Methods 0.000 claims abstract description 18
- 229920001817 Agar Polymers 0.000 claims abstract description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 15
- 239000008272 agar Substances 0.000 claims abstract description 15
- 230000001580 bacterial effect Effects 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000012895 dilution Substances 0.000 claims abstract description 12
- 238000010790 dilution Methods 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 239000000725 suspension Substances 0.000 claims abstract description 9
- 239000011248 coating agent Substances 0.000 claims abstract description 6
- 238000000576 coating method Methods 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 238000005303 weighing Methods 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 11
- 239000003085 diluting agent Substances 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 17
- 238000012216 screening Methods 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 8
- 238000000265 homogenisation Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- 241000272525 Anas platyrhynchos Species 0.000 description 3
- 241000272814 Anser sp. Species 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 235000019764 Soybean Meal Nutrition 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000004455 soybean meal Substances 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000000529 probiotic effect Effects 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting bacillus subtilis in feed, which comprises the following steps: weighing a sample, adding sterilized normal saline, homogenizing, and preparing an initial suspension; sucking the initial suspension, adding sterilized normal saline, and fully and uniformly mixing to prepare a diluent; s2 is repeated continuously according to the bacterial content of the sample, and further ten times serial incremental dilution is carried out to prepare inoculation liquid; 2-3 inoculating liquid water baths are selected, a pipette is used for sucking, then a coating rod is used for coating the agar surfaces of different nutrient agar plates respectively, the nutrient agar plates are placed in a biosafety cabinet for 4-6 min in a cover opening manner, and the nutrient agar plates are cultured in an incubator; after culturing, the colony count on each nutrient agar plate is C, characteristic colony A is selected for a confirmation test, and the bacillus subtilis colony count a on each nutrient agar plate is counted. The invention shortens the detection time, fully and accurately reflects the actual pollution condition of the sample by the bacterial colony, and improves the accuracy of detection data.
Description
Technical Field
The invention relates to the field of detection, in particular to a method for detecting bacillus subtilis in feed.
Background
The bacillus subtilis is widely applied to aquatic feeds, has strong protease, lipase and amylase activities, can promote the degradation of nutrients in the feeds, and enables the aquatic animals to fully absorb and utilize the feeds. Bacillus subtilis is added to animal feed in the form of spores. The spore is a living cell in a dormant state, can endure the bad environment in the feed processing process, is stable and easy to store after being prepared into a microbial inoculum, and can be quickly revived and propagated after entering the intestinal tract of animals. The bacillus subtilis can exert the probiotic characteristics after resuscitating and proliferating in animal intestinal tracts, including improving animal intestinal flora, enhancing organism immunity, providing enzymes required by various animals, and the like, can make up for the deficiency of endogenous enzymes of animal bodies, promote the growth and development of animals, and has remarkable probiotic effect. The current common detection method of bacillus subtilis in feed can not fully and accurately reflect the actual pollution condition of a sample by bacillus subtilis colony, the pollution condition of the sample or the pollution condition of the surrounding environment by the sample can occur in the detection process, and the pretreatment steps are required to be repeated, so that the experiment time is wasted.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the method for detecting the bacillus subtilis in the feed, which shortens the detection time, fully and accurately reflects the actual pollution condition of the sample by bacterial colonies and improves the accuracy of detection data.
In order to solve the technical problems, the invention adopts the following technical scheme: a method for detecting bacillus subtilis in feed comprises the following steps:
s1, weighing a sample, adding sterilized normal saline, homogenizing, and preparing into 1: 10;
s2, sucking the initial suspension, adding sterilized normal saline, and fully and uniformly mixing to prepare 1: a dilution of 100;
s3, continuously repeating the step S2 according to the bacterial content of the sample, and performing further ten times serial incremental dilution to prepare an inoculation liquid;
s4, selecting 2-3 inoculating liquid water baths prepared in the step S3, sucking by using a pipette, respectively coating the inoculating liquid water baths on the agar surfaces of different nutrient agar plates by using a coating rod, uniformly uncovering the nutrient agar plates, placing the nutrient agar plates in a biosafety cabinet for 4-6 min, and culturing in an incubator;
s5, after culturing, counting the bacterial colony on each nutrient agar plate as C, selecting a characteristic bacterial colony A for a confirmation test, and counting the bacterial colony number a of the bacillus subtilis on each nutrient agar plate.
In the present invention, the dilution of the sample is determined according to the type of sample and the degree of contamination.
When the bacillus subtilis is inoculated and cultured on a sample, the biological safety cabinet is selected, so that the inoculation liquid is only required to be absorbed by agar completely for 4-6 min, and the absorption of the inoculation liquid can be quickened due to the flowing of air flow in the biological safety cabinet. And meanwhile, the inoculation liquid is prevented from being polluted by the external environment, and the situation that the inoculation liquid pollutes the external environment is avoided.
In a preferred embodiment of the present invention, the homogenizing time in S1 is 10 to 20min. The homogenizing time is prolonged to more than 10 minutes and less than 20 minutes, the samples can be mixed as uniformly as possible in the optimal time range, so that the colony growth condition of the samples in the post-treatment process is more stable, the actual contamination condition of the samples by bacillus subtilis colonies can be reflected more fully and accurately, and the accuracy of detection data is improved.
In a preferred embodiment of the present invention, the concentration of the sterilized normal saline is 0.85%.
In a preferred embodiment of the present invention, the mass to volume ratio of the sample to the sterilized normal saline in S1 is 25g (mL): 225mL.
In a preferred embodiment of the invention, the mass to volume ratio of the initial suspension to the sterile saline in S2 is 1mL:9mL.
In a preferred embodiment of the invention, the temperature of the water bath in S4 is 79-81℃and the time of the water bath is 9-11min. Removing mixed bacteria in the sample through water bath, activating and inducing spore germination.
In a preferred embodiment of the present invention, the temperature of the culture in S4 is 36 to 38℃and the time of the culture is 46 to 50 hours.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the optimal homogenizing time is selected, so that the actual pollution condition of the sample by bacillus subtilis bacterial colonies can be reflected more fully and accurately, and the accuracy of detection data is improved.
2. When the bacillus subtilis is inoculated and cultured on a sample, the biological safety cabinet is selected, so that the inoculation liquid is only required to be absorbed by agar completely for 4-6 min, and the absorption of the inoculation liquid can be quickened due to the flowing of air flow in the biological safety cabinet. Meanwhile, the inoculation liquid is prevented from being polluted by the external environment, the experimental time is saved, and the waste of reagents and the like in the pretreatment process is avoided.
Drawings
FIG. 1 is a photograph showing the total number of colonies for 10min and 15min of homogenization in examples 1 and 2 and 5min of placement in a biosafety cabinet;
FIG. 2 is a photograph showing the comparison of the samples of the present invention for homogenization for 2min, 5min, 10min and 15 min;
FIG. 3 is a photograph of a sample of examples 1-2 of the present invention placed in a biosafety cabinet for 5 minutes;
FIG. 4 is a photograph showing the colony count of comparative example 3 of the present invention after homogenizing for 10min and leaving it at room temperature for 15 min.
Detailed Description
Example 1
Weighing 25g of sample Sheng Yi Ben Cao kang (chicken, duck and goose fermented soybean meal raw material) by aseptic operation, adding 225ml of 0.85% sterilized normal saline, homogenizing for 10min to prepare 1: 10; 1mL of the initial suspension of 1:10 is sucked, 9mL of 0.85% sterilized physiological saline is added, and the mixture is fully and uniformly mixed to prepare a 1:100 diluent.
According to the bacterial content of the sample, only two dilutions of 1:10 and 1:100 are selected in the embodiment, and 1 is selected in the embodiment: 10. 1:100 two inoculation solutions (the last inoculation solution is selected after the preparation, the embodiment does not need to be further increased by ten times, so that the 1:100 dilution solution prepared after full mixing is the inoculation solution) are maintained for 9min in a water bath at 80 ℃, 0.1mL of each inoculation solution is respectively absorbed by a sterile pipette, the agar surfaces of the two nutrient agar plates are respectively coated, the coated two nutrient agar plates are opened and placed in a biosafety cabinet for 5min, the inoculation solution is completely absorbed by agar, and the nutrient agar plates are turned over and placed in a 37 ℃ incubator for culturing for 48h.
(1) Nutrient agar plate 1:10, and screening and confirmation test of colony count and screening of inoculation liquid
After culturing, the colony count on the plate is calculated to be 70, namely C, 5 colony A in the characteristic colonies are selected for a confirmation test, and the confirmation test proves that 5 colonies are all bacillus subtilis, namely b.
The number of colonies of Bacillus subtilis a on the plate was counted at 70CFU/g (see Table 1).
(2) Nutrient agar plate 1:100, and screening and validation tests
After the culture, the colony count on the plate is calculated to be 7, namely C, 5 colony A in the characteristic colonies are selected for a confirmation test, and the confirmation test proves that 5 colonies are all bacillus subtilis, namely b.
The number of colonies of Bacillus subtilis a on the plate was counted at 7CFU/g (see Table 1).
Wherein, the calculation formula is: a=b/a×c.
a, counting the colony number of bacillus subtilis on each flat plate;
b, the colony number of bacillus subtilis after picking is proved;
a, picking the colony number used for verification on the flat plate;
c-number of colonies of all features on the plate.
Example 2
Weighing 25g of sample Sheng Yi Ben Cao kang (chicken, duck and goose fermented soybean meal raw material) by aseptic operation, adding 225ml of 0.85% sterilized normal saline, homogenizing for 15min to prepare 1: 10; 1mL of the initial suspension of 1:10 is sucked, 9mL of 0.85% sterilized physiological saline is added, and the mixture is fully and uniformly mixed to prepare a 1:100 diluent.
According to the bacterial content of the sample, only two dilutions of 1:10 and 1:100 are selected in the embodiment, and 1 is selected in the embodiment: 10. 1:100 two inoculation solutions (the last inoculation solution is selected after the preparation, the embodiment does not need to be further increased by ten times, so that the 1:100 dilution solution prepared after full mixing is the inoculation solution) are maintained for 9min in a water bath at 80 ℃, 0.1mL of each inoculation solution is respectively absorbed by a sterile pipette, the agar surfaces of the two nutrient agar plates are respectively coated, the coated two nutrient agar plates are opened and placed in a biosafety cabinet for 5min, the inoculation solution is completely absorbed by agar, and the nutrient agar plates are turned over and placed in a 37 ℃ incubator for culturing for 48h.
(1) Nutrient agar plate 1:10, and screening and confirmation test of colony count and screening of inoculation liquid
After the culture, the colony count on the plate is calculated to be 71, namely C, 5 colony A in the characteristic colonies is selected for a confirmation test, and the confirmation test proves that 5 colonies are all bacillus subtilis, namely b.
The number of colonies of Bacillus subtilis a on the plate was counted at 71CFU/g (see Table 1).
(2) Nutrient agar plate 1:100, and screening and validation tests
After the culture, the colony count on the plate is calculated to be 7, namely C, 5 colony A in the characteristic colonies are selected for a confirmation test, and the confirmation test proves that 5 colonies are all bacillus subtilis, namely b.
The number of colonies of Bacillus subtilis a on the plate was counted at 7CFU/g (see Table 1).
Comparative example 1
This example differs from example 1 in that the homogenization time is 2min, calculated as 1 on nutrient agar plates: 10, the number of dead colonies a on each plate of the inoculation liquid is 52CFU/g; the nutrient agar plates were calculated as 1: the number of colonies per plate of 100 colonies per plate was 5CFU/g (see Table 1).
Comparative example 2
This example differs from example 1 in that the homogenization time is 5min, calculated as 1 on nutrient agar plates: 10, the number of dead colonies a on each plate of the inoculation liquid is 58CFU/g; the nutrient agar plates were calculated as 1: the number of colonies per plate of 100 colonies per plate was 6CFU/g (see Table 1).
Comparative example 3
Weighing 25g of sample Sheng Yi Ben Cao kang (chicken, duck and goose fermented soybean meal raw material) by aseptic operation, adding 225ml of 0.85% sterilized normal saline, homogenizing for 10min to prepare 1: 10; 1mL of the initial suspension of 1:10 is sucked, 9mL of 0.85% sterilized physiological saline is added, and the mixture is fully and uniformly mixed to prepare a 1:100 diluent.
According to the bacterial content of the sample, only two dilutions of 1:10 and 1:100 are selected in the embodiment, and 1 is selected in the embodiment: 10. 1:100 two inoculation solutions (the last inoculation solution is selected after the preparation, the embodiment does not need to be further increased by ten times, so that the 1:100 dilution solution prepared after full mixing is the inoculation solution) are maintained for 9min in a water bath at 80 ℃, 0.1mL of each inoculation solution is respectively sucked by a sterile pipette, the two nutrition agar plates are respectively coated on the agar surfaces of the two nutrition agar plates, the coated two nutrition agar plates are covered, and the mixture is placed in room temperature for 15min to enable the inoculums to be completely absorbed by the agar. The nutrient agar plates are turned over and placed in a 37 ℃ incubator for culturing for 48 hours.
(1) Nutrient agar plate 1:10, and screening and confirmation test of colony count and screening of inoculation liquid
After the culture, the colony count on the plate is calculated to be 69, namely C, 5 colony A in the characteristic colonies is selected for a confirmation test, and the confirmation test proves that 5 colonies are all bacillus subtilis, namely b.
The number of colonies of Bacillus subtilis a on the plate was counted at 69CFU/g (see Table 1).
(2) Nutrient agar plate 1:100, and screening and validation tests
After the culture, the colony count on the plate is calculated to be 7, namely C, 5 colony A in the characteristic colonies are selected for a confirmation test, and the confirmation test proves that 5 colonies are all bacillus subtilis, namely b.
The number of colonies of Bacillus subtilis a on the plate was counted at 7CFU/g (see Table 1).
Table 1: colony meter for samples with different homogenization time
The samples of examples 1 and 2 and comparative examples 1 and 2 were found to have a homogenization time of 10 to 20 minutes, and the total number of colonies did not substantially change, so that the actual colony growth of the samples could be more accurately shown.
By implementing 1 and 2 and comparing examples 3 and 4, the biological safety cabinet is selected to ensure that only 4-6 min is needed when the inoculation liquid is completely absorbed by the agar, and the absorption of the inoculation liquid is quickened, the detection time is reduced, and the detection precision is improved because of the airflow in the biological safety cabinet.
Claims (7)
1. The method for detecting the bacillus subtilis in the feed is characterized by comprising the following steps of:
s1, weighing a sample, adding sterilized normal saline, homogenizing, and preparing into 1: 10;
s2, sucking the initial suspension, adding sterilized normal saline, and fully and uniformly mixing to prepare 1: a dilution of 100;
s3, continuously repeating the step S2 according to the bacterial content of the sample, and performing further ten times serial incremental dilution to prepare an inoculation liquid;
s4, selecting 2-3 inoculating liquid water baths prepared in the step S3, sucking by using a pipette, respectively coating the inoculating liquid water baths on the agar surfaces of different nutrient agar plates by using a coating rod, uniformly uncovering the nutrient agar plates, placing the nutrient agar plates in a biosafety cabinet for 4-6 min, and culturing in an incubator;
s5, after culturing, counting the bacterial colony on each nutrient agar plate as C, selecting a characteristic bacterial colony A for a confirmation test, and counting the bacterial colony number a of the bacillus subtilis on each nutrient agar plate.
2. The method for detecting bacillus subtilis in feed according to claim 1, wherein the homogenizing time in S1 is 10 to 20min.
3. The method for detecting bacillus subtilis in feed according to claim 1, wherein the concentration of the sterilized normal saline is 0.85%.
4. The method for detecting bacillus subtilis in feed according to claim 1, wherein the mass-to-volume ratio of the sample in S1 to the sterilized normal saline is 25g (mL): 225mL.
5. The method for detecting bacillus subtilis in feed according to claim 1, wherein the mass-to-volume ratio of the initial suspension to the sterilized normal saline in S2 is 1mL:9mL.
6. The method for detecting bacillus subtilis in feed according to claim 1, wherein the temperature of the water bath in S4 is 79-81 ℃, and the time of the water bath is 9-11min.
7. The method for detecting bacillus subtilis in feed according to claim 1, wherein the temperature of the culture in S4 is 36-38 ℃ and the time of the culture is 46-50 h.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311372659.5A CN117165652A (en) | 2023-10-23 | 2023-10-23 | Method for detecting bacillus subtilis in feed |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311372659.5A CN117165652A (en) | 2023-10-23 | 2023-10-23 | Method for detecting bacillus subtilis in feed |
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| CN117165652A true CN117165652A (en) | 2023-12-05 |
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| CN202311372659.5A Pending CN117165652A (en) | 2023-10-23 | 2023-10-23 | Method for detecting bacillus subtilis in feed |
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- 2023-10-23 CN CN202311372659.5A patent/CN117165652A/en active Pending
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