Method for determination of functional microorganism in biologic products
Some standard content:
ICS07.080
National Standard of the People's Republic of China
GB/T34224—2017
Method for determination of functional microorganism in biologic products2017-09-07Issued
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaStandardization Administration of China
2018-04-01Implementation
This standard was drafted in accordance with the rules given in GB/T1.1—2009. This standard was proposed and managed by the China National Institute of Standardization. GB/T34224—2017
The drafting organizations of this standard are: Hebei Food Inspection Institute, Zhejiang Gongshang University, Yellow Sea Fisheries Research Institute of Chinese Academy of Fishery Sciences, China National Institute of Standardization, Zhejiang Liziyuan Food Co., Ltd., Hebei Medical University, Beijing Institute of Food Science, Hefei University of Technology, Henan University, Jiangnan University, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang Junlebao Dairy Co., Ltd., Rongcheng Huatong Marine Biotechnology Co., Ltd.
The main drafters of this standard are: Zhang Yan, Wang Yanbo, Fu Linglin, Zhao Feng, Ma Aijin, Wang Lixia, Wang Shunyu, Lv Pin, Zhou Wei, Sun Yong, Zheng Lei, Kang Wenyi, Zhou Peng, Liu Daoliang, Chai Yanbing, Zhang Yaoguang, Bu Xiaofeng, Liu Zhongfu, Li Yongbo. 1 Scope
Detection of functional microorganisms in biological products
GB/T34224—2017
This standard specifies the basic principles, general requirements and test methods for the detection of functional microorganisms in biological products. This standard applies to the detection of Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus subtilis, Bacillus licheniformis, Enterococcus faecalis, Enterococcus faecium, Candida utilis and Saccharomyces cerevisiae in biological products. Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For any undated referenced documents, the latest version (including all amendments) shall apply to this document GB4789.1 National food safety standard General principles for food microbiology examination GB4789.28 National food safety standard Quality requirements for food microbiology examination culture media and reagents GB19489 General requirements for laboratory biosafety GB/T27405 Laboratory quality control specification Food microbiology testing RB/T151
Guide for assessment of measurement uncertainty for quantitative detection of food microorganisms SN/T0330
SN/T2632
SN/T2660
3 Terms and definitions
General principles for microbiology examination of exported foods
Technical regulations for routine preservation of microbial strains
Methods for preservation of strains in food microbiology laboratories The following terms and definitions apply to this document. 3.1
biologic products
Biological products
Industrial products related to organisms.
Note: The biological products in this standard specifically refer to live bacterial preparations made by functional microorganisms after industrial production and expansion, and products added with such preparations.
functional microorganism
Functional microorganism
Live microbial strains with definite regulatory and improvement functions under certain conditions. 3.3
Colony-forming units
colony-formingunits; CFU
In the counting of live bacterial cultures, the colonies formed by a single bacterium or multiple bacteriums aggregated into a mass growing and multiplying on a solid culture medium. 1
GB/T34224—2017
4 General
Functional microbiological tests carried out in accordance with this standard shall comply with the provisions of SN/T0330. 4.1
Laboratory biosafety requirements shall comply with the provisions of GB19489. Laboratory personnel shall meet the requirements of GB4789.1. 4.4
The preservation of strains shall comply with the provisions of SN/T2632 and SN/T2660. The quality requirements of culture media and reagents shall comply with the provisions of GB4789.28. Laboratory quality control shall comply with the provisions of GB/T27405. The quantitative test values shall be rounded off according to the principle of "rounding up to the nearest integer", and the reporting unit is CFU/g/(CFU/mL). The quantitative test measurement uncertainty assessment shall be carried out in accordance with the requirements of RB/T151. 4.8
The sample under test can only be processed after the test result is reported. The sample shall be harmlessly treated. Plant Lactobacillus
Equipment and Materials
In addition to the routine sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 5.1.1 Constant temperature anaerobic incubator: 37℃±1℃. 5.1.2 Refrigerator: 2℃~5℃.
5.1.3 Balance: sensitivity is 0.1g.
5.1.4 Homogenizer and sterile homogenizing bag and homogenizing cup. 5.1.5 Oscillator
5.1. 6 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and pipette tip. 5.1.7 Sterile conical flask: 250mL, 500mL capacity. 5.1.8 Sterile culture dish: 90mm diameter.
5.1.9 Microscope: 10x to 100x.
pH meter or pH colorimetric tube or precision pH test paper. 5.1.10
Culture medium and reagents
MRS (ManRogosaSharpe) agar culture medium: see A.1 in Appendix A. MRS broth culture medium: see A.2.
Sterile physiological saline: see A.10.
5.2.4 Gram staining solution: see A.11.
5.2.5 Bacterial biochemical identification kit.
Test procedure
The test procedure for Lactobacillus plantarum is shown in Figure 1.
5.4 Operation steps
5.4.1 Sample preparation
1 Solid and semi-solid samples
Sample 25g (mL) + 225mL sterile saline 10-fold serial dilution
Select 2 to 3 consecutive appropriate dilutions, and take 0.1mL of the sample solution for each dilution and inoculate it on MRS agar for anaerobic culture at 37℃±1℃ for 48h2h
Colony count
Select 5 colonies with transparent and neat edges on the plate and transfer them to MRS meat culture medium respectively
Anaerobic culture at 37℃±1℃ for 24h±2hMorphological identification
Figure 1 Plant Lactobacillus test procedure
Biochemical identification
GB/T34224—2017
Weigh 25g of sample, place it in a sterile homogenizing bag containing 225mL of normal saline, and beat it with a slapping homogenizer for 1min~2min to make a 1:10 sample solution; or place it in a sterile homogenizing cup containing 225mL of sterile normal saline, and homogenize it at 8000r/min~10000r/min for 1min~2min to make a 1:10 sample solution. 5.4.1.2 Liquid sample
First, shake it thoroughly, then use a sterile pipette to draw 25mL of sample and put it into a sterile conical flask containing 225mL of normal saline (with an appropriate number of sterile glass beads pre-placed in the flask), shake it thoroughly, and make a 1:10 sample solution. 5.4.2 Sample dilution
5.4.2.1 Use a 1mL sterile pipette or micropipette to draw 1mL of 1:10 sample solution. Slowly pour it into a sterile test tube containing 9mL of physiological saline along the tube wall (be careful not to let the tip of the pipette touch the diluent). Shake the test tube or use a sterile pipette to blow and beat repeatedly to mix it evenly to make a 1:100 sample solution. 5.4.2.2 Take another 1mL sterile pipette or micropipette tip and make 10-fold incremental sample solution according to the above operation sequence. Each time the incremental dilution is performed, the 1mL sterile pipette or pipette tip is used once. 5.4.3 Culture
Based on the estimated bacterial content of the sample to be tested, select 2 to 3 consecutive appropriate dilutions, and draw 3
GB/T34224—2017
0.Inoculate 1mL into the MRS agar plate, and use a spreading stick to spread the inoculation solution on the agar surface as carefully and quickly as possible. The spreading stick must not touch the edge of the plate. Inoculate 2 plates for each dilution. After spreading, let the plate stand for 10 minutes to allow the inoculum to be completely absorbed by the culture medium. Turn the plate over and place it in an anaerobic incubator at 37℃±1℃ for 48h±2h. 5.4.4 Colony count
5.4.4.1 Select the plates with characteristic colony counts between 30CFU and 300CFU and no spreading colony growth to count the total number of colonies. For plates with less than 30CFU, record the specific colony count, and for plates with more than 300CFU, record as too many to count. The colony count for each dilution should be the average of the two plates.
5.4.4.2 If one of the plates has large flake colonies, it should not be used. Instead, the plate without flake colonies should be used as the colony count for the dilution. If the flake colonies are less than half of the plate, and the colonies in the other half are evenly distributed, half of the plate can be counted and multiplied by 2 to represent the colony count of one plate.
5.4.4.3 When chain growth without obvious boundaries between colonies appears on the plate, each single chain is counted as a colony. 5.4.5 Identification
5.4.5.1 Colony selection and pure culture
Select 5 colonies with transparent and neat edges on the plate and transfer them to MRS broth medium and culture them in an anaerobic incubator at 37℃±1℃ for 24h±2h for morphological and biochemical identification. 5.4.5.2 Morphological identification
Gram staining and microscopic examination. Lactobacillus plantarum is a Gram-positive bacillus, straight rod-shaped, 3.0 μm to 8.0 μm in length, single, paired or chain-shaped, usually lacking flagella and without spores. 5.4.5.3 Biochemical Identification
Biochemical identification was performed using a bacterial biochemical identification kit. The identification characteristics are shown in Table B.1 in Appendix B. The main identification characteristics of common species of Lactobacillus are shown in Table C.1 in Appendix C.
5.5 Result Calculation
5.5.1 Number of Lactobacillus plantarum on each plate The calculation of the number of Lactobacillus plantarum colonies on each plate is shown in formula (1): bxc
Where:
The number of Lactobacillus plantarum colonies on each plate; b
The number of colonies confirmed to be Lactobacillus plantarum after picking; A
The number of colonies on the plate for verification: C
The number of all characteristic colonies on the plate.
5.5.2 Calculation method of total colony count
.. (1)
5.5.2.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the average number of Lactobacillus plantarum colonies on the two plates, and then multiply the average by the corresponding dilution factor to obtain the total number of colonies per gram (ml). 5.5.2.2 If the number of colonies on two consecutive dilution plates is within the appropriate counting range, calculate according to formula (2): N
(n+0.1nz)d
(2)
Where:
5.6 Report
The number of colonies in the sample;
The sum of the number of colonies on the plates (including the plates with the number of colonies in the appropriate range): the number of plates with the first dilution (low dilution factor); the number of plates with the second dilution (high dilution factor): the dilution factor (first dilution).
Result determination
If the sample colonies meet the characteristics of morphological and biochemical identification of Lactobacillus plantarum, it can be determined to be Lactobacillus plantarum. 5.6.2 Result report
Qualitative report
Whether Lactobacillus plantarum is detected or not detected in 25g (mL) sample. 5.6.2.2
Quantitative report
GB/T34224—2017
When the colony count is less than 100CFU, report it as an integer. When the colony count is greater than or equal to 100CFU, round off the third digit, take the first two digits, and replace the digit with 0: it can also be expressed in the form of 10 exponentials, using two significant digits. Lactobacillus acidophilus
Equipment and materials
In addition to the conventional sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 6.1.1 Constant temperature anaerobic incubator: 37℃±1℃. 6.1.2 Refrigerator: 2℃~5℃.
6.1.3 Balance: Sensitivity is 0.1g.
6.1.4 Homogenizer and sterile homogenizing bag and cup. 6.1.5 Oscillator.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and pipette tip. 6.1.7
Sterile conical flask: capacity 250mL, 500mL. Sterile culture dish: diameter 90mm.
Microscope: 10x~100x.
pH meter or pH colorimetric tube or precision pH test paper. 6.1.10
Culture medium and reagents
MRS agar culture medium: see A.1.
MRS broth culture medium: see A.2.
6.2.3 Sterile physiological saline: see A.10.
6.2.4 Gram staining solution: see A.11.
6.2.5 Bacterial biochemical identification kit.
GB/T34224—2017
6.3 Test procedure
The test procedure for Lactobacillus acidophilus is shown in Figure 2.
Sample 25g (mL) + 225mL sterile saline 10-fold serial dilution
Select 2 to 3 consecutive appropriate dilutions, take 0.1mL of sample solution for each dilution and inoculate it on MRS agar for anaerobic culture, 37℃±1℃, 48h2h
Colony count
Select 5 convex, rough-surfaced and curled-edge colonies on the plate and transfer them to MRS broth medium
Anaerobic culture, 37℃±1℃.24h±2h
Morphological identification
6. 4 Operation steps
6.4.1 Sample preparation
Solid and semi-solid samples
Figure 2 Lactobacillus acidophilus test procedure
Biochemical identification
Weigh 25g of sample, place it in a sterile homogenizing bag containing 225mL of normal saline, and beat it with a slapping homogenizer for 1min~2min to make a 1:10 sample solution; or place it in a sterile homogenizing cup containing 225mL of sterile normal saline, and homogenize it at 8000r/min~10000r/min for 1min~2min to make a 1:10 sample solution. 6.4.1.2 Liquid samples
First, shake it thoroughly to make a sample solution, then use a sterile pipette to draw 25mL of the sample and put it into a sterile conical flask containing 225mL of normal saline (with an appropriate number of sterile glass beads pre-placed in the flask), shake it thoroughly to make a 1:10 sample solution. 6.4.2 Sample dilution
6.4.2.1 Use a 1mL sterile pipette or micropipette to draw 1mL of 1:10 sample solution, and slowly inject it into a sterile test tube containing 9mL of physiological saline along the tube wall (be careful not to let the tip of the pipette touch the diluent), shake the test tube or use a sterile pipette to blow and beat repeatedly to mix evenly, and make a 1:100 sample solution. 6.4.2.2 Take another 1mL sterile pipette or micropipette tip, and make 10-fold incremental sample solution according to the above operation sequence. Each time the incremental dilution is performed, the 1mL sterile pipette or tip is used once. 6
6.4.3 Culture
GB/T34224—2017
Based on the estimation of the bacterial content of the sample to be tested, select 2 to 3 consecutive appropriate dilutions. For each dilution, take 0.1mL of the sample homogenate and inoculate it into the MRS agar plate. Use a spreading stick to spread the inoculum on the agar surface as carefully and quickly as possible. The spreading stick must not touch the edge of the plate. Inoculate 2 plates for each dilution. After spreading, let the plate stand for 10 minutes to allow the inoculum to be completely absorbed by the culture medium. Turn the plate over and place it in an anaerobic incubator at 37℃±1℃ for 48h±2h. 6.4.4 Colony count
6.4.4.1 Select the plates with characteristic colony counts between 30CFU and 300CFU and no spreading colony growth to count the total number of colonies. For plates with less than 30CFU, record the specific colony count. For plates with more than 300CFU, record as too many to count. The colony count for each dilution should be the average of the two plates.
6.4.4.2 If one of the plates has large flake colonies, it should not be used, and the plate without flake colonies should be used as the colony count for that dilution; if the flake colonies are less than half of the plate, and the colonies in the remaining half are evenly distributed, half of the plate can be counted and multiplied by 2 to represent the colony count of one plate.
6.4.4.3 When there is chain growth without obvious boundaries between colonies on the plate, each single chain should be counted as a colony. 6.4.5 Identification
Colony selection and pure culture
Select 5 colonies with protrusions, rough surfaces and curled edges on the plate and transfer them to MRS broth medium in an anaerobic incubator at 37℃±1℃ for 24h±2h for morphological and biochemical identification. 6.4.5.2 Morphological identification
Gram staining and microscopic examination. Lactobacillus acidophilus is a Gram-positive rod-shaped bacillus with round ends and a length of 0.9μm to 6.0um. It is single, paired or chain-shaped, without flagella and spores. 6.4.5.3 Biochemical identification
Use a bacterial biochemical identification kit for biochemical identification. See Table B.2 in Appendix B for identification characteristics. See Table C.1 in Appendix C for the main identification characteristics of common species of Lactobacillus.
6.5 Calculation of results
6.5.1 Number of Lactobacillus acidophilus on each plate The calculation of the number of Lactobacillus acidophilus colonies on each plate is shown in formula (3): b
Wherein:
The number of Lactobacillus acidophilus colonies on each plate; a
bThe number of colonies confirmed to be Lactobacillus acidophilus after picking; A——The number of colonies on the picked plate for verification; the number of all characteristic colonies on the plate,
6.5.2 Calculation method of total colony count
(3)
6.5.2.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the number of Lactobacillus acidophilus colonies on both plates. 7
GB/T34224—2017
average value, then multiply the average value by the corresponding dilution factor as the total number of colonies per gram (ml) Result 2 If the number of colonies on two consecutive dilution plates is within the appropriate counting range, calculate according to formula (4): 6.5.2.2
Where:
6.6 Report
Number of colonies in the sample;
(n+0.1nz)d
The sum of the number of colonies on the plate (including the plate with the number of colonies in the appropriate range); the number of plates at the first dilution (low dilution factor); the number of plates at the second dilution (high dilution factor); the dilution factor (first dilution).
Result judgment
If the sample colonies meet the characteristics of morphology and biochemical identification of Lactobacillus acidophilus, it can be determined to be Lactobacillus acidophilus. 6.6.2
Result report
6.6.2.1 Qualitative report
Lactobacillus acidophilus was detected or not detected in 25g (mL) sample. 6.6.2.2
Quantitative report
. (4)
When the colony count is less than 100 CFU, report it as an integer. When the colony count is greater than or equal to 100 CFU, round off the third digit and take the first two digits, and replace the digit with 0; it can also be expressed in the form of 10 exponentials with two significant digits. 7 Bacillus subtilis
Equipment and materials
In addition to the routine sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 7.1.1 Constant temperature incubator: 37℃±1℃. 7.1.2 Refrigerator: 2℃~5℃.
7.1.3 Constant temperature water bath: 80℃±1℃. 7.1.4 Balance: Sensitivity is 0.1g.
7.1.5 Homogenizer and sterile homogenizing bag and homogenizing cup. 7.1.6 Oscillator.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and pipette tip. 7.1.7
7.1.8 Sterile conical flask: capacity 250mL, 500mL. 7.1.9 Sterile culture dish: diameter 90mm.
Microscope: 10x~100x.
pH meter or pH colorimetric tube or precision pH test paper. 7.2 Culture medium and reagents
Nutrient agar culture medium: see A.3.
Nutrient broth culture medium: see A.4.
7.2.3 Sterile physiological saline: see A.10.
7.2.4 Gram staining solution: see A.11.
7.2.5 Bacterial biochemical identification kit.
Test procedure
The test procedure for Bacillus subtilis is shown in Figure 3. Sample 25g (mL) + 225mL sterile physiological saline 10-fold serial dilution
Select 2 to 3 consecutive appropriate dilutions, maintain each dilution in a water bath at 80℃±1℃ for 10 minutes, draw 0.2mL of bacterial solution and inoculate it on a nutrient agar plate
37 C±1 C, 48 h±2 h
Colony counting
Pick 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them into nutrient broth culture medium at 37℃±1℃.24h±2h
Morphological identification
Biochemical identification
Figure 3 Bacillus subtilis test procedure
7.4 Operation steps
7.4.1 Sample preparation
Solid and semi-solid samples
GB/T34224—2017
Weigh 25g of sample, put it in a sterile homogenizing bag containing 225mL of normal saline, and beat it with a slapping homogenizer for 1min2min to make a 1:10 sample solution; or put it in a sterile homogenizing cup containing 225mL of sterile normal saline, and homogenize it at 8000r/min~10000/min for 1min~2min to make a 1:10 sample solution. 7.4.1.2 Liquid samples should be shaken thoroughly before mixing. Use a sterile pipette to draw 25 mL of the sample into a sterile conical flask containing 225 mL of normal saline (with an appropriate number of sterile glass beads pre-placed in the flask), shake thoroughly to make a 1:10 sample homogenate. 7.4.2 Sample dilution 7.4.2.1 Use a 1 mL sterile pipette or micropipette to draw 1 mL of 1:10 sample homogenate and slowly inject it along the tube wall into a sterile test tube containing 9 mL of normal saline (be careful not to let the tip of the pipette touch the diluent), shake the test tube or use a sterile pipette to blow and beat repeatedly to mix it evenly, and make a 1:100 sample homogenate. 7.4.2.2 Take another 1mL sterile pipette or micropipette tip and make 10-fold incremental sample dilution according to the above operation sequence. Each time the dilution is incrementally diluted, use a 1mL sterile pipette or pipette tip. 7.4.3 Culture
According to the estimation of the bacterial content of the sample to be tested, select 2 to 3 consecutive appropriate dilutions. Each dilution is maintained in a water bath at 80℃±1℃ for 10 minutes. Pipette 0.2mL of bacterial solution and inoculate it into the nutrient agar plate. Use a spreading stick to spread the inoculum on the agar surface as carefully and quickly as possible. The spreading stick must not touch the edge of the plate. Inoculate 2 plates for each dilution. After spreading, let the plate stand for 10 minutes to allow the inoculum to be completely absorbed by the culture medium. Turn the plate over and place it in a 37℃±1℃ incubator for 48h±2h. 7.4.4 Colony Count
7.4.4.1 Select plates with characteristic colony counts between 20CFU and 200CFU and no spreading colony growth to count the total number of colonies. Plates with less than 20CFU should record the specific colony count, and those with more than 200CFU can be recorded as too many to count. The colony count of each dilution should be the average of the two plates.
7.4.4.2 If one of the plates has large flake colonies growing, it should not be used, and the plate without flake colonies should be used as the colony count of the dilution; if the flake colonies are less than half of the plate, and the colony distribution in the remaining half is very uniform, half of the plate can be counted and multiplied by 2 to represent the colony count of one plate.
7.4.4.3 When chain growth without obvious boundaries between colonies appears on the plate, each single chain is counted as a colony. 7.4.5 Identification
7.4.5.1 Colony selection and pure culture
Select 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them to nutrient broth medium and culture them in an incubator at 37℃±1℃ for 24h±2h for morphological and biochemical identification. 7.4.5.2 Morphological identification
Gram staining and microscopic examination. Bacillus subtilis is a Gram-positive rod, rod-shaped, 1.5um to 3.0μm in length, without capsule, with flagella around the periphery, and buds. The buds are oval, mesogenic or submesogenic, and the bud capsule is not obviously swollen. 7.4.5.3 Biochemical identification
Use a bacterial biochemical identification kit for biochemical identification. The identification characteristics are shown in Table B.3 in Appendix B. 7.5 Calculation of results
7.5.1 Number of Bacillus subtilis on each plate The calculation of the number of Bacillus subtilis colonies on each plate is shown in formula (5): b
Wherein:
The number of Bacillus subtilis colonies on each plate; a
The number of colonies confirmed to be Bacillus subtilis after picking; The number of colonies on the plate for verification; C——The number of all characteristic colonies on the plate. 10
·(5)2 Morphological identification
Gram staining, microscopic examination. Lactobacillus acidophilus is a Gram-positive rod-shaped bacillus with rounded ends, 0.9μm to 6.0um in length, single, paired or chain-shaped, without flagella and spores. 6.4.5.3 Biochemical identification
Biochemical identification is performed using a bacterial biochemical identification kit. The identification characteristics are shown in Table B.2 in Appendix B. The main identification characteristics of common species of Lactobacillus are shown in Table C.1 in Appendix C.
6.5 Calculation of results
6.5.1 Number of Lactobacillus acidophilus on each plate The calculation of the number of Lactobacillus acidophilus colonies on each plate is shown in formula (3): b
Wherein:
The number of Lactobacillus acidophilus colonies on each plate; a
bThe number of colonies confirmed to be Lactobacillus acidophilus after picking; A——The number of colonies on the picked plate for verification; the number of all characteristic colonies on the plate,
6.5.2 Calculation method of total colony count
(3)
6.5.2.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the number of Lactobacillus acidophilus colonies on both plates. 7
GB/T34224—2017
average value, then multiply the average value by the corresponding dilution factor as the total number of colonies per gram (ml) Result 2 If the number of colonies on two consecutive dilution plates is within the appropriate counting range, calculate according to formula (4): 6.5.2.2
Where:
6.6 Report
Number of colonies in the sample;
(n+0.1nz)d
The sum of the number of colonies on the plate (including the plate with the number of colonies in the appropriate range); the number of plates at the first dilution (low dilution factor); the number of plates at the second dilution (high dilution factor); the dilution factor (first dilution).
Result judgment
If the sample colonies meet the characteristics of morphology and biochemical identification of Lactobacillus acidophilus, it can be determined to be Lactobacillus acidophilus. 6.6.2
Result report
6.6.2.1 Qualitative report
Lactobacillus acidophilus was detected or not detected in 25g (mL) sample. 6.6.2.2
Quantitative report
. (4)
When the colony count is less than 100 CFU, report it as an integer. When the colony count is greater than or equal to 100 CFU, round off the third digit and take the first two digits, and replace the digit with 0; it can also be expressed in the form of 10 exponentials with two significant digits. 7 Bacillus subtilis
Equipment and materials
In addition to the routine sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 7.1.1 Constant temperature incubator: 37℃±1℃. 7.1.2 Refrigerator: 2℃~5℃.
7.1.3 Constant temperature water bath: 80℃±1℃. 7.1.4 Balance: Sensitivity is 0.1g.
7.1.5 Homogenizer and sterile homogenizing bag and homogenizing cup. 7.1.6 Oscillator.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and pipette tip. 7.1.7
7.1.8 Sterile conical flask: capacity 250mL, 500mL. 7.1.9 Sterile culture dish: diameter 90mm.
Microscope: 10x~100x.
pH meter or pH colorimetric tube or precision pH test paper. 7.2 Culture medium and reagents
Nutrient agar culture medium: see A.3.
Nutrient broth culture medium: see A.4.
7.2.3 Sterile physiological saline: see A.10.
7.2.4 Gram staining solution: see A.11.
7.2.5 Bacterial biochemical identification kit.
Test procedure
The test procedure for Bacillus subtilis is shown in Figure 3. Sample 25g (mL) + 225mL sterile physiological saline 10-fold serial dilution
Select 2 to 3 consecutive appropriate dilutions, maintain each dilution in a water bath at 80℃±1℃ for 10 minutes, draw 0.2mL of bacterial solution and inoculate it on a nutrient agar plate
37 C±1 C, 48 h±2 h
Colony count
Pick 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them into nutrient broth culture medium at 37℃±1℃.24h±2h
Morphological identification
Biochemical identification
Figure 3 Bacillus subtilis test procedure
7.4 Operation steps
7.4.1 Sample preparation
Solid and semi-solid samples
GB/T34224—2017
Weigh 25g of sample, put it in a sterile homogenizing bag containing 225mL of normal saline, and beat it with a slapping homogenizer for 1min2min to make a 1:10 sample solution: or put it in a sterile homogenizing cup containing 225mL of sterile normal saline, and homogenize it at 8000r/min~10000/min for 1min~2min to make a 1:10 sample solution. 7.4.1.2 Liquid samples should be shaken thoroughly before mixing. Use a sterile pipette to draw 25 mL of the sample into a sterile conical flask containing 225 mL of normal saline (with an appropriate number of sterile glass beads pre-placed in the flask), shake thoroughly to make a 1:10 sample homogenate. 7.4.2 Sample dilution 7.4.2.1 Use a 1 mL sterile pipette or micropipette to draw 1 mL of 1:10 sample homogenate and slowly inject it along the tube wall into a sterile test tube containing 9 mL of normal saline (be careful not to let the tip of the pipette touch the diluent), shake the test tube or use a sterile pipette to blow and beat repeatedly to mix it evenly, and make a 1:100 sample homogenate. 7.4.2.2 Take another 1mL sterile pipette or micropipette tip and make 10-fold incremental sample dilution according to the above operation sequence. Each time the dilution is incrementally diluted, use a 1mL sterile pipette or pipette tip. 7.4.3 Culture
According to the estimation of the bacterial content of the sample to be tested, select 2 to 3 consecutive appropriate dilutions. Each dilution is maintained in a water bath at 80℃±1℃ for 10 minutes. Pipette 0.2mL of bacterial solution and inoculate it into the nutrient agar plate. Use a spreading stick to spread the inoculum on the agar surface as carefully and quickly as possible. The spreading stick must not touch the edge of the plate. Inoculate 2 plates for each dilution. After spreading, let the plate stand for 10 minutes to allow the inoculum to be completely absorbed by the culture medium. Turn the plate over and place it in a 37℃±1℃ incubator for 48h±2h. 7.4.4 Colony Count
7.4.4.1 Select plates with characteristic colony counts between 20CFU and 200CFU and no spreading colony growth to count the total number of colonies. Plates with less than 20CFU should record the specific colony count, and those with more than 200CFU can be recorded as too many to count. The colony count of each dilution should be the average of the two plates.
7.4.4.2 If one of the plates has large flake colonies growing, it should not be used, and the plate without flake colonies should be used as the colony count of the dilution; if the flake colonies are less than half of the plate, and the colony distribution in the remaining half is very uniform, half of the plate can be counted and multiplied by 2 to represent the colony count of one plate.
7.4.4.3 When chain growth without obvious boundaries between colonies appears on the plate, each single chain is counted as a colony. 7.4.5 Identification
7.4.5.1 Colony selection and pure culture
Select 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them to nutrient broth medium and culture them in an incubator at 37℃±1℃ for 24h±2h for morphological and biochemical identification. 7.4.5.2 Morphological identification
Gram staining and microscopic examination. Bacillus subtilis is a Gram-positive rod, rod-shaped, 1.5um to 3.0μm in length, without capsule, with flagella around the periphery, and buds. The buds are oval, mesogenic or submesogenic, and the bud capsule is not obviously swollen. 7.4.5.3 Biochemical identification
Use a bacterial biochemical identification kit for biochemical identification. The identification characteristics are shown in Table B.3 in Appendix B. 7.5 Calculation of results
7.5.1 Number of Bacillus subtilis on each plate The calculation of the number of Bacillus subtilis colonies on each plate is shown in formula (5): b
Wherein:
The number of Bacillus subtilis colonies on each plate; a
The number of colonies confirmed to be Bacillus subtilis after picking; The number of colonies on the plate for verification; C——The number of all characteristic colonies on the plate. 10
·(5)2 Morphological identification
Gram staining, microscopic examination. Lactobacillus acidophilus is a Gram-positive rod-shaped bacillus with rounded ends, 0.9μm to 6.0um in length, single, paired or chain-shaped, without flagella and spores. 6.4.5.3 Biochemical identification
Biochemical identification is performed using a bacterial biochemical identification kit. The identification characteristics are shown in Table B.2 in Appendix B. The main identification characteristics of common species of Lactobacillus are shown in Table C.1 in Appendix C.
6.5 Calculation of results
6.5.1 Number of Lactobacillus acidophilus on each plate The calculation of the number of Lactobacillus acidophilus colonies on each plate is shown in formula (3): b
Wherein:
The number of Lactobacillus acidophilus colonies on each plate; a
bThe number of colonies confirmed to be Lactobacillus acidophilus after picking; A——The number of colonies on the picked plate for verification; the number of all characteristic colonies on the plate,
6.5.2 Calculation method of total colony count
(3)
6.5.2.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the number of Lactobacillus acidophilus colonies on both plates. 7
GB/T34224—2017
average value, then multiply the average value by the corresponding dilution factor as the total number of colonies per gram (ml) Result 2 If the number of colonies on two consecutive dilution plates is within the appropriate counting range, calculate according to formula (4): 6.5.2.2
Where:
6.6 Report
Number of colonies in the sample;
(n+0.1nz)d
The sum of the number of colonies on the plate (including the plate with the number of colonies in the appropriate range); the number of plates at the first dilution (low dilution factor); the number of plates at the second dilution (high dilution factor); the dilution factor (first dilution).
Result judgment
If the sample colonies meet the characteristics of morphology and biochemical identification of Lactobacillus acidophilus, it can be determined to be Lactobacillus acidophilus. 6.6.2
Result report
6.6.2.1 Qualitative report
Lactobacillus acidophilus was detected or not detected in 25g (mL) sample. 6.6.2.2
Quantitative report
. (4)
When the colony count is less than 100 CFU, report it as an integer. When the colony count is greater than or equal to 100 CFU, round off the third digit and take the first two digits, and replace the digit with 0; it can also be expressed in the form of 10 exponentials with two significant digits. 7 Bacillus subtilis
Equipment and materials
In addition to the routine sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 7.1.1 Constant temperature incubator: 37℃±1℃. 7.1.2 Refrigerator: 2℃~5℃.
7.1.3 Constant temperature water bath: 80℃±1℃. 7.1.4 Balance: Sensitivity is 0.1g.
7.1.5 Homogenizer and sterile homogenizing bag and homogenizing cup. 7.1.6 Oscillator.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and pipette tip. 7.1.7
7.1.8 Sterile conical flask: capacity 250mL, 500mL. 7.1.9 Sterile culture dish: diameter 90mm.
Microscope: 10x~100x.
pH meter or pH colorimetric tube or precision pH test paper. 7.2 Culture medium and reagents
Nutrient agar culture medium: see A.3.
Nutrient broth culture medium: see A.4.
7.2.3 Sterile physiological saline: see A.10.
7.2.4 Gram staining solution: see A.11.
7.2.5 Bacterial biochemical identification kit.
Test procedure
The test procedure for Bacillus subtilis is shown in Figure 3. Sample 25g (mL) + 225mL sterile physiological saline 10-fold serial dilution
Select 2 to 3 consecutive appropriate dilutions, maintain each dilution in a water bath at 80℃±1℃ for 10 minutes, draw 0.2mL of bacterial solution and inoculate it on a nutrient agar plate
37 C±1 C, 48 h±2 h
Colony count
Pick 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them into nutrient broth culture medium at 37℃±1℃.24h±2h
Morphological identification
Biochemical identification
Figure 3 Bacillus subtilis test procedure
7.4 Operation steps
7.4.1 Sample preparation
Solid and semi-solid samples
GB/T34224—2017
Weigh 25g of sample, put it in a sterile homogenizing bag containing 225mL of normal saline, and beat it with a slapping homogenizer for 1min2min to make a 1:10 sample solution: or put it in a sterile homogenizing cup containing 225mL of sterile normal saline, and homogenize it at 8000r/min~10000/min for 1min~2min to make a 1:10 sample solution. 7.4.1.2 Liquid samples should be shaken thoroughly before mixing. Use a sterile pipette to draw 25 mL of the sample into a sterile conical flask containing 225 mL of normal saline (with an appropriate number of sterile glass beads pre-placed in the flask), shake thoroughly to make a 1:10 sample homogenate. 7.4.2 Sample dilution 7.4.2.1 Use a 1 mL sterile pipette or micropipette to draw 1 mL of 1:10 sample homogenate and slowly inject it along the tube wall into a sterile test tube containing 9 mL of normal saline (be careful not to let the tip of the pipette touch the diluent), shake the test tube or use a sterile pipette to blow and beat repeatedly to mix it evenly, and make a 1:100 sample homogenate. 7.4.2.2 Take another 1mL sterile pipette or micropipette tip and make 10-fold incremental sample dilution according to the above operation sequence. Each time the dilution is incrementally diluted, use a 1mL sterile pipette or pipette tip. 7.4.3 Culture
According to the estimation of the bacterial content of the sample to be tested, select 2 to 3 consecutive appropriate dilutions. Each dilution is maintained in a water bath at 80℃±1℃ for 10 minutes. Pipette 0.2mL of bacterial solution and inoculate it into the nutrient agar plate. Use a spreading stick to spread the inoculum on the agar surface as carefully and quickly as possible. The spreading stick must not touch the edge of the plate. Inoculate 2 plates for each dilution. After spreading, let the plate stand for 10 minutes to allow the inoculum to be completely absorbed by the culture medium. Turn the plate over and place it in a 37℃±1℃ incubator for 48h±2h. 7.4.4 Colony Count
7.4.4.1 Select plates with characteristic colony counts between 20CFU and 200CFU and no spreading colony growth to count the total number of colonies. Plates with less than 20CFU should record the specific colony count, and those with more than 200CFU can be recorded as too many to count. The colony count of each dilution should be the average of the two plates.
7.4.4.2 If one of the plates has large flake colonies growing, it should not be used, and the plate without flake colonies should be used as the colony count of the dilution; if the flake colonies are less than half of the plate, and the colony distribution in the remaining half is very uniform, half of the plate can be counted and multiplied by 2 to represent the colony count of one plate.
7.4.4.3 When chain growth without obvious boundaries between colonies appears on the plate, each single chain is counted as a colony. 7.4.5 Identification
7.4.5.1 Colony selection and pure culture
Select 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them to nutrient broth medium and culture them in an incubator at 37℃±1℃ for 24h±2h for morphological and biochemical identification. 7.4.5.2 Morphological identification
Gram staining and microscopic examination. Bacillus subtilis is a Gram-positive rod, rod-shaped, 1.5um to 3.0μm in length, without capsule, with flagella around the periphery, and buds. The buds are oval, mesogenic or submesogenic, and the bud capsule is not obviously swollen. 7.4.5.3 Biochemical identification
Use a bacterial biochemical identification kit for biochemical identification. The identification characteristics are shown in Table B.3 in Appendix B. 7.5 Calculation of results
7.5.1 Number of Bacillus subtilis on each plate The calculation of the number of Bacillus subtilis colonies on each plate is shown in formula (5): b
Wherein:
The number of Bacillus subtilis colonies on each plate; a
The number of colonies confirmed to be Bacillus subtilis after picking; The number of colonies on the picked plate for verification; C——The number of all characteristic colonies on the plate. 10
·(5)5 Calculation of results
6.5.1 Number of Lactobacillus acidophilus on each plate The calculation of the number of Lactobacillus acidophilus colonies on each plate is shown in formula (3): b
Wherein:
The number of Lactobacillus acidophilus colonies on each plate; a
bThe number of colonies confirmed to be Lactobacillus acidophilus after picking; A——The number of colonies on the picked plate for verification; The number of all characteristic colonies on the plate,
6.5.2 Calculation method of total colony count
(3)
6.5.2.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the 7| of the number of Lactobacillus acidophilus colonies on the two plates. |tt||GB/T34224—2017
Average value, then multiply the average value by the corresponding dilution factor as the total number of colonies per gram (ml) Result 2 If the number of colonies on two consecutive dilution plates is within the appropriate counting range, calculate according to formula (4): 6.5.2.2
Where:
6.6 Report
The number of colonies in the sample;
(n+0.1nz)d
The sum of the number of colonies on the plate (including the plate with the number of colonies in the appropriate range); the number of plates at the first dilution (low dilution factor); the number of plates at the second dilution (high dilution factor); the dilution factor (first dilution).
Result judgment
If the sample colonies meet the characteristics of morphology and biochemical identification of Lactobacillus acidophilus, it can be determined to be Lactobacillus acidophilus. 6.6.2
Result report
6.6.2.1 Qualitative report
Lactobacillus acidophilus was detected or not detected in 25g (mL) sample. 6.6.2.2
Quantitative report
. (4)
When the colony count is less than 100 CFU, report it as an integer. When the colony count is greater than or equal to 100 CFU, round off the third digit and take the first two digits, and replace the digit with 0; it can also be expressed in the form of 10 exponentials with two significant digits. 7 Bacillus subtilis
Equipment and materials
In addition to the routine sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 7.1.1 Constant temperature incubator: 37℃±1℃. 7.1.2 Refrigerator: 2℃~5℃.
7.1.3 Constant temperature water bath: 80℃±1℃. 7.1.4 Balance: Sensitivity is 0.1g.
7.1.5 Homogenizer and sterile homogenizing bag and homogenizing cup. 7.1.6 Oscillator.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and pipette tip. 7.1.7
7.1.8 Sterile conical flask: capacity 250mL, 500mL. 7.1.9 Sterile culture dish: diameter 90mm.
Microscope: 10x~100x.
pH meter or pH colorimetric tube or precision pH test paper. 7.2 Culture medium and reagents
Nutrient agar culture medium: see A.3.
Nutrient broth culture medium: see A.4.
7.2.3 Sterile physiological saline: see A.10.
7.2.4 Gram staining solution: see A.11.
7.2.5 Bacterial biochemical identification kit.
Test procedure
The test procedure for Bacillus subtilis is shown in Figure 3. Sample 25g (mL) + 225mL sterile physiological saline 10-fold serial dilutionwww.bzxz.net
Select 2 to 3 consecutive appropriate dilutions, maintain each dilution in a water bath at 80℃±1℃ for 10 minutes, draw 0.2mL of bacterial solution and inoculate it on a nutrient agar plate
37 C±1 C, 48 h±2 h
Colony count
Pick 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them into nutrient broth culture medium at 37℃±1℃.24h±2h
Morphological identification
Biochemical identification
Figure 3 Bacillus subtilis test procedure
7.4 Operation steps
7.4.1 Sample preparation
Solid and semi-solid samples
GB/T34224—2017
Weigh 25g of sample, put it in a sterile homogenizing bag containing 225mL of normal saline, and beat it with a slapping homogenizer for 1min2min to make a 1:10 sample solution: or put it in a sterile homogenizing cup containing 225mL of sterile normal saline, and homogenize it at 8000r/min~10000/min for 1min~2min to make a 1:10 sample solution. 7.4.1.2 Liquid samples should be shaken thoroughly before mixing. Use a sterile pipette to draw 25 mL of the sample into a sterile conical flask containing 225 mL of normal saline (with an appropriate number of sterile glass beads pre-placed in the flask), shake thoroughly to make a 1:10 sample homogenate. 7.4.2 Sample dilution 7.4.2.1 Use a 1 mL sterile pipette or micropipette to draw 1 mL of 1:10 sample homogenate and slowly inject it along the tube wall into a sterile test tube containing 9 mL of normal saline (be careful not to let the tip of the pipette touch the diluent), shake the test tube or use a sterile pipette to blow and beat repeatedly to mix it evenly, and make a 1:100 sample homogenate. 7.4.2.2 Take another 1mL sterile pipette or micropipette tip and make 10-fold incremental sample dilution according to the above operation sequence. Each time the dilution is incrementally diluted, use a 1mL sterile pipette or pipette tip. 7.4.3 Culture
According to the estimation of the bacterial content of the sample to be tested, select 2 to 3 consecutive appropriate dilutions. Each dilution is maintained in a water bath at 80℃±1℃ for 10 minutes. Pipette 0.2mL of bacterial solution and inoculate it into the nutrient agar plate. Use a spreading stick to spread the inoculum on the agar surface as carefully and quickly as possible. The spreading stick must not touch the edge of the plate. Inoculate 2 plates for each dilution. After spreading, let the plate stand for 10 minutes to allow the inoculum to be completely absorbed by the culture medium. Turn the plate over and place it in a 37℃±1℃ incubator for 48h±2h. 7.4.4 Colony Count
7.4.4.1 Select plates with characteristic colony counts between 20CFU and 200CFU and no spreading colony growth to count the total number of colonies. Plates with less than 20CFU should record the specific colony count, and those with more than 200CFU can be recorded as too many to count. The colony count of each dilution should be the average of the two plates.
7.4.4.2 If one of the plates has large flake colonies growing, it should not be used, and the plate without flake colonies should be used as the colony count of the dilution; if the flake colonies are less than half of the plate, and the colony distribution in the remaining half is very uniform, half of the plate can be counted and multiplied by 2 to represent the colony count of one plate.
7.4.4.3 When chain growth without obvious boundaries between colonies appears on the plate, each single chain is counted as a colony. 7.4.5 Identification
7.4.5.1 Colony selection and pure culture
Select 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them to nutrient broth medium and culture them in an incubator at 37℃±1℃ for 24h±2h for morphological and biochemical identification. 7.4.5.2 Morphological identification
Gram staining and microscopic examination. Bacillus subtilis is a Gram-positive rod, rod-shaped, 1.5um to 3.0μm in length, without capsule, with flagella around the periphery, and buds. The buds are oval, mesogenic or submesogenic, and the bud capsule is not obviously swollen. 7.4.5.3 Biochemical identification
Use a bacterial biochemical identification kit for biochemical identification. The identification characteristics are shown in Table B.3 in Appendix B. 7.5 Calculation of results
7.5.1 Number of Bacillus subtilis on each plate The calculation of the number of Bacillus subtilis colonies on each plate is shown in formula (5): b
Wherein:
The number of Bacillus subtilis colonies on each plate; a
The number of colonies confirmed to be Bacillus subtilis after picking; The number of colonies on the plate for verification; C——The number of all characteristic colonies on the plate. 10
·(5)5 Calculation of results
6.5.1 Number of Lactobacillus acidophilus on each plate The calculation of the number of Lactobacillus acidophilus colonies on each plate is shown in formula (3): b
Wherein:
The number of Lactobacillus acidophilus colonies on each plate; a
bThe number of colonies confirmed to be Lactobacillus acidophilus after picking; A——The number of colonies on the picked plate for verification; The number of all characteristic colonies on the plate,
6.5.2 Calculation method of total colony count
(3)
6.5.2.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the 7| of the number of Lactobacillus acidophilus colonies on the two plates. |tt||GB/T34224—2017
Average value, then multiply the average value by the corresponding dilution factor as the total number of colonies per gram (ml) Result 2 If the number of colonies on two consecutive dilution plates is within the appropriate counting range, calculate according to formula (4): 6.5.2.2
Where:
6.6 Report
The number of colonies in the sample;
(n+0.1nz)d
The sum of the number of colonies on the plate (including the plate with the number of colonies in the appropriate range); the number of plates at the first dilution (low dilution factor); the number of plates at the second dilution (high dilution factor); the dilution factor (first dilution).
Result judgment
If the sample colonies meet the characteristics of morphology and biochemical identification of Lactobacillus acidophilus, it can be determined to be Lactobacillus acidophilus. 6.6.2
Result report
6.6.2.1 Qualitative report
Lactobacillus acidophilus was detected or not detected in 25g (mL) sample. 6.6.2.2
Quantitative report
. (4)
When the colony count is less than 100 CFU, report it as an integer. When the colony count is greater than or equal to 100 CFU, round off the third digit and take the first two digits, and replace the digit with 0; it can also be expressed in the form of 10 exponentials with two significant digits. 7 Bacillus subtilis
Equipment and materials
In addition to the routine sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 7.1.1 Constant temperature incubator: 37℃±1℃. 7.1.2 Refrigerator: 2℃~5℃.
7.1.3 Constant temperature water bath: 80℃±1℃. 7.1.4 Balance: Sensitivity is 0.1g.
7.1.5 Homogenizer and sterile homogenizing bag and homogenizing cup. 7.1.6 Oscillator.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and pipette tip. 7.1.7
7.1.8 Sterile conical flask: capacity 250mL, 500mL. 7.1.9 Sterile culture dish: diameter 90mm.
Microscope: 10x~100x.
pH meter or pH colorimetric tube or precision pH test paper. 7.2 Culture medium and reagents
Nutrient agar culture medium: see A.3.
Nutrient broth culture medium: see A.4.
7.2.3 Sterile physiological saline: see A.10.
7.2.4 Gram staining solution: see A.11.
7.2.5 Bacterial biochemical identification kit.
Test procedure
The test procedure for Bacillus subtilis is shown in Figure 3. Sample 25g (mL) + 225mL sterile physiological saline 10-fold serial dilution
Select 2 to 3 consecutive appropriate dilutions, maintain each dilution in a water bath at 80℃±1℃ for 10 minutes, draw 0.2mL of bacterial solution and inoculate it on a nutrient agar plate
37 C±1 C, 48 h±2 h
Colony count
Pick 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them into nutrient broth culture medium at 37℃±1℃.24h±2h
Morphological identification
Biochemical identification
Figure 3 Bacillus subtilis test procedure
7.4 Operation steps
7.4.1 Sample preparation
Solid and semi-solid samples
GB/T34224—2017
Weigh 25g of sample, put it in a sterile homogenizing bag containing 225mL of normal saline, and beat it with a slapping homogenizer for 1min2min to make a 1:10 sample solution: or put it in a sterile homogenizing cup containing 225mL of sterile normal saline, and homogenize it at 8000r/min~10000/min for 1min~2min to make a 1:10 sample solution. 7.4.1.2 Liquid samples should be shaken thoroughly before mixing. Use a sterile pipette to draw 25 mL of the sample into a sterile conical flask containing 225 mL of normal saline (with an appropriate number of sterile glass beads pre-placed in the flask), shake thoroughly to make a 1:10 sample homogenate. 7.4.2 Sample dilution 7.4.2.1 Use a 1 mL sterile pipette or micropipette to draw 1 mL of 1:10 sample homogenate and slowly inject it along the tube wall into a sterile test tube containing 9 mL of normal saline (be careful not to let the tip of the pipette touch the diluent), shake the test tube or use a sterile pipette to blow and beat repeatedly to mix it evenly, and make a 1:100 sample homogenate. 7.4.2.2 Take another 1mL sterile pipette or micropipette tip and make 10-fold incremental sample dilution according to the above operation sequence. Each time the dilution is incrementally diluted, use a 1mL sterile pipette or pipette tip. 7.4.3 Culture
According to the estimation of the bacterial content of the sample to be tested, select 2 to 3 consecutive appropriate dilutions. Each dilution is maintained in a water bath at 80℃±1℃ for 10 minutes. Pipette 0.2mL of bacterial solution and inoculate it into the nutrient agar plate. Use a spreading stick to spread the inoculum on the agar surface as carefully and quickly as possible. The spreading stick must not touch the edge of the plate. Inoculate 2 plates for each dilution. After spreading, let the plate stand for 10 minutes to allow the inoculum to be completely absorbed by the culture medium. Turn the plate over and place it in a 37℃±1℃ incubator for 48h±2h. 7.4.4 Colony Count
7.4.4.1 Select plates with characteristic colony counts between 20CFU and 200CFU and no spreading colony growth to count the total number of colonies. Plates with less than 20CFU should record the specific colony count, and those with more than 200CFU can be recorded as too many to count. The colony count of each dilution should be the average of the two plates.
7.4.4.2 If one of the plates has large flake colonies growing, it should not be used, and the plate without flake colonies should be used as the colony count of the dilution; if the flake colonies are less than half of the plate, and the colony distribution in the remaining half is very uniform, half of the plate can be counted and multiplied by 2 to represent the colony count of one plate.
7.4.4.3 When chain growth without obvious boundaries between colonies appears on the plate, each single chain is counted as a colony. 7.4.5 Identification
7.4.5.1 Colony selection and pure culture
Select 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them to nutrient broth medium and culture them in an incubator at 37℃±1℃ for 24h±2h for morphological and biochemical identification. 7.4.5.2 Morphological identification
Gram staining and microscopic examination. Bacillus subtilis is a Gram-positive rod, rod-shaped, 1.5um to 3.0μm in length, without capsule, with flagella around the periphery, and buds. The buds are oval, mesogenic or submesogenic, and the bud capsule is not obviously swollen. 7.4.5.3 Biochemical identification
Use a bacterial biochemical identification kit for biochemical identification. The identification characteristics are shown in Table B.3 in Appendix B. 7.5 Calculation of results
7.5.1 Number of Bacillus subtilis on each plate The calculation of the number of Bacillus subtilis colonies on each plate is shown in formula (5): b
Wherein:
The number of Bacillus subtilis colonies on each plate; a
The number of colonies confirmed to be Bacillus subtilis after picking; The number of colonies on the plate for verification; C——The number of all characteristic colonies on the plate. 10
·(5)1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the average of the number of Lactobacillus acidophilus colonies on the two plates, and then multiply the average by the corresponding dilution factor to obtain the total number of colonies per gram (ml). 2 If the number of colonies on two consecutive dilution plates is within the appropriate counting range, calculate according to formula (4): 6.5.2.2
Where:
6.6 Report
The number of colonies in the sample;
(n+0.1nz)d
The sum of the number of colonies on the plates (including the plates with the number of colonies in the appropriate range); the number of plates with the first dilution (low dilution factor); the number of plates with the second dilution (high dilution factor); the dilution factor (first dilution).
Result determination
If the sample colonies meet the morphological and biochemical characteristics of Lactobacillus acidophilus, they can be determined to be Lactobacillus acidophilus. 6.6.2
Result report
6.6.2.1 Qualitative report
Whether Lactobacillus acidophilus is detected or not detected in 25g (mL) sample. 6.6.2.2
Quantitative report
. (4)
When the colony count is less than 100 CFU, report it as an integer. When the colony count is greater than or equal to 100 CFU, round off the third digit and take the first two digits, and replace the digit with 0; it can also be expressed in the form of 10 exponentials with two significant digits. 7 Bacillus subtilis
Equipment and materials
In addition to the conventional sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 7.1.1 Constant temperature incubator: 37℃±1℃. 7.1.2 Refrigerator: 2℃~5℃.
7.1.3 Constant temperature water bath: 80℃±1℃. 7.1.4 Balance: Sensitivity is 0.1g.
7.1.5 Homogenizer and sterile homogenizing bag and homogenizing cup. 7.1.6 Oscillator.
Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or micropipette and pipette tip. 7.1.7
7.1.8 Sterile conical flask: capacity 250mL, 500mL. 7.1.9 Sterile culture dish: diameter 90mm.
Microscope: 10x~100x.
pH meter or pH colorimetric tube or precision pH test paper. 7.2 Culture medium and reagents
Nutrient agar culture medium: see A.3.
Nutrient broth culture medium: see A.4.
7.2.3 Sterile physiological saline: see A.10.
7.2.4 Gram staining solution: see A.11.
7.2.5 Bacterial biochemical identification kit.
Test procedure
The test procedure for Bacillus subtilis is shown in Figure 3. Sample 25g (mL) + 225mL sterile physiological saline 10-fold serial dilution
Select 2 to 3 consecutive appropriate dilutions, maintain each dilution in a water bath at 80℃±1℃ for 10 minutes, aspirate 0.2mL of bacterial solution and inoculate it into a nutrient agar plate
37 C±1 C, 48 h±2 h
Colony counting
Pick 5 colonies with rough, opaque, gray or slightly yellowish surfaces on the plate and transfer them into nutrient broth culture medium at 37℃±1℃.24h±2h
Morphological identification
Biochemical identification
Figure 3 Bacillus subtilis test procedure
7.4 Operation steps
7.4.1 Sample preparation
Solid and semi-solid samples
GB/T34224—2017
Weigh 25g of sample, put it in a sterile homogenizing bag containing 225mL of normal saline, and beat it with a slapping homogenizer for 1min2min to make a 1:10 sample solution; or put it in a sterile homogenizing cup containing 225mL of sterile normal saline, and homogenize it at 8000r/min~10000/min for 1min~2min to make a 1:10 sample solution. 7.4.1.2 Liquid samples should be shaken thoroughly before mixing. Use a sterile pipette to draw 25 mL of the sample into a sterile conical flask containing 225 mL of normal saline (with an appropriate number of sterile glass beads pre-placed in the flask), shake thoroughly to make a 1:10 sample homogenate. 7.4.2 Sample dilution 7.4.2.1 Use a 1 mL sterile pipette or micropipette to draw 1 mL of 1:10 sample homogenate and slowly inject it along the tube wall into a sterile test tube containing 9 mL of normal saline (be careful not to let the tip of the pipette touch the diluent), shake the test tube or use a sterile pipette to blow and beat repeatedly to mix it evenly, and make a 1:100 sample homogenate. 7.4.2.2 Take another 1mL sterile pipette or micropipette tip and make 10-fold incremental sample dilution according to the above operation sequence. Each time the dilution is incrementally diluted, use a 1mL sterile pipette or pipette tip. 7.4.3 Culture
According to the estimation of the bacterial content of the sample to be tested, select 2 to 3 consecutive appropriate dilutions. Each dilution is maintained in a water bath at 80℃±1℃ for 10 minutes. Pipette 0.2mL of bacterial solution and inoculate it into the nutrient agar plate. Use a spreading stick to spread the inoculum on the agar surface as carefully and quickly as possible. The spreading stick must not touch the edge of the plate. Inoculate 2 plates for each dilution. After spreading, let the plate stand for 10 minutes to allow the inoculum to be completely absorbed by the culture medium. Turn the plate over and place it in a 37℃±1℃ incubator for 48h±2h. 7.4.4 Colony Count
7.4.4.1 Select plates with characteristic colony counts between 20CFU and 200CFU and no spreading colony growth to count the total number of colonies. Plates with less than 20CFU should record the specific colony count, and those with more than 200CFU can be recorded as too many to count. The colony count of each dilution should be the average of the two plates.
7.4.4.2 If one of the plates has large flake colonies growing, it
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