3 thoughts on “What method is the national standard of E. coli detection method?”
Hazel
E. coli detection methods are divided into three types: 1, bacterial separation identification method separation culture and identification: stool specimen directly inocularly inocularly inocularly inocated intestinal selective medium. Blood first uses the broth to increase the bacteria, and then plant the blood agar tablet. Other specimens are inoculated at the same time to inoculate the blood agar tablet and intestinal mobilizer selective medium. 37 ℃ incubated for 18-24 hours, observed the colonies of the colonies and dyeing mirror inspection. Intestinal pathogenic E. coli must be first tested for serum science. When necessary, check the intestinal mold toxin. 2, sanitary bacterial examination method E. coli will continue to be excreted with feces, polluting the surrounding environmental water sources and food. During the sampling examination, the more E. coli in the sample, the more serious the sample is polluted by the stool, indicating that the possibility of pathogenic bacteria in the sample is also greater. Neramus index: refers to the number of colonobacteriae in each rising, and the lactose fermentation method is detected. China ’s health standards are not more than 3 colonobacteria every 1000ml of drinking water, and bottle soda and fruit juice should not exceed 5 of the colonic flora per 100ml. 3, the test method of fully automatic microbial quantitative analyzer (TEMPO) The automatic microbial quantitative analysis (TEMPO) messengobacteriae counting test has two methods: TEMPOTC and TEMPOCC. tempotc development is to obtain a considerable performance level to obtain the NF ISO4832 standard. tempocc method is a method for counting the E. coli group method specifically on the Tempo instrument according to the BAM method. The expansion information: The application of E. coli in biotechnology This strains can no longer grow under natural conditions because they lose important components such as cell walls. Even ordinary detergents can easily kill such strains. Even if the operation is accidentally flowing out of the laboratory due to accidental operation, it is not easy to lead to a biochemical crisis. The genomes for biological engineering have been optimized to make it with different genotypes (such as β -galactogenase defect type), which can be better used for molecular cloning experiments. The nucleus genes are expressed in E. coli, and there must be a suitable expression carrier (VECTOR), commonly used carriers: PBV220, PET system. Thestable genes expressed in E. coli: E. coli is more suitable for the expression of prokaryotic genes. The output output of exogenous genes is positively related to the unit volume of the unit. There is a dynamic balance between the between a single cell, which is related to the number of copies of the exogenous genes, the efficiency of gene expression, the stability of the expression of the product, and the cell metabolic load. Reference materials: Baidu Encyclopedia-E. coli
Coli is usually referred to as E. coli and was discovered by Escherich in 1885. For a long period of time, it has been regarded as an integral part of the normal intestinal flora and believes that it is non -pathogenic. It was not until the middle of the 20th century that some special serum type E. coli had pathogenicity to humans and animals, especially for babies and young animals (poultry), which often caused severe diarrhea and sepsis. Different biological characteristics are divided into 6 categories of pathogenic E. coli: E. coli (EPEC), bowel toxic E. coli (ETEC), intestinal invasion E. coli (EIEC), intestinal hemorrhagic E. coli ( EHEC), intestinal adhesion E. coli (EAEC), and diffuse adhesive E. coli (DAEC). E. coli belongs to Gram-negative bacteria (G-). If of the separation of bacteria 1. Simagonia: Outbelary infection of the intestinal tract takes the middle urine, blood, pus, cerebrospinal fluid, etc., diarrhea takes feces. 2. Separation Cultivation and Appraisal: Selective medium of intestinal bacteria in the stool specimen directly. Blood need to use broth to increase bacteria first, and then plant blood agar tablets. Other specimens can be inoculated at the same time to inoculate the selected medium of blood agar and intestinal bacteria. After incubation at 37 ° C for 18 to 24 hours, observe the colonies and paint -dyed mirror inspection. Appraisal with a series of biochemical reactions. Intestinal pathogenic E. coli must be first tested for serum science. If necessary, check the intestinal mold toxin. If the urinary system, in addition to the determination of E. coli, it should also count. When the amount of bacteria contains ≥100,000 per ml, the diagnostic value is only ≥100,000. The sanitary bacterial examination Nebacterium E. coli is constantly excreted with feces, polluting the surrounding environment and water sources, food, etc. During the sampling examination, the more E. coli in the sample, the more serious the sample is polluted by the stool, and it also shows that the possibility of pathogenic bacteria in the sample. Therefore, the sanitary bacterial examination should be performed in response to drinking water, food, and beverages. 1. Total of bacteria: detect the number of bacteria contained in each ml or sample, and use the calculation calculation. The sanitary standards stipulated in China are that the total number of bacteria per ml drinking water must not exceed 100. . The number of colorectal bacteria: refers to the number of colonobacteriae in each rising, and the lactose fermentation method is used to detect. China's sanitary standards must not exceed 3 colonobacteria every 1000ml of drinking water; bottle soda, fruit juice, etc. per 100ml of colonobacteria must not exceed 5. The automatic microbial quantitative analyzer (TEMPO) detection The E. coli alignment analysis (TEMPO) rhobacteriae counting method has two methods: TC and CC. The development of TEMPO TC is to obtain the NF ISO4832 standard quite performance level. The TEMPO CC method is a method that is specially used in Tempo instruments based on the BAM method for 24H to count the E. coli group. The method is to obtain the effect of consistent with AOAC's official method 966.24 and the US Public Health Federation to detect dairy testers (SMEDP). The TEMPO system calculates the number of E. coli groups in the original sample based on the size and number of the positive empty. food detection method The colonobacteria mpn counting method 1 dilute of samples 1.1 solid and semi -solid sample: weigh 25 g samples, put in 225 ml phosphate, In the sterile average cup of buffer or physiological saline, 8000 R/min ～ 10000 R/min average 1 min to 2 min, or placed in 225 mL phosphate buffer or physiological saline In the of the bacteria , beat 1 min ~ 2 min with a hit mortal average, and make a 1:10 sample uniform solution. 1.2 Line sample: Axicated straws with 25 mL samples are placed with 225 mL phosphate buffer or sterile cone bottle with physiological saline ) In the middle, mix it in full and make a 1:10 sample liquid. 1.3 The pH value of the uniform fluid of the sample should be between 6.5 and 7.5. If necessary, use 1 mol/l naOH or 1 mol/L HCL. .4 Use 1 mL sterile straw or trace pipette to absorb 1:10 Sample uniform liquid 1 ml, slowly inject 9 ml phosphate buffer along the tube wall (Note that the straw or the tip of the head should not touch the diluted liquid surface), shake the tube or use 1 mL sterile The bleeding tube repeatedly to make it mixed evenly to make a 1: 100 sample uniform solution. 1.5 According to the estimation of the sample pollution status, according to the above operations, the uniform liquid of the sample is diluted by ten times in order. Dilute each time , and use 1 mL sterile straw or suction head. From the preparation sample liquid to the sample vaccination, the whole process must not exceed 15 min. 2. Preliminary fermentation test Each sample, select 3 suitable continuous dilution sample uniform liquids (can choose raw liquid in liquid samples), each dilution degree 3 Sulfate Tiespel (LST) soup, 1ml per pipe (if the amount of inoculation exceeds 1 ml, use double material lston soup), 36 ℃ ± 1 ℃ to cultivate 24 h ± 2 h to observe whether the inverse tube is There are air bubbles. Those who produce gas at 24 h ± 2 h conducted a recurrence test. Those who are not produced are negative of colonobacteria. 3. Copy and permeable test In the inoculation ring from the LST soup tube of the gas -produced ring. ± 1 ℃ Cultivate 48 h ± 2 h to observe the gas production situation. For those who produce qi, it is included in the colonic colonic group positive tube. . The report of the most likely (MPN) of the colon flora In the number of positive tubes of LST LST by the colonies confirmed by 3, retrieve the MPN table (see Appendix B), and report per G (ml) sample The MPN value of the E. coli. Themes of the colorectal flora tablet 1 Dilute the sample In the previous method to dilute. 2 tablet count 2.1 Select 2 to 3 suitable continuous dilution degree, each dilution degree is vaccinated with 2 sterile dishes, each dish is 1 ml. At the same time, take 1 mL physiological salt This water to add sterile dish for blank control. 2.2 Cold 15 ml to 20 ml of crystalline purple neutral red gallbladder salt agar (VRBA) in time to pour in each flat dish. Be careful Rotate the flat dish, mix the medium with the sample solution. After the agar is solidified, add 3 ml to 4 mlvrba to cover the surface layer. Flip flat plate and place it at 36 ℃ ± 1 ℃ for 18 h to 24 h. 3 Selection of the number of tablet colonies The tablets between the number of colonies between 15 CFU and 150 CFU are selected, and the typical and suspicious colonobacteria icons appear on the tablet. The typical colonies are purple -red, and there is a red gall salt precipitation ring around the colonies. The diameter of the colonies is 0.5 mm or larger. 4 Confirmation of test The typical and suspected colonies of 10 different types of typical and suspected bacteria from the VRBA tablet, migrated in the BGLB soup tube, 36 ℃ ± 1 ℃ H, observe the gas production situation. When the BGLB broth is produced, it can be reported to the colonic population positive.
Separational identification of bacteria 1. Specifications: The intestinal tract This infection takes the middle urine, blood, pus, cerebrospinal fluid, etc., and diarrhea takes feces. 2. Separation Cultivation and Appraisal: Selective medium of intestinal bacteria in the stool specimen directly. Blood need to use broth to increase bacteria first, and then plant blood agar tablets. Other specimens can be inoculated at the same time to inoculate the selected medium of blood agar and intestinal bacteria. After incubation at 37 ° C for 18 to 24 hours, observe the colonies and paint -dyed mirror inspection. Appraisal with a series of biochemical reactions. Intestinal pathogenic E. coli must be first tested for serum science. If necessary, check the intestinal mold toxin. If the urinary system, in addition to the determination of E. coli, it should also count. When the amount of bacteria contains ≥100,000 per ml, the diagnostic value is only ≥100,000. The sanitary bacterial examination Nebacterium E. coli is constantly excreted with feces, polluting the surrounding environment and water sources, food, etc. During the sampling examination, the more E. coli in the sample, the more serious the sample is polluted by the stool, and it also shows that the possibility of pathogenic bacteria in the sample. Therefore, the sanitary bacterial examination should be performed in response to drinking water, food, and beverages. 1. Total of bacteria: detect the number of bacteria contained in each ml or sample, and use the calculation calculation. The sanitary standards stipulated in China are that the total number of bacteria per ml drinking water must not exceed 100. . The number of colorectal bacteria: refers to the number of colonobacteriae in each rising, and the lactose fermentation method is used to detect. China's sanitary standards must not exceed 3 colonobacteria every 1000ml of drinking water; bottle soda, fruit juice, etc. per 100ml of colonobacteria must not exceed 5.
E. coli detection methods are divided into three types:
1, bacterial separation identification method
separation culture and identification: stool specimen directly inocularly inocularly inocularly inocated intestinal selective medium. Blood first uses the broth to increase the bacteria, and then plant the blood agar tablet. Other specimens are inoculated at the same time to inoculate the blood agar tablet and intestinal mobilizer selective medium.
37 ℃ incubated for 18-24 hours, observed the colonies of the colonies and dyeing mirror inspection. Intestinal pathogenic E. coli must be first tested for serum science. When necessary, check the intestinal mold toxin.
2, sanitary bacterial examination method
E. coli will continue to be excreted with feces, polluting the surrounding environmental water sources and food. During the sampling examination, the more E. coli in the sample, the more serious the sample is polluted by the stool, indicating that the possibility of pathogenic bacteria in the sample is also greater.
Neramus index: refers to the number of colonobacteriae in each rising, and the lactose fermentation method is detected. China ’s health standards are not more than 3 colonobacteria every 1000ml of drinking water, and bottle soda and fruit juice should not exceed 5 of the colonic flora per 100ml.
3, the test method of fully automatic microbial quantitative analyzer (TEMPO)
The automatic microbial quantitative analysis (TEMPO) messengobacteriae counting test has two methods: TEMPOTC and TEMPOCC.
tempotc development is to obtain a considerable performance level to obtain the NF ISO4832 standard.
tempocc method is a method for counting the E. coli group method specifically on the Tempo instrument according to the BAM method.
The expansion information:
The application of E. coli in biotechnology
This strains can no longer grow under natural conditions because they lose important components such as cell walls. Even ordinary detergents can easily kill such strains. Even if the operation is accidentally flowing out of the laboratory due to accidental operation, it is not easy to lead to a biochemical crisis.
The genomes for biological engineering have been optimized to make it with different genotypes (such as β -galactogenase defect type), which can be better used for molecular cloning experiments.
The nucleus genes are expressed in E. coli, and there must be a suitable expression carrier (VECTOR), commonly used carriers: PBV220, PET system.
Thestable genes expressed in E. coli:
E. coli is more suitable for the expression of prokaryotic genes. The output output of exogenous genes is positively related to the unit volume of the unit. There is a dynamic balance between the between a single cell, which is related to the number of copies of the exogenous genes, the efficiency of gene expression, the stability of the expression of the product, and the cell metabolic load.
Reference materials: Baidu Encyclopedia-E. coli
Coli is usually referred to as E. coli and was discovered by Escherich in 1885. For a long period of time, it has been regarded as an integral part of the normal intestinal flora and believes that it is non -pathogenic. It was not until the middle of the 20th century that some special serum type E. coli had pathogenicity to humans and animals, especially for babies and young animals (poultry), which often caused severe diarrhea and sepsis. Different biological characteristics are divided into 6 categories of pathogenic E. coli: E. coli (EPEC), bowel toxic E. coli (ETEC), intestinal invasion E. coli (EIEC), intestinal hemorrhagic E. coli ( EHEC), intestinal adhesion E. coli (EAEC), and diffuse adhesive E. coli (DAEC). E. coli belongs to Gram-negative bacteria (G-).
If of the separation of bacteria
1. Simagonia: Outbelary infection of the intestinal tract takes the middle urine, blood, pus, cerebrospinal fluid, etc., diarrhea takes feces.
2. Separation Cultivation and Appraisal: Selective medium of intestinal bacteria in the stool specimen directly. Blood need to use broth to increase bacteria first, and then plant blood agar tablets. Other specimens can be inoculated at the same time to inoculate the selected medium of blood agar and intestinal bacteria. After incubation at 37 ° C for 18 to 24 hours, observe the colonies and paint -dyed mirror inspection. Appraisal with a series of biochemical reactions. Intestinal pathogenic E. coli must be first tested for serum science. If necessary, check the intestinal mold toxin.
If the urinary system, in addition to the determination of E. coli, it should also count. When the amount of bacteria contains ≥100,000 per ml, the diagnostic value is only ≥100,000.
The sanitary bacterial examination
Nebacterium E. coli is constantly excreted with feces, polluting the surrounding environment and water sources, food, etc. During the sampling examination, the more E. coli in the sample, the more serious the sample is polluted by the stool, and it also shows that the possibility of pathogenic bacteria in the sample. Therefore, the sanitary bacterial examination should be performed in response to drinking water, food, and beverages.
1. Total of bacteria: detect the number of bacteria contained in each ml or sample, and use the calculation calculation. The sanitary standards stipulated in China are that the total number of bacteria per ml drinking water must not exceed 100.
. The number of colorectal bacteria: refers to the number of colonobacteriae in each rising, and the lactose fermentation method is used to detect. China's sanitary standards must not exceed 3 colonobacteria every 1000ml of drinking water; bottle soda, fruit juice, etc. per 100ml of colonobacteria must not exceed 5.
The automatic microbial quantitative analyzer (TEMPO) detection
The E. coli alignment analysis (TEMPO) rhobacteriae counting method has two methods: TC and CC. The development of TEMPO TC is to obtain the NF ISO4832 standard quite performance level. The TEMPO CC method is a method that is specially used in Tempo instruments based on the BAM method for 24H to count the E. coli group. The method is to obtain the effect of consistent with AOAC's official method 966.24 and the US Public Health Federation to detect dairy testers (SMEDP). The TEMPO system calculates the number of E. coli groups in the original sample based on the size and number of the positive empty.
food detection method
The colonobacteria mpn counting method
1 dilute of samples
1.1 solid and semi -solid sample: weigh 25 g samples, put in 225 ml phosphate, In the sterile average cup of buffer or physiological saline,
8000 R/min ～ 10000 R/min average 1 min to 2 min, or placed in 225 mL phosphate buffer or physiological saline In the
of the bacteria
, beat 1 min ~ 2 min with a hit mortal average, and make a 1:10 sample uniform solution.
1.2 Line sample: Axicated straws with 25 mL samples are placed with 225 mL phosphate buffer or sterile cone bottle with physiological saline
) In the middle, mix it in full and make a 1:10 sample liquid.
1.3 The pH value of the uniform fluid of the sample should be between 6.5 and 7.5. If necessary, use 1 mol/l naOH or 1 mol/L HCL.
.4 Use 1 mL sterile straw or trace pipette to absorb 1:10 Sample uniform liquid 1 ml, slowly inject 9 ml phosphate buffer along the tube wall
(Note that the straw or the tip of the head should not touch the diluted liquid surface), shake the tube or use 1 mL sterile
The bleeding tube repeatedly to make it mixed evenly to make a 1: 100 sample uniform solution.
1.5 According to the estimation of the sample pollution status, according to the above operations, the uniform liquid of the sample is diluted by ten times in order. Dilute each time
, and use 1 mL sterile straw or suction head. From the preparation sample liquid to the sample vaccination, the whole process must not exceed 15 min.
2. Preliminary fermentation test
Each sample, select 3 suitable continuous dilution sample uniform liquids (can choose raw liquid in liquid samples), each dilution degree 3
Sulfate Tiespel (LST) soup, 1ml per pipe (if the amount of inoculation exceeds 1 ml, use double material lston soup), 36
℃ ± 1 ℃ to cultivate 24 h ± 2 h to observe whether the inverse tube is There are air bubbles. Those who produce gas at 24 h ± 2 h conducted a recurrence test. Those who are not produced are negative of colonobacteria.
3. Copy and permeable test
In the inoculation ring from the LST soup tube of the gas -produced ring. ± 1
℃ Cultivate 48 h ± 2 h to observe the gas production situation. For those who produce qi, it is included in the colonic colonic group positive tube.
. The report of the most likely (MPN) of the colon flora
In the number of positive tubes of LST LST by the colonies confirmed by 3, retrieve the MPN table (see Appendix B), and report per G (ml) sample The
MPN value of the E. coli.
Themes of the colorectal flora tablet
1 Dilute the sample
In the previous method to dilute.
2 tablet count
2.1 Select 2 to 3 suitable continuous dilution degree, each dilution degree is vaccinated with 2 sterile dishes, each dish is 1 ml. At the same time, take 1 mL physiological salt
This water to add sterile dish for blank control.
2.2 Cold 15 ml to 20 ml of crystalline purple neutral red gallbladder salt agar (VRBA) in time to pour in each flat dish. Be careful
Rotate the flat dish, mix the medium with the sample solution. After the agar is solidified, add 3 ml to 4 mlvrba to cover the surface layer. Flip flat
plate and place it at 36 ℃ ± 1 ℃ for 18 h to 24 h.
3 Selection of the number of tablet colonies
The tablets between the number of colonies between 15 CFU and 150 CFU are selected, and the typical and suspicious colonobacteria icons appear on the tablet.
The typical colonies are purple -red, and there is a red gall salt precipitation ring around the colonies. The diameter of the colonies is 0.5 mm or larger.
4 Confirmation of test
The typical and suspected colonies of 10 different types of typical and suspected bacteria from the VRBA tablet, migrated in the BGLB soup tube, 36 ℃ ± 1 ℃
H, observe the gas production situation. When the BGLB broth is produced, it can be reported to the colonic population positive.
Separational identification of bacteria
1. Specifications:
The intestinal tract
This infection takes the middle urine, blood, pus, cerebrospinal fluid, etc., and diarrhea takes feces.
2. Separation Cultivation and Appraisal: Selective medium of intestinal bacteria in the stool specimen directly. Blood need to use broth to increase bacteria first, and then plant blood agar tablets. Other specimens can be inoculated at the same time to inoculate the selected medium of blood agar and intestinal bacteria. After incubation at 37 ° C for 18 to 24 hours, observe the colonies and paint -dyed mirror inspection. Appraisal with a series of biochemical reactions. Intestinal pathogenic E. coli must be first tested for serum science. If necessary, check the intestinal mold toxin.
If the urinary system, in addition to the determination of E. coli, it should also count. When the amount of bacteria contains ≥100,000 per ml, the diagnostic value is only ≥100,000.
The sanitary bacterial examination
Nebacterium E. coli is constantly excreted with feces, polluting the surrounding environment and water sources, food, etc. During the sampling examination, the more E. coli in the sample, the more serious the sample is polluted by the stool, and it also shows that the possibility of pathogenic bacteria in the sample. Therefore, the sanitary bacterial examination should be performed in response to drinking water, food, and beverages.
1. Total of bacteria: detect the number of bacteria contained in each ml or sample, and use the calculation calculation. The sanitary standards stipulated in China are that the total number of bacteria per ml drinking water must not exceed 100.
. The number of colorectal bacteria: refers to the number of colonobacteriae in each rising, and the lactose fermentation method is used to detect. China's sanitary standards must not exceed 3 colonobacteria every 1000ml of drinking water; bottle soda, fruit juice, etc. per 100ml of colonobacteria must not exceed 5.