Multi -tube fermentation method. Mascular fermentation method is a traditional method for detecting E. coli in water bodies. This method is to continuously cultivate 24h in a medium containing fluorescent substrates at 44.5 ° C, and then it can produce a decomposed fluorescent substrate -glucose glucose The aldehydase, thereby release the fluorescent product, and then cause the medium to produce characteristic fluorescence under ultraviolet light. This method can be used to make statistical estimates on the number of colonies in the original sample. Ferring in the fermentation tube of single or double lactose gallbladder salt, separation training test, use Yonghongmei blue diner, and then perform secondary fermentation in lactose fermentation pipes. Finally , Microscope observation, etc. to complete. The multi -tube fermentation method does not require high test conditions and low cost, but the testing time is long, which is easily disturbed by other conditions, which will affect the accuracy of the measurement results. Extension information: Note: 1. Pay attention to sterile operation when testing. 2. The diluted solution used during dilution can be used with phosphate buffer or physiological saline. The nine tube method can be used during vaccination. The test tube of each dilution should be fully mixed. 3, each experiment requires negative control. rn4、使用小倒管培养基配制时，为排净气泡，灭菌时不要把试管的盖子盖的太紧，灭菌后注意观察一下倒管中的气体是否排完全、r N5, the pressure gauge of the high -pressure sterilization cooker after high pressure sterilization reaches 0, remove the medium and put it in the refrigerator to cool, the effect will be better. Reference information Source: Baidu Encyclopedia-E. coli test tablet Reference materials Source: Baidu Encyclopedia-E. coli n
I. Separational identification of bacteria 1, specimen: Endochemical infections take mid -septum urine, blood, pus, cerebrospinal fluid, etc., diarrhea takes feces. 2. Separation training and identification: Specifications of the stools directly inoculate the selective medium of intestinal bacteria. 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. 37 ° C incubated for 18 to 24 hours, observed colonies and painting and dyeing 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 examination of sanitary bacterial science Perobacterium E. coli is constantly excreted with the 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 number of bacteria: detect the number of bacteria contained in per gram of samples per gram of samples, 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. 2, the number of colorectal bacteria: refers to the number of colonobacteriae in each rising, and the lactose fermentation method is detected. 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 test of the entire automatic microbial quantitative analyzer (TEMPO) n The colonobacteriae counting of automatic microbial quantitative analysis (TEMPO) instrument has two methods: TC and CC. The development of TEMPO TC is to obtain the NF ISO4832 standard quite performance level. tempo CC method is a method for the E. colon population in the Tempo instrument according to the BAM method. 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. Pucting information: Mascular fermentation method is a traditional method for detecting E. coli in water bodies. This method is to continuously cultivate 24h in a medium containing fluorescent substrates at 44.5 ° C, which can be generated after it can be generated. The fluorescence substrate -glucosalinase is released, thereby release the fluorescent product, and then the medium produces characteristic fluorescence under ultraviolet light. This can be used as a statistical estimate of the colonies in the original sample. The tests are generally involved in the following tests: first is fermentation, fermentation in the fermentation tube of single or double lactose gallbladder salt; then separate training tests, use Yonghongmei Blue Drin to separate; then perform secondary in the lactose fermentation tube. Fermented, finally completed through spore dyeing, Gram -dyeing, and microscope observation. Mepomorphic fermentation method does not require high test conditions and low cost, but the testing time is long, which is easily interfered by other conditions, which will affect the accuracy of the measurement result. Reference information Source: Baidu Encyclopedia-E. coli
E. coli group refers to a group of Gram -negative germiosol that can ferment lactose, acidic gas, oxygen and concentrated anaerobic. The bacteria mainly come to human and animal feces, so it is used as an index of feces to evaluate the hygiene quality of food, and it is inferred that the possibility of contaminating intestinal pathogenic bacteria in food is inferred. The inferior of the colorectal flora in food is most likely expressed by 100ml (g) in -sample colorectal flora (MPN). 1 device and material 1.1 temperature box: 36 ± 1 ℃. 1.2 Refrigerator: 0 ～ 4 ℃. 1.3 Constitution water bath: 44.5 ± 0.5 ℃. 1.4 balance. 1.5 Microscope. 1.6 Modifier or milk bowl. 1.7 flat dish: diameter is 90mm. 1.8 test tube. 1.9 straws. 1.10 wide -mouth bottle or triangular bottle: The capacity is 500ml. 1.11 glass beads: diameter is about 5mm. 1.12 glass. 1.13 alcohol lamp. 1.14 IVF. 2 The medium and reagents 2.1 Lava gallbladder salt fermentation tubes: 4.9 in GB 4789.28. 2.2 Yongmei blue agar tablets: 4.25 in GB 4789.28. 2.3 Lactan fermentation tubes: 4.10 in GB 4789.28. 2.4 EC broth: 4.11 in GB 4789.28. . 2.5 phosphate buffer dilution solution: 3.22 in GB 4789.28. 2.6 physiological saline. 2.7 Gram -dyeing solution: 2.2 in GB 4789.28 2.2. 3.1 Dilute sample 3.1.1 Put the sample of 25ml (or g) with a sterile operation in a sterilized glass bottle with 225ml sterilized physiological salt water or other diluted fluid In the appropriate amount of glass beads) or sterilized milk bowl, it is fully shaken or grinded into a uniform diluted solution of 1:10. It is best to use a homogeneous device for solid testing, and treat it at a speed of 8000-10 000 R/min to make 1min and make a uniform diluted solution of 1:10. 3.1.2 Use a 1ml sterilization straw to absorb 1:10 diluted solution and 1ml, inject the test tube containing 9ml sterilized physiological saline or other diluted solution, and shake the tube mix and make 1: 100 diluted solution. . 3.1.3 Take a 1ml sterilization straw, add 10 times the diluted solution according to the above operation, dilute each time, and use 1 ml sterilization straw. 3.1.4 According to the requirements of food hygiene standards or estimates of the pollution of the test, choose three dilution degree, each dilution degree, and inoculate 3 tubes. 3.2 lactose fermentation test In the sample to be checked in the lactose gallbladder fermentation pipe, the amount of inoculation is above 1ml, the two lactose gallbladder salt fermentation tubes, 1ml and 1ml below Salt fermentation tube. Each dilution is inoculated and 36 ± 1 ° C thermostat, and 24 ± 2h. If all lactose gallbladder salt fermentation tubes do not produce gas, it can be reported to the colonic flora negative. Press according to the following procedures. 3.3 Separation Cultivation The fermentation tube of Qi-produced gas is inacheted on Yonghongmei Blue Graphon Flate, set up 36 ± 1 ℃ thermal box, cultivate 18-24h, then take it out to observe the formula of the colonies, and observe the formula shape. And do Gram -dyeing and confirming tests. 3.4 Confirmation of the test On the above tablet, pick up 1-2 suspicious colonobacterial bacteria for Gram-dyeing, and at the same time inoculate the lactose fermentation tube, set up 36 ± 1 ° C in the temperature of the temperature of 24 ± ± ± ± ± 2h, observe gas production. For lactose tubes and Gram -dyeing as negative germ bacon, it can be reported as positive of colonbaccinal groups. 3.5 Report In the number of pipes that are confirmed to be the positive pipeline of colonobacter flora, check the MPN retrieval table, and report the MPN value of each 100ml (g) colon flora. 4 FAECAL Coliform 4.1 Use the inoculation ring to turn all the gas -producing lactose biliary salt fermentation tubes (see 3.2) in the EC broth tube, and place 44.5 ± 0.2 ℃ The water bath (the water in the water bath should be higher than the EC broth liquid surface), and cultivate 24 ± 2h. After training, if all EC broth tubes do not produce gas, it can be reported as negative; For those, the EC broth tubes of Qi produced are inoculated on Yonghongmei Blue Graphon Flate, and they are cultivated for 18-24h. Those who have typical colonies on the tablet are confirmed to be positive for the fecal colonobacterus. 4.2 Results Report In the confirmation of the positive tube of the dung colon flora, check the MPN retrieval form, and report the MPN value of each 100ml (g) the mpn of the dung colon flora.
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If the total number of colonies in food measurement can be measured by reference to GB4789.2, the total number of colonies is measured with a tablet to count agar, and the large intestine is used in VRBA tiger red agar can not measure the colon flora. Growth If materials or dual materials, depending on the product you measured, the amount of samples you need to add, general sample volume> 10ml can choose dual materials
Multi -tube fermentation method.
Mascular fermentation method is a traditional method for detecting E. coli in water bodies. This method is to continuously cultivate 24h in a medium containing fluorescent substrates at 44.5 ° C, and then it can produce a decomposed fluorescent substrate -glucose glucose The aldehydase, thereby release the fluorescent product, and then cause the medium to produce characteristic fluorescence under ultraviolet light. This method can be used to make statistical estimates on the number of colonies in the original sample.
Ferring in the fermentation tube of single or double lactose gallbladder salt, separation training test, use Yonghongmei blue diner, and then perform secondary fermentation in lactose fermentation pipes. Finally , Microscope observation, etc. to complete. The multi -tube fermentation method does not require high test conditions and low cost, but the testing time is long, which is easily disturbed by other conditions, which will affect the accuracy of the measurement results.
Extension information:
Note:
1. Pay attention to sterile operation when testing.
2. The diluted solution used during dilution can be used with phosphate buffer or physiological saline. The nine tube method can be used during vaccination. The test tube of each dilution should be fully mixed.
3, each experiment requires negative control. rn4、使用小倒管培养基配制时，为排净气泡，灭菌时不要把试管的盖子盖的太紧，灭菌后注意观察一下倒管中的气体是否排完全、r N5, the pressure gauge of the high -pressure sterilization cooker after high pressure sterilization reaches 0, remove the medium and put it in the refrigerator to cool, the effect will be better.
Reference information Source: Baidu Encyclopedia-E. coli test tablet
Reference materials Source: Baidu Encyclopedia-E. coli n
I. Separational identification of bacteria
1, specimen: Endochemical infections take mid -septum urine, blood, pus, cerebrospinal fluid, etc., diarrhea takes feces.
2. Separation training and identification: Specifications of the stools directly inoculate the selective medium of intestinal bacteria. 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.
37 ° C incubated for 18 to 24 hours, observed colonies and painting and dyeing 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 examination of sanitary bacterial science
Perobacterium E. coli is constantly excreted with the 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 number of bacteria: detect the number of bacteria contained in per gram of samples per gram of samples, 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.
2, the number of colorectal bacteria: refers to the number of colonobacteriae in each rising, and the lactose fermentation method is detected. 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 test of the entire automatic microbial quantitative analyzer (TEMPO) n The colonobacteriae counting of automatic microbial quantitative analysis (TEMPO) instrument has two methods: TC and CC. The development of TEMPO TC is to obtain the NF ISO4832 standard quite performance level.
tempo CC method is a method for the E. colon population in the Tempo instrument according to the BAM method. 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.
Pucting information:
Mascular fermentation method is a traditional method for detecting E. coli in water bodies. This method is to continuously cultivate 24h in a medium containing fluorescent substrates at 44.5 ° C, which can be generated after it can be generated. The fluorescence substrate -glucosalinase is released, thereby release the fluorescent product, and then the medium produces characteristic fluorescence under ultraviolet light.
This can be used as a statistical estimate of the colonies in the original sample.
The tests are generally involved in the following tests: first is fermentation, fermentation in the fermentation tube of single or double lactose gallbladder salt; then separate training tests, use Yonghongmei Blue Drin to separate; then perform secondary in the lactose fermentation tube. Fermented, finally completed through spore dyeing, Gram -dyeing, and microscope observation.
Mepomorphic fermentation method does not require high test conditions and low cost, but the testing time is long, which is easily interfered by other conditions, which will affect the accuracy of the measurement result.
Reference information Source: Baidu Encyclopedia-E. coli
E. coli group refers to a group of Gram -negative germiosol that can ferment lactose, acidic gas, oxygen and concentrated anaerobic. The bacteria mainly come to human and animal feces, so it is used as an index of feces to evaluate the hygiene quality of food, and it is inferred that the possibility of contaminating intestinal pathogenic bacteria in food is inferred.
The inferior of the colorectal flora in food is most likely expressed by 100ml (g) in -sample colorectal flora (MPN).
1 device and material
1.1 temperature box: 36 ± 1 ℃. 1.2 Refrigerator: 0 ～ 4 ℃. 1.3 Constitution water bath: 44.5 ± 0.5 ℃. 1.4 balance. 1.5 Microscope. 1.6 Modifier or milk bowl. 1.7 flat dish: diameter is 90mm. 1.8 test tube. 1.9 straws. 1.10 wide -mouth bottle or triangular bottle: The capacity is 500ml. 1.11 glass beads: diameter is about 5mm. 1.12 glass. 1.13 alcohol lamp. 1.14 IVF.
2 The medium and reagents
2.1 Lava gallbladder salt fermentation tubes: 4.9 in GB 4789.28.
2.2 Yongmei blue agar tablets: 4.25 in GB 4789.28.
2.3 Lactan fermentation tubes: 4.10 in GB 4789.28.
2.4 EC broth: 4.11 in GB 4789.28.
. 2.5 phosphate buffer dilution solution: 3.22 in GB 4789.28.
2.6 physiological saline.
2.7 Gram -dyeing solution: 2.2 in GB 4789.28 2.2.
3.1 Dilute sample
3.1.1 Put the sample of 25ml (or g) with a sterile operation in a sterilized glass bottle with 225ml sterilized physiological salt water or other diluted fluid In the appropriate amount of glass beads) or sterilized milk bowl, it is fully shaken or grinded into a uniform diluted solution of 1:10. It is best to use a homogeneous device for solid testing, and treat it at a speed of 8000-10 000 R/min to make 1min and make a uniform diluted solution of 1:10.
3.1.2 Use a 1ml sterilization straw to absorb 1:10 diluted solution and 1ml, inject the test tube containing 9ml sterilized physiological saline or other diluted solution, and shake the tube mix and make 1: 100 diluted solution.
. 3.1.3 Take a 1ml sterilization straw, add 10 times the diluted solution according to the above operation, dilute each time, and use 1 ml sterilization straw.
3.1.4 According to the requirements of food hygiene standards or estimates of the pollution of the test, choose three dilution degree, each dilution degree, and inoculate 3 tubes.
3.2 lactose fermentation test
In the sample to be checked in the lactose gallbladder fermentation pipe, the amount of inoculation is above 1ml, the two lactose gallbladder salt fermentation tubes, 1ml and 1ml below Salt fermentation tube. Each dilution is inoculated and 36 ± 1 ° C thermostat, and 24 ± 2h. If all lactose gallbladder salt fermentation tubes do not produce gas, it can be reported to the colonic flora negative. Press according to the following procedures.
3.3 Separation Cultivation
The fermentation tube of Qi-produced gas is inacheted on Yonghongmei Blue Graphon Flate, set up 36 ± 1 ℃ thermal box, cultivate 18-24h, then take it out to observe the formula of the colonies, and observe the formula shape. And do Gram -dyeing and confirming tests.
3.4 Confirmation of the test
On the above tablet, pick up 1-2 suspicious colonobacterial bacteria for Gram-dyeing, and at the same time inoculate the lactose fermentation tube, set up 36 ± 1 ° C in the temperature of the temperature of 24 ± ± ± ± ± 2h, observe gas production. For lactose tubes and Gram -dyeing as negative germ bacon, it can be reported as positive of colonbaccinal groups.
3.5 Report
In the number of pipes that are confirmed to be the positive pipeline of colonobacter flora, check the MPN retrieval table, and report the MPN value of each 100ml (g) colon flora.
4 FAECAL Coliform
4.1 Use the inoculation ring to turn all the gas -producing lactose biliary salt fermentation tubes (see 3.2) in the EC broth tube, and place 44.5 ± 0.2 ℃ The water bath (the water in the water bath should be higher than the EC broth liquid surface), and cultivate 24 ± 2h. After training, if all EC broth tubes do not produce gas, it can be reported as negative; For those, the EC broth tubes of Qi produced are inoculated on Yonghongmei Blue Graphon Flate, and they are cultivated for 18-24h. Those who have typical colonies on the tablet are confirmed to be positive for the fecal colonobacterus.
4.2 Results Report
In the confirmation of the positive tube of the dung colon flora, check the MPN retrieval form, and report the MPN value of each 100ml (g) the mpn of the dung colon flora.
n00:00 / 00: 3070% shortcut keys to describe space: Play / pause ESC: Exit full screen ↑: increase volume 10% ↓: decreases by 10% →: Single fast forward 5 seconds studio Here you can drag no longer appear in the player settings to reopen the small window shortcut key description
If the total number of colonies in food measurement can be measured by reference to GB4789.2, the total number of colonies is measured with a tablet to count agar, and the large intestine is used in VRBA
tiger red agar can not measure the colon flora. Growth
If materials or dual materials, depending on the product you measured, the amount of samples you need to add, general sample volume> 10ml can choose dual materials