How to identify E. coli? What is the national standard for E. coli testing

E. coli assay.

Typical E. coli colonies on eosin blue plates were inoculated into nutrient agar plates and cultured at (36 soil 1) for 18 h-24 h. Pick at least 2 colonies per plate. The pure culture of the above colonies was selected and inoculated with biochemical medium and lactose fermentation tube, and the results were observed after (36 Shi 1) 0C culture for 24 h.

Test method for distinguishing Escherichia coli from non-coliform vaccines.

Indigo matrix test:

Add kovacs' indigo matrix reagent dropwise to mix in indigo matrix test medium, let stand, and observe the results.

Those with a red ring are positive; Those with yellow rings are negative.

Methyl Red (MR) test:

Add methyl red indicator M 1 dropwise and mix in MR-VP medium and observe the results.

The presence of red color is positive; The presence of yellow is negative.

VP test:

Add VP reagent solution 0 dropwise2 ml was mixed in MR-VP medium, and then VP reagent B solution was added dropwise to mix, let stand and observe the results. If there is a red color within 15 minutes, it is a positive reaction; Those with no color change are negative.

If the negative result is observed again after 1 hour, the appearance of red color is also a positive reaction.

Simonson's citrate test: observe the color change of the medium, and the blue color is positive; No discoloration is negative.

E. coli identification results.

Indigo matrix MR VP Simonson's citrate identification.

Typical Escherichia coli.

Atypical Escherichia coli.

Typical citrate bacillus.

Atypical citrate bacillus.

Typical Enterobacter aerogenes.

Atypical aerogenic Enterobacter.

If there is a type of biochemical reaction other than that in Table 1, it is an indication that the culture may be impure and should be re-streaked and separated, and repeated tests should be performed if necessary.

Report on the results. The result of the 1m VIC test is ++one.

1. For those who produce gas in 1+11 and lactose fermentation tubes, check the number of trachea-producing tubes of EC broth and its dilution ratio.

number, and reported the Escherichia coli MPN A (ml) value in the sample against the MPN search table in Appendix B (Informational Appendix).

Echina-Melin medium is a type of identification medium used to identify E. coli, which turns purple and has a metallic sheen if E. coli is present.

The red and blue reagent can identify Enterobacteriaceae.

Enterobacteriaceae assay.

Eosin blue plates typical Enterobacter colonies inoculated nutrient agar plates (36 soil 1) culture for 18 h per plate at least.

Legal Analysis]: International Organization for Standardization, Standards for Enterobacter Bacteria in Food.

ISO 16654 AMD 1-2017 Microbiology of food and animal feed. Method for detecting levels of E. coli 0157. Modification 1: Appendix B: Results of laboratory dust research.

ISO 16654 AMD 1-2017 Microbiology of food and animal feed. Method for detecting levels of E. coli 0157. Modification 1: Appendix B: Results of the laboratory's research.

ISO TS 13136-2012 Microbiology of Food and Animal Food. Real-time polymerase chain reaction (PCR)-based methods are used for the detection of foodborne pathogens. Determination of O157, O111, O26, O103 and O145 serogroups and Shiga toxin-producing Escherichia coli (STEC) levels were used.

ISO 16654-2001 Microbiology of Food and Animal Feed Method for Detecting Levels of E. coli 0157.

About food E. coli testing criteria.

MSZ 3620 1-1972 Canned Food Testing: E. coli Content Testing.

MSZ 3612 E. coli test for canned food.

Industry Standard - Commodity Inspection, a standard for food E. coli testing.

SN T 3640-2013 Methods for the detection of diarrhea-causing Escherichia coli in export food by PCR method.

Loop-mediated isothermal amplification (LAMP) detection of pathogenic bacteria in SNT export foods. Part 2: Escherichia coli O157

SN T 0169-2010 Method for the detection of coliforms, fecal coliforms and E. coli in imported and exported foods.

SN T 0973-2010 Method for the detection of enterohaemorrhagic Escherichia coli O157:H7 in imported and exported meat, meat products and other foods.

SNT Methods for the detection of E. coli O157 in food and animal feed. Immunomagnetic bead method.

Legal basis]: Standardization Law of the People's Republic of China Article 2 The standards (including standard samples) mentioned in this law refer to the technical requirements that need to be unified in the fields of agriculture, industry, service industry and social undertakings.

Standards include national standards, banquet industry standards, local standards, group standards, and enterprise standards. National standards are divided into mandatory standards and recommended standards, and industry standards and local standards are recommended standards.

Mandatory standards must be enforced. The State encourages the adoption of recommended standards.

1. Fermentation method.

This method is mainly used to culture E. coli on the lower medium, which contains a fluorescent substrate and needs to be cultured for 24 h. However, the release of the fluorescent substrate requires glucuronic acid to allow the medium to fluoresce under ultraviolet light.

In this way, it is also possible to statistically estimate the colonies in the original sample. The main steps include fermentation, separation and culture, secondary fermentation, microscopic observation, etc.

2. Membrane method.

The main process of the method is to add about 10 ml of sterile water to the filter, then add some sterile water to clean the inner wall of the filter, and then filter, put the filter Kaizai alarm film in M-FC medium, there can be no bubbles between the two, and then seal, store at a temperature of about 24 h, until the flora of E. coli turns blue or blue-green.

The data is then recorded, the number of microflora per unit of aqueous solution is estimated, and the E. coli value is converted.

1.E. coli assay method: Inoculate typical E. coli colonies on eosin blue plates into nutrient agar plates at 36 degrees Celsius.

Incubate for 18 to 24 h.

2.Pick at least 2 colonies per plate. Pretend to be a brother.

3.Pick the pure culture of the above colonies and connect the hall cryptospecies 363 biochemical medium.

and lactose fermentation tubes, incubated at 36 degrees Celsius for 24 h to observe the results.

4.Escherichia coli and non-coli seedlings identification test method Indigo matrix test: Add dropwise add a milliliter of kovacs indigo matrix reagent to mix in indigo matrix test medium, stand still, and observe the results.

5.Those with a red ring are positive;

6.The appearance of yellow rings is the purity of the reaction dust.

7.Escherichia coli is a representative bacterium of the genus Escherichia.

8.Generally, it is not pathogenic, and it is a resident bacteria in the intestines of humans and animals, and can cause extraintestinal infections under certain conditions.

9.Some serotypes are highly pathogenic and cause diarrhea, collectively known as pathogenic E. coli.

Isolation and identification of bacteria.

1. Specimens: Urine, blood, pus, cerebrospinal fluid, etc. are taken from the middle of the intestinal infection, and feces are taken from those with diarrhea.

2. Separation, culture and identification: fecal specimens are directly inoculated with Enterobacteriaceae selective medium. The blood needs to be enriched by broth before transplanting into blood agar plates.

Other specimens can be inoculated with both blood agar plates and Enterobacteriaceae selective medium. 37 After 18 24 h of incubation, observe the colonies and microscopic examination of smear staining. Identification was performed using a series of biochemical reactions.

Enteropathogenic E. coli should be preceded by serotypology testing. Testify for enterotoxins if necessary.

In addition to identifying E. coli, the urinary system should be counted at a level of 100,000 bacteria per milliliter of urine for diagnosis.

Hygienic bacteriological examination.

E. coli is constantly excreted with feces, contaminating the surrounding environment, water sources, food, etc. The more E. coli in the sample, the more likely it is that the sample is contaminated with feces and the greater the likelihood of intestinal pathogens being present in the sample. Therefore, hygienic bacteriological examination should be carried out on drinking water, food, and beverages.

1. Total number of bacteria: the number of bacteria contained in each milliliter or gram of sample is detected, and the pouring culture is used to calculate. China's health standard is that the total number of bacteria per milliliter of drinking water should not exceed 100.

2. Coliform number index: refers to the number of coliform bacteria per liter, which is detected by lactose fermentation method. China's health standard is no more than 3 coliform bacteria per 1000ml of drinking water; Bottled sodas, juices, etc., should not exceed 5 coliform bacteria per 100ml.

Detected with eosin indigo stain, the E. coli community showed a deep purplish-black, smooth, moist, metallic round colony. It has a strong rancid taste.