E. coli detection method

E. coli is a group of fermented lactose, acid-producing gas, aerobic and anaerobic Glucose-negative spore-free bacteria, the bacteria mainly derived from human and animal faeces, so as an indicator of fecal contamination to evaluate the hygienic quality of food, infer whether there is contamination of the intestinal pathogenic bacteria in food. The number of E. coli groups in food is expressed as the most likely number of E. coli groups (MPN) in a 100 ml (g) sample.

2. Instruments and appliances:

2.1 < a href"> thermostat culture box: 36±1 degrees C.

2.2 1000ml triangular cans, 1ml, 10ml pipe.

2.3 vaccination needles.

2.4 < a href"" > microscope .

2.5 super net work table.

2.6 slides, alcohol lamps, ear wash balls, test tube racks.

2.7 < a href"" > balance : sense 0.01g.

2.8 sterilization kettle.

2.9 Petri dish (90mm diameter), test tube, 121 degrees C, 30 minutes sterilization.

2.10 test tube clip, alcohol lamp, stopwatch, slide 3. Culture basereagents:

3.1 lactose bile salt fermentation tube: according to the manufacturer's instructions for preparation, sterilization.

3.2 Ihong Meilanagar tablet: according to the manufacturer's instructions for use to make, sterilization.

3.3 lactose fermentation tube: according to the manufacturer's instructions for preparation, sterilization.

3.4 Terran's staining fluid.

3.4.1 crystalline amethyst:

crystalline purple 1g

95% ethanol 20 ml

1% ammonium grassate aqueous solution 80ml

crystalline azithromy solution dissolved in ethanol, and then mixed with ammonium grass acid solution.

3.4.2 Grady iodine:

iodine 1g

Potassium iodide 2g

Distilled water 300ml

3.4.3 Sand yellow re-dyeing liquid:

sand yellow 0.25g

95% ethanol 10ml

Distilled water 90ml

dissolves sand yellow in ethanol and dilutes it with distilled water.

3.5 physiological saline.

4. Inspection procedures:

5. Measurement method:

5.1 sample dilution:

5.1.1 sterile operation of the sample 25 ml (or 25g) in a sterile plastic berries containing 225 ml of sterilized physiological saline water, fully shaken into 1:10 uniform dilution.

5.1.2 Suck 1:10 dilution 1 ml with a 1 ml sterilization straw and inject it into a test tube containing 9 ml of sterilized physiological saline water, shaking it into 1:100 dilution.

5.1.3 repeat the operation of 5.1.2 to form a 1:1000 dilution.

5.2 Lactose bile salt fermentation test: according to the requirements of food hygiene standards or the estimation of sample contamination, select two dilution, each dilution inoculated 3 tubes, the amount of vaccination in more than 1 ml, with double lactose bile salt fermentation tube. 1 ml and less, fermentation tube with single lactose bile salt. It is then placed in a 36±1 ±C

aquaculture tank and cultured for 24±2hr, which can be reported as coli

syspole negative if all lactose bile salt fermentation tubes are not gas-producing, and if gas-producing persons are produced, they can follow the following procedures.

5.3 separation culture: the gas-producing fermentation tube was inoculated on the Ihong Meilan agar plate, placed in a temperature box of 36±1 degrees C, cultured 24hr and removed, observed the pattern of germs, and made Grady staining.

5.4 confirmed the test: on the above-mentioned plate, pick out 1 to 2 suspected E. coli colonies for Grady dyeing and lactose fermentation tube, placed in a temperature tank of 36±1 degrees C culture 24±2hr, to observe the gas production. Lactose fermentation tube gas production, Glominic dyeing negative spore-free bacteria, can be reported as E. coli group positive.

5.5 report: Based on the number of tubes confirmed to be positive for E. coli, check the MPN search form and report the most likely number per 100 ml (g) E. coli group.

detection method of Staphylococcus acobacteria in food

Staphylococcus aelobacter is an important pathogenic bacteria that causes human and animalized pus infection, and is also one of the common pathogenic bacteria that cause human food poisoning. The bacteria are widely distributed in nature, such as air, soil, water and other environments. The bacteria are also common in the skin of humans and animals and in the cavities connected to the outside world. It is reported that in the normal population of bacteria rate of up to 30% to 80%, of which the skin bacteria rate of 8 to 22%, nasal cavity and throat and other upper respiratory tract bacteria rate of more than 40 to 50%, so it can be through various ways and means, especially by the staff's hands and upper respiratory tract and contaminated food. Because the disease-causing Staphylococcus aracidosis can produce enterotoxins, once bacteria contaminate food, and in the right temperature environment, bacteria can multiply and produce enterotoxins, resulting in food poisoning of consumers.

food poisoning caused by Staphylococcus acobacteria is very common all over the world. In particular, the incidence is higher in North America and Europe. In the above-mentioned countries, every year there are cases of food poisoning caused by Staphylococcus acobacteria, second only to salmonella, and bacterial food poisoning cases ranked 2nd to 3rd, the economic losses caused by this are also quite heavy, in our country caused by Staphylococcus acobacteria food poisoning cases are also reported from time to time, so at present all countries in the world have Staphylococcus acuity for food hygiene statutory testing items.

the current method of Staphylococcus auspsia in China is based on the national standard GB.4789-10-84 and the inspection and quarantine system industry standard SN.0172-92. The final testing process takes about 5 days, which is time-timed and labor-efforts, and causes a backlog of goods, but also affects the timely shipment of goods, and the storage costs of cargo owners increase, resulting in greater economic losses.

many foodmicrobiology laboratories have been exploring and looking for some highly accurate and fast detection methods, and recently we obtained two rapid detection of pathogenic Staphylococcus acurvye media, named:

(1) Petrifilm RSA. Count Plate (developed and produced by 3M Corporation of the United States) is a thin-film, rapid detection of Staphylococcus acobacteria counting plate.

(2) Baird-Parker and RPF Agar. (Staphylococcus abigaus detection count plate developed by French biomerieres with Baird-Parker agar-free plasma fibrinogen).

principle: Petrifilm. Rsa. The test film is made up of two parts. The first part is made up of gold-colored staphylococcus culture tablets, which contain modified Barid-Parker, with nutrients plus gel soluble in cold water. The second part is a thermoenatase (TNase) reaction film containing DNA and toluidine Blue -O and tetraeolium. This indicator helps to count the bacterium and determine the presence of staphylococcus thermoclytic nuclease.

heat-resistant DNAs are typical of the production of toxic glucobacterium, which is very heat-resistant, heats 30min at 100C, is not prone to loss of activity, and its D The value is

16.6min, the molecular weight of this enzyme is 16800, the isoelectric point PH is 9.6, heat-resistant DNA is similar to the detection of plasma coagulation enzyme, is a method of identifying Staphylococcus austratic, in Petrifilm. On the RSA test sheet, the heat-resistant DNA enzyme reaction appears to be a pink band surrounded by a red or blue bacteria.

Petrifilm.RSA test tablets must be used with Peyrifilm thermonase reaction tablets, and use alone will not show the bacterium, as the indicator with auxiliary counting of the bacterium is on the reaction tablet, not on the Petrifilm.RSA test sheet.

experiment implementation:

materials and methods:

this experiment uses the following three reagents:

1. Petrifilm. Rapid