Method for detection of coliforms, fecal coliforms and Escherichia coli in food for export

Serial number 0169-92

Replace zbx09 002-86.

1 Subject content and scope of application

This standard specifies the inspection methods for coliforms, fecal coliforms and Escherichia coli in exported food.

This standard is applicable to the inspection of exported food.

2 Equipment and materials

2. 1 pipette: 1mL with a scale of 0.1ml; 5mL and 10mL with 1mL scale.

2 2 Water bath box: 44.5 0.5℃.

2.3 Incubator: 36 65 438 0℃, 44.5 0.5℃.

2.4 refrigerator: 0 ~ 5℃ and-15 ~-20℃.

2.5 homogenizer.

2.6 mortar and grinding rod.

2.7 Plate: 90 mm in diameter.

2.8 balance: sensitivity 0.1g.

2.9 microscope.

2. 10 dilution bottles: 100mL, 200mL and 500mL triangular flasks and jars.

2. 1 1 glass beads: about 5mm in diameter.

2. 12 colony counter.

3 media and reagents

3. 1 dodecyl sulfate pancreatic protein (LST) broth.

3.2 Bright green lactose bile salt (BGLB) broth.

3.3 EC broth.

3.4 Eosin methylene blue agar (EMB).

3.5 nutrient agar slant.

3.6 tryptophan broth.

3.7 MR-VP is medium.

3.8 Coriolis citric acid broth.

3.9 Crystal violet neutral red bile salt agar (VRBA).

3. 10 butterfield phosphate buffer diluent.

3. 1 1 physiological saline.

3. 12 Gram staining.

3. 13 Kovacs indigo matrix reagent.

3. 14 methyl red indicator.

3. 15 Vogus-Proskauer (v-p) reagent.

4 sample preparation

4. 1 Take representative samples for aseptic operation. If there is a package, wipe it with 75% ethanol at the opening and take a sample. If it cannot be inspected in time, the frozen sample should be kept at-65438 05℃; Non-frozen and perishable foods should be stored in a refrigerator at 4℃. Samples frozen before inspection can be thawed within 65438 08 hours at 2-5℃ or within 65438 05 minutes at 45℃.

4.2 Preparation of Homogeneous Solutions of Different Food Samples

4.2. 1 liquid food

Take 25mL sample with sterile straw, put it into a sterile glass bottle with 25ml diluent (glass beads are preset in the bottle), and shake it 25 times within 7s with an amplitude of 30cm (or shake it with a mechanical oscillator) to prepare a homogeneous sample solution of 1: 10.

4.2.2 Solid and Semi-solid Foods

Aseptic operation: Take 25g samples, put them into a sterilized homogenization cup filled with 225mL diluent, and homogenize them at 8000r/min 1 ~ 2min to make 1: 10 sample homogenate (which can also be replaced by grinding in a sterilized mortar).

4.3 Dilution of sample homogenate According to the estimation of sample pollution, the sample homogenate is made into a series of sample dilutions with ten times the amount, such as 10-2, 10-3 and 10-4. The whole process from sample preparation to dilution should not exceed 15 minutes.

5 determination of coliform bacteria

5. Determination of MPN value of1coliforms

5. 1. 1 For each sample, select an appropriate sample dilution and dilute it three times in succession. Inoculate three tubes of LST broth at each dilution, each tube is inoculated with 1 ml.

5.65438 0.2 Incubate the inoculation tube at 36 65438 0℃ for 48±2h.

5. 1.3 Observe the gas production in the test tube: check whether there are bubbles in the inverted tube, and record the number of LST broth tubes that produce gas within 24h and 48h. If all LST broth tubes do not produce gas, they can be reported as Escherichia coli negative; If gas is produced, further confirmation tests will be conducted.

5.2 Confirmation test of coliform bacteria

5.2. 1 transplant all the gas production tubes into bright green lactose bile salt (BGLB) broth tubes with a diameter of 3 mm ..

5.2.2 Place BGLB broth tube and culture at 36 65438 0℃ for 48±2h.

5.2.3 Record the number of gas production pipes of all BGLB broth pipes.

5.2.4 Report of results: According to the number of trachea produced by BGLB broth, check MPN table (see Appendix B (supplement)) and report MPN value of coliform bacteria in each gram (milliliter) sample.

6 Determination of fecal coliforms

6. 1 transplant all LST broth tube cultures that generate gas within 48+/-2h into EC broth tubes with inoculation rings with a diameter of 3 mm ..

6.2 All inoculated EC broth tubes were put into a covered 44.5 0.5℃ water bath box within 30 minutes and cultured for 24±2h hours. The water level of the water bath box should be higher than that of the broth medium. It is known that Escherichia coli that produces gas at 44.5℃ and Enterobacter aerogenes or other coliforms that do not produce gas at 44.5℃ should be used as positive and negative controls.

6.3 Record the gas output of EC broth pipe. Tracheal fecal coliform test was positive; Unproductive tracheal fecal coliform test was negative.

6.4 Result report: According to the number of gas production pipes, check the MPN table and report the MPN value of fecal coliforms in each gram (ml) sample.

7 Determination of Escherichia coli

7. 1 Continue to culture the EC broth tube in 6.3 for 24 hours, take its tracheal culture, streak and inoculate it on EMB plate, and culture it at 36 1℃ for 242 hours.

7.2 Check whether there are typical colonies with black, shiny or dull center on the plate.

7.3 If there are typical colonies, select at least 2 typical colonies from each plate; If there are no typical colonies, at least 2 suspicious colonies should be selected from each plate. Contact the center of the colony with an inoculation needle, transplant it on the inclined plane of nutrient agar, and culture it at 36 1℃ 18 ~ 24h.

7.4 Transfer the slant culture to the following culture medium for biochemical test.

7.4. 1 Tryptophan Broth: After culturing at 36 65438 0℃ for 24±2h hours, add 0.2 ~ 0.3 ml of kovac's reagent, and the upper layer is positive for indigo matrix.

7.4.2 Mr-VP medium; Cultured at 3665438 0℃ for 48 2h. Aseptic operation: transfer the culture from 1mL to 13mm× 100mm, add 0.6mL of 5%ι- naphthol ethanol solution, 0.2mL of 40% potassium hydroxide solution and a small amount of creatine crystals, shake the test tube and stand for 2 hours. If it is red, the VP test is positive.

The remaining Mr-VP cultures were cultured for another 48 hours, and 5 drops of methyl red solution were added dropwise. When the culture turns red, the methyl red test is positive, and when it turns yellow, it is negative.

7.4.3 Kosel Citric Acid Broth: Cultured at 36 65438 0℃ for 96h, and recorded whether it grows or not.

7.4.4 LST broth: incubate at 30 65438 0℃ for 48±2h, and observe whether there is gas in the test tube.

7.4.5 Gram staining: Take 18h nutrient agar slant culture for Gram staining. E. coli is gram-negative.

7.4.6 Biochemical identification of Escherichia coli and non-Escherichia coli is as follows:

Identification of indigo matrix MR VP citrate (type)

++-Typical Escherichia coli

-+-atypical Escherichia coli

+++typical intermediate type

-+-+atypical intermediate type

-++Typical Enterobacter aerogenes

++++atypical Enterobacter aerogenes

If there are biochemical reaction types other than those in the above table, it means that the culture may be impure, so it should be re-streaked and separated, and repeated testing should be carried out if necessary.

7.5 Results Report: Escherichia coli is a Gram-negative bacterium, which ferments lactose to produce acid and gas, and the IMViC test is++-or-+-. Then look up MPN table according to the number of positive tubes in LST broth, and report the MPN value of Escherichia coli in each gram (ml) sample.

Determination of coliform bacteria in solid culture medium

8. 1 Sample preparation is the same as in Chapter 4.

8.2 Counting

8.2. 1 Select an appropriate sample solution diluted three times in succession and inoculate it on two sterilization plates, each time diluting 1 ml. Another lmL diluent was added to the sterile Petri dish as a blank control.

8.2.2 Pour 10 ~ 15 ml of crystallized purple neutral red bile salt agar (VRBA) cooled to 45 0.5℃ into each Petri dish. Carefully rotate the Petri dish and thoroughly mix the culture medium and sample solution.

8.2.3 After the agar is solidified, add 3 ~ 4ml VRBA to cover the surface of the plate.

8.2.4 Turn over the plate and culture at 36 L℃ 18 ~ 24h.

8.2.5 Select the plate with the colony number of 30 ~ 150, and count the typical coliforms appearing on the plate. Typical colonies are purplish red, surrounded by red bile salt precipitation rings. The colony diameter is 0.5 mm or more.

confirm

8.2.6. 1 Select 10 different types of typical and suspicious colonies from VRBA plates, transplant them into BGLB broth tubes, and culture them at 36+1℃ for 24h and 48h to observe the gas production.

8.2.6.2 judged that the broth tube producing gas was positive for Escherichia coli. Gram staining should be carried out on the positive tube forming bacterial membrane to exclude Gram-positive bacilli.

Result report

The percentage of test tubes that are finally confirmed as positive (aerogenes, Gram-negative bacilli) is multiplied by the number of plate colonies counted in Article 8.2.5, and then multiplied by the dilution multiple, that is, the number of coliforms in each gram (milliliter) sample.

Example: 10-4 sample diluent lmL, there are 100 typical and suspicious colonies on VRBA plate, of which 10 is inoculated with BGLB broth tubes, and it is confirmed that there are 6 positive tubes, then the number of coliforms in this sample is:

100× 6/10×104/g (ml) = 6.0× 105/ g (ml)

Annex a

Culture medium and reagents

(Supplement)

A 1 dodecyl sulfate pancreatic protein (LST) broth

Pancreatin or trypsin.

20 grams

Sodium chloride 5.0g.

Lactose 5.0 g

2.75g of dipotassium hydrogen phosphate (K2HPO4)

2.75g of potassium dihydrogen phosphate (KH2P04)

Sodium dodecyl sulfate 0. 1 g

Distilled water 1000.0 ml

Dissolve the components in distilled water. Inverted fermentation tubes were put into 20mm× 150mm test tubes, each tube was 10mL. 12 1℃ autoclaving 15 minutes. The final ph value was 6.8 0.2.

A2 bright green lactose bile salt (BGAB) broth

Peptone10.0g

Lactose 10.0 g

Bovine bile powder (bovine bile or bovine bile) solution 200.0 ml

0. 1% bright green aqueous solution13.3ml.

distilled water

Dissolve peptone lactose in 500mL distilled water, add 200mL bovine bile powder solution (dissolve 20.0g dehydrated bovine bile powder in 200mL distilled water, pH 7.0 ~ 7.5), dilute with distilled water to 975mL, and adjust the pH to 7.4. Then add 0. 1% bright green water solution 13.3mL, make it up to 1000mL with distilled water, filter it with cotton, and pack it in 20 mm× 150 mm test tubes (with inverted small fermentation tubes), each tube is 10mL. 12 1℃ autoclaving 15 minutes. The final ph value was 7.2 0.1.

A3 EC broth

Pancreatic protein or pancreatic cheese

20.0 grams

No.3 bile salt or mixed bile salt 1.5g

Lactose 5.0 g

4.0g of potassium hydrogen phosphate

Potassium dihydrogen phosphate (KH2P04) 1.5g

Sodium chloride 5.0g.

Distilled water 1000.0 ml

Dissolve the above components in distilled water, and subpackage them into 16mm× 150mm test tubes (with inverted small fermentation tubes), each with 8mL.

121autoclaved at℃ 15 minutes, and the final pH was 6.9 0. 1。

A4 eosin methylene blue agar (EMB)

Peptone10.0g

Lactose 10.0 g

2.0g dipotassium hydrogen phosphate (KH2P04)

Agar15.0g

Eosin γ (water-soluble) 0.4g or 20mL 2% aqueous solution.

Methylene blue 0.065g or 0.5% aqueous solution 13mL.

Distilled water 1000.0 ml

Boil peptone, phosphate and agar in 1 000mL distilled water, and add water to make up the original amount. Packed in triangular bottles. 100mL or 200mL per bottle, autoclaved15min. The final ph was 7.10.2. Melt the agar before use, add 5mL of sterilized 20% lactose aqueous solution, 2ml of 2% eosin γ aqueous solution and1.3ml of 0.5% methylene blue aqueous solution to each 100mL agar, shake well, cool to 45 ~ 50℃, and pour into the dish.

A5 nutrient agar slant

3.0 grams of beef sauce

Peptone 5.0 g

Agar15.0g

Distilled water 1000.0 ml

Adding the above ingredients into distilled water, and boiling to dissolve. Sub-package appropriate test tubes. 12 1℃ autoclaving 15 minutes. The final ph value was 7.3 0.1. After sterilization, put it on the inclined plane for later use.

A6 tryptophan broth

Tryptone or Tryptone10.0g.

Distilled water 1000.0 ml

Heating and stirring to dissolve tryptone or tryptone in distilled water. Independent test tubes, 5 ml each. 12 1℃ autoclaving 15 minutes. The final ph value was 6.9 0.2.

A7 MR-VP medium

peptone

7.0 g

Glucose 5.0 g

5.0g of potassium hydrogen phosphate

Distilled water 1000.0 ml

Dissolve the components in distilled water, package them in test tubes, and sterilize at 0℃ 12 15 min, and the final pH is 6.9 0.2.

A8 Kose's citric acid broth

Ammonium hydrogen phosphate sodium (NaNH4HPO4? 6? 14H2O)

Potassium hydrogen phosphate (K2HPO4)1.0g.

Magnesium sulfate (MgSO4? 6? 17H2O) 0.2g

3.0g of sodium citrate (including 2h2o)

Distilled water 1000.0 ml

Dissolve all the components in distilled water, package them in test tubes, and autoclave at 0℃ 10mL, each tube 12 15 min. The final ph value was 6.7 0.2.

A9 crystal violet neutral red bile salt agar (VRBA)

Peptone 7.0 g

Yeast extract 3.0g.

Lactose 10.0 g

Sodium chloride 5.0g.

Bile salt or No.3 bile salt 1.5g

Neutral 0.03 g

Crystal violet 0.002g.

Agar 15 ~ 18g

Distilled water 1000.0 ml

Dissolve the above components in distilled water, stand for a few minutes, fully stir, and adjust the pH to 7.4 0.1. Boil for 2 minutes, cool the culture medium to 45 ~ 50℃ and pour it into a flat plate. When in use, the preparation time should not exceed 3h.

A 1O butterfield phosphate buffer diluent

Store liquid

34.0g of potassium dihydrogen phosphate

500 ml distilled water

Dissolve potassium dihydrogen phosphate in distilled water, and adjust the pH to pH7.2 with about 175ml 1mol/L sodium hydroxide. Add distilled water to 1000mL and store it in the refrigerator. Diluent is 1.25mL stock solution, diluted to 1000mL with distilled water, packed in appropriate containers, and autoclaved at12/℃ 15min.

A 1 1 physiological saline

8.5g sodium chloride

Distilled water 1000.0 ml

Dissolve sodium chloride in distilled water and autoclave at 0℃ 12 15 minutes.

A 12 gram staining

Crystal violet staining solution:

Crystal violet 1.0g

95% ethanol 20.0 ml

1% ammonium oxalate aqueous solution 80. OmL

Crystal violet was completely dissolved in ethanol and then mixed with ammonium oxalate solution.

Gram iodine solution:

Iodine 1.0 g

2.0g of potassium iodide

Distilled water 300.0 ml

First, mix iodine and potassium iodide, add a little distilled water and shake well. After complete dissolution, add distilled water to 300 ml.

Sand yellow dye solution:

Sand yellow 0.25g.

95% ethanol 10.0 ml

Distilled water 90.0 ml

Dissolve yellow sand in ethanol, and then dilute with distilled water.

Dye

A. Fix the smear on the flame, drop crystal violet dye solution, dye for 65438±0min, and wash with water.

B, dropping gram iodine solution for 1 min, and washing.

C, dropwise adding 95% ethanol to decolorize for about15-30s until the dye solution is washed away. Don't over-decolorize, wash with water.

D, dripping a counterstain solution, counterstain for 1 min, washing, drying and microscopic examination.

Results: Gram-positive bacteria were purple and Gram-negative bacteria were red.

Indigo matrix reagent for A 13 Kovacs

P-dimethylaminobenzaldehyde 5.0g.

Pentanol 75.0 ml

Hydrochloric acid (concentrated) 25.0 ml

Dissolve p-dimethylaminobenzaldehyde in amyl alcohol, and then slowly add concentrated hydrochloric acid.

A 14 methyl red indicator

Methyl red 0. 1g

300 ml of 95% ethanol

Dissolve methyl red in 300 ml of ethanol and dilute it to 500 ml with water.

A 15 Vogus-Proskauer (v-p) reagent

Nail fluid

5.0g of A- naphthol

Anhydrous ethanol100.0ml

Liquid b

Potassium hydroxide 40.0g.

Add 100. Soak in distilled water.

Additional record b

The nearest value (MPN) is listed in the 1g sample.

(Supplement)

The inoculation amount was 0. 1, 0.0 1 and 0.00 μ g respectively by three-tube method.

Positive tube number MPN positive tube number MPN

0. 1 0.0 1 0.00 1 0. 1 0.0 1 0.00 1

0 0 0 & lt3 2 0 0 9. 1

0 0 1 3 2 0 1 14

0 0 2 6 2 0 2 20

0 0 3 9 2 0 3 26

0 1 0 3 2 1 0 15

0 1 1 6. 1 2 1 1 20

0 1 2 9.2 2 1 2 27

0 1 3 12 2 1 3 34

0 2 0 6.2 2 2 0 2 1

0 2 1 9.3 2 2 1 28

0 2 2 12 2 2 2 35

0 2 3 16 2 2 3 42

0 3 0 9.4 2 3 0 29

0 3 1 13 2 3 1 36

0 3 2 16 2 3 2 44

0 3 3 19 2 3 3 53

1 0 0 3.6 3 0 0 23

1 0 1 7.2 3 0 1 39

1 0 2 1 1 3 0 2 64

1 0 3 15 3 0 3 95

1 1 0 7.3 3 1 0 43

1 1 1 1 1 3 1 1 75

1 1 2 15 3 1 2 120

1 1 3 19 3 1 3 160

1 2 0 1 1 3 2 0 93

1 2 1 15 3 2 1 150

1 2 2 20 3 2 2 2 10

1 2 3 24 3 2 3 290

1 3 0 16 3 3 0 240

1 3 1 20 3 3 1 460

1 3 2 24 3 3 2 1 100

13329333 >； 1 100

Note: ① Three dilutions [0. 1g(mL), 0.0lg(mL) and 0.00 1g(mL)] were used in this table, and three tubes were inoculated at each dilution.

② If the samples listed in the table are changed to 1g(mL), 0. 18(mL) and 0.0 1g(mL), the numbers in the table should be reduced by 10 times: if it is changed to 0.0 1g(mL)