Biochemical incubator escherichia coli detection method

Operational details of coliform group determination Coliform group refers to a class of Gram-negative non-spore-forming bacilli that can ferment lactose, produce acid and gas, and are aerobic and facultatively anaerobic. The bacteria mainly come from human and animal feces, so as a fecal contamination indicator, it is used to evaluate the sanitary quality of food and infer whether the food is likely to be contaminated by intestinal pathogens.

The number of coliform groups in food is expressed as the most probable number (MPN) of coliform groups in 100mL (g) of sample.

1 Equipment and materials 1.1 Biochemical incubator: constant temperature 36±1℃.

1.2 Refrigerator: 0～4℃.

1.3 Constant temperature water bath: 44.5±0.5℃.

1.4 Balance.

1.5 Microscope.

1.6 Homogenizer or mortar.

1.7 Plate: diameter 90mm.

1.8 Test tube.

1.9 Pipette.

1.10 Wide-mouth bottle or conical flask: capacity 500mL.

1.11 Glass beads: diameter about 5mm.

1.12 Glass slide.

1.13 Alcohol lamp.

1.14 Test tube rack.

2 Culture medium and reagents 2.1 Lactose bile salt fermentation tube: in accordance with 4.9 of GB 4789.28.

2.2 Eosin-methylene blue agar plate: in accordance with 4.25 of GB 4789.28.

2.3 Lactose fermentation tube: in accordance with 4.10 of GB 4789.28.

2.4 EC broth: in accordance with 4.11 of GB 4789.28.

2.5 Physiological saline: in accordance with 3.22 of GB 4789.28.

2.6 Physiological saline.

2.7 Gram staining solution: as specified in 2.2 of GB 4789.28.

3 Operation steps

3.1 Sample dilution 3.1.1 Place 25 mL (or g) of sample in a sterile glass bottle (with an appropriate number of glass beads) or sterile mortar under sterile conditions, add 225 mL of sterile saline or other diluent, shake or grind thoroughly to make a 1:10 uniform dilution. Solid samples are best treated with a homogenizer at a speed of 8000-10000 r/min for 1 min to make a 1:10 uniform dilution.

3.1.2 Use a 1 mL sterile pipette to draw 1 mL of the 1:10 dilution, inject it into a test tube containing 9 mL of sterile saline or other diluent, shake the test tube to mix, and make a 1:100 dilution.

3.1.3 Take another 1 mL sterile pipette and make 10-fold incremental dilutions according to the above operation. Use a 1 mL sterile pipette to pipette each time the dilution is increased.

3.1.4 According to the requirements of food hygiene standards or the estimation of sample contamination, select 3 dilutions and inoculate 3 tubes for each dilution.

3.2 Lactose fermentation test Inoculate the sample to be tested into a lactose bile salt fermentation tube. If the inoculation volume is above 1 mL, use a double-layer lactose bile salt fermentation tube. If the inoculation volume is below 1 mL, use a single-layer lactose bile salt fermentation tube. Inoculate 3 tubes for each dilution, place them in a 36±1℃ incubator, and culture for 24±2h. If all lactose bile salt fermentation tubes do not produce gas, the coliform group can be reported as negative. If gas is produced, follow the steps below.

3.3 Isolation and culture: Transfer the gas-producing fermentation tube to the eosin-methylene blue agar plate, place it in a 36±1℃ incubator, take it out after culturing for 18-24h, observe the colony morphology, and perform Gram staining and confirmation test.

3.4 Confirmation test: Pick 1-2 suspected coliform colonies on the above plate for Gram staining, and inoculate the lactose fermentation tube at the same time, place it in a 36±1℃ incubator for 24±2h, and observe the gas production. All lactose tubes that produce gas and are Gram-negative and free of bacilli can be reported as coliform-positive.

3.5 Report: According to the number of tubes confirmed to be coliform-positive, check the MPN retrieval table and report the MPN value per 100mL (g) of coliform.

4.1 Use an inoculation loop to transfer all gas-producing lactose bile salt fermentation tube cultures (see 3.2) into EC broth tubes, place them in a 44.5±0.2℃ water bath (the water level in the water bath should be higher than the EC broth liquid level), and culture for 24±2h. After culture, if all EC broth tubes do not produce gas, they can be reported as negative; if there is gas, transfer all gas-producing EC broth tubes to eosin-methylene blue agar plates and culture for 18 to 24h. Any plate with typical colonies is confirmed to be positive for fecal coliforms.

4.2 Result report According to the number of positive tubes confirmed as fecal coliforms, check the MPN retrieval table and report the MPN value per 100mL (g) of fecal coliforms.