Rapid detection of coliform bacteria (MPN method and plate method) (1)

The coliform group refers to a group of gram-negative non-bacillus bacteria that can ferment lactose, produce acid or aldehyde, and produce β-galactosidase aerobic or facultative anaerobic

Coliform - coliform group is a gram-negative, non-spore, oxidase negative rod-shaped bacteria, aerobic and facultative anaerobic, bile salts can have (or have other surfactants inhibition of growth) is present In the case of growth, it can usually ferment lactose at 36±2°C and produce acid and aldehydes, with β-galactosidase

[Detection principle]

The most probable number (MPN) method-the coliform can produce β-galactosidase, which decomposes the enzyme substrate in the liquid medium-4-methylumbelliferone-β-D-galactoside (hereinafter referred to as MUGal) to free 4-methylumbelliferone

Plate method-coliforms can produce β-galactosidase, which decomposes the enzyme substrate in the medium-Alizarin β-D-galactosidase (hereinafter referred to as Aliz-gal), frees Alizarin and solids The aluminum, potassium, iron, and ammonium ions in the medium combine to form a purple (or red) chelate, which makes the colony appear the corresponding color

[Equipment and materials]

Incubator: (37±1)℃;

Freezer: (0±4)℃;

Balance: Sensitivity 0.

Flat III: Diameter 90mm;

Test tube: 20mm×150mm;

Straws: 1.

Jars, triangular flasks, test tube racks, glass beads, homogenizers, mortars, ultraviolet lamps (wavelength 366nm)

[Medium and Reagents]

(1) Phosphate buffer is prepared according to "CB4789.

(2) Dissolve sodium chloride (8.

(3)MUGal broth

① tryptone peptone 20.

② Sodium chloride 5.

③Anhydrous dipotassium hydrogen phosphate 2.

④ Anhydrous potassium dihydrogen phosphate 2.

⑤ Sodium lauryl sulfate 0.

⑥MUCal (purity not less than 99%) 0.

⑦1000mL of distilled water

Heat the ingredients and dissolve them in distilled water, adjust the pH with 15%-20% sodium hydroxide solution, and divide them into 20mm×150mm test tubes, 9mL per tube, and steam sterilize at 116°C for 10min.
The final pH is 7.
0~7.
2
.
After the culture medium is cooled, add 0.
1 mL of 500ug/mL cefsulodin solution diluted with sterile water to each tube of culture solution or add 1 mL of 5 mg/mL diluted with sterile water to 1000ml sterile culture solution.
mL cefsulodin solution, and dispense test tubes aseptically
.

(4) Aliz-gal agar

① Tryptone or tryptone 20.
0g;

② Sodium chloride 5.
0g;

③Anhydrous dipotassium hydrogen phosphate 2.
75g;

④ Anhydrous potassium dihydrogen phosphate 2.
75g;

⑤ Sodium lauryl sulfate 0.
1g;

⑥Aliz-gal (purity not less than 97%) 0.
05g;

⑦Isopropylthiogalactoside 0.
03g;

⑧0.
5g of potassium aluminum sulfate ;

⑨0.
5g of ferric ammonium citrate ;

⑩Agar 15.
0g;

⑪1000mL of distilled water
.

Put each component into distilled water, heat it to melt, adjust the pH with 15%-20% NaOH, divide into flasks, steam fire at 116°C for 10 minutes, and the final pH is 7.
0-7.
2
.

[Detection Procedure]

See Figure 1-2
.