4.52 Coliform bacteria
4.52.1 Multiple-tube fermentation (MTF)
4.52.1.1 Measurement range
This method is applicable to determination of coliform bacteria.
4.52.1.2 Principle
According to the biological characteristics of coliform bacteria, like negative Gram sporeless bacterium, the cultivation of 24h at 37℃ fermenting lactose and generating gas; inoculating water sample of different amounts into culture medium containing lactose; the positive reaction after cultivation can indicate the MPN of coliform bacteria in the original water samples.
4.52.1.3 Culture medium and reagent
4.52.1.3.1 Lactose bile salt fermenting culture solution
4.52.1.3.1.1 Ingredients
Single-fold Two-fold
Peptone 20.0 g 40.0 g
Pig bile salts (or cattle, sheep bile salts) 5.0 g 10.0 g
Lactose 10.0 g 20.0 g
Bromcresol purple solution (4g/L) 2.5 mL 5.0 mL
Distilled water 1 000 mL 1 000 mL
4.52.1.3.1.2 Preparation: dissolve peptone, bile salt and lactose in distilled water, regulate pH value to 7.4, add bromcresol purple solution and mix; shift 10mL into test tubes separately (each test tube has a small inverted tube). Sterilize at 115℃ for 20min.
4.52.1.3.2 Brilliant green lactose bile salt culture solution (BGB)
4.52.1.3.2.1 Ingredients
Peptone 10.0 g
Lactose 10.0 g
Bile salt 2.0 g
Brilliant green, BGB 0.011 3g
Distilled water 1 000 mL
4.52.1.3.2.2 Preparation: besides brilliant green, add dissolve ingredients into distilled water, regulate pH value to 7.2, add brilliant green and mix; shift 10mL into test tubes separately (each test tube has a small inverted tube); and sterilize at 115℃ for 20min.
4.52.1.4 Apparatus
4.52.1.4.1 Test tube: 150mm×20mm, 140mm×15mm.
4.52.1.4.2 Small inverted tube.
4.52.1.4.3 Quantitive portioner.
4.52.1.4.4 High-pressure sterilizer
4.52.1.4.5 Incubator: 36℃±1℃.
4.52.1.4.6 Refrigerator: 0℃~8℃.
4.52.1.4.7 Balance.
4.52.1.4.8 Microscope.
4.52.1.4.9 Electric oven.
4.52.1.5 Procedure
4.52.1.5.1 The procedures are as shown in Figure 10.
