Determination of Coliforms in Water by Filter Membrane Method
First, the purpose of the request
The colorectal flora in water was determined by the membrane method.
Second, the basic principles
The filter membrane method uses a filter to filter the water sample, and the bacteria are trapped on the filter membrane, and then the filter membrane is placed on an appropriate medium for cultivation, and the coliform group can be directly grown on the membrane, so that it can be directly counted. The filter membrane used is a porous nitrocellulose membrane or a cellulose acetate membrane, which is water-based filtration membrane and has a pore diameter of about 0.45 μm.
Third, test raw materials and equipment
Experimental materials: Yihongmei blue agar plate or Endo’s agar plate, lactose peptone fermentation tube with inverted small casing), sterilized water, river water or lake water;
Equipment: tweezers; clamps; beakers; vacuum pumps; filter membrane; filters, etc.
Fourth, the operation steps
1. Filter membrane sterilization
The filter membrane was placed in a beaker containing distilled water, boiled for 15 minutes by heating, and boiled three times in total. After boiling twice, the water was washed 2-3 times and then boiled to wash away the residual solvent on the filter membrane.
2. Filter device
The sterile filter base, filter membrane, funnel and filter bottle are installed in a sterile procedure, and the filter membrane was transferred to the base of the filter with sterile tweezers (immersed in 95% alcohol and sterilized by flame). Others can be operated directly by hand or with a clamp, but do not touch the rubber stopper part that protrudes into the filter bottle to avoid contamination.
3. Connect the filter bottle to the vacuum pump.
4. Add water sample filtration
For a filter with a diameter of 50 mm, the amount of water to be filtered generally requires a larger number of filter devices, and more than 50 filters are suitable. Therefore, it is preferable to use a multi-filter, which can reduce errors. Generally, 300-500 ml of deep well water or treated river water and lake water can be sampled; 1-100ml can be sampled for relatively clean river water or lake water; severely contaminated water samples can be diluted first; unknown water samples can be sampled. Make three dilutions and choose the appropriate dilution for the number of colonies to calculate. In this experiment, 100 ml of relatively clean river water or lake water was added to the filter funnel and capped.
5. Start the vacuum pump for oil filtration.
6. After pumping, add the same amount of sterile water and continue to filter, in order to flush the funnel wall.
7. After the filtration, close the vacuum pump, use the sterile tweezers to take the edge of the filter, and place the side without bacteria on the Endo agar plate or the Espresso blue agar plate. There must be no air bubbles between the filter and the medium.
8. The plate was inverted and incubated at 37 °C for 22-24 hours.
9. Colonies that meet the characteristics of coliform colonies are picked and subjected to Gram staining and microscopic examination.
10. Microscopically, Gram-negative, Bacillus-free colonies were inoculated with a lactose-protein fermentation tube, and cultured at 37 °C for 24 hours. The acid-producing and gas-producing group was positive for coliform bacteria.