Detection of E. coli in water bodies

Exploitation principle
the so-called E. coli group, refers to the 37 degrees C
culture
24h can ferment lactose-producing acid, gas-producing and anaerobic glucose-negative Bacillus spores. It is mainly composed of bacteria in four genus of E. coli, namely E. coli genus, Bacillus citric acid genus, Crebb's genus and E. coli genus.
of E. coli in water is the actual number of E. coli groups contained in 100 mL water samples, expressed as the most recent number of E. coli groups (MPN). Under normal circumstances, the intestines are mainly E. coli, streptococcus and anaerobic spores and other bacteria. These bacteria can enter the water source with human and animal excreta, because of the large number of E. coli groups in the intestines, so the number of E. coli groups in water sources is a direct reflection of water pollution by animal excreta an important indicator. At present, it has been recognized internationally that E. coli is an indicator of fecal contamination. Therefore, the drinking water must be examined for E. coli.
detection method of E. coli in water, multi-tube fermentation and membrane method are commonly used. Multi-tube fermentation method can be applied to the inspection of various water samples, but the operation is cumbersome and takes a long time. Membrane method is only applicable to tap water and deep well water, simple and fast operation, but not for water samples with more impurities and easy to clog the filter holes.materials and utensils
(-)
culture base
1, common lactose proteinculture liquid
protein 10g, beef paste 3g, lactose 5g, sodium chloride 5g, 1.6% bromophenol purple ethanol liquid 1.0mL, distilled water 1000mL, pH 7.2 to 7.4.
protein , beef paste, lactose, sodium chloride
heating
dissolved in 1000mL distilled water, adjusted pH of 7.2 to 7.4, and added 1.4 6% bromophenol purple ethanol liquid 1.0mL, fully mixed, divided into duhan's tube
tation tube
, each tube 10mL, 115 degrees C sterilization 20min.
2, triple concentrated lactose proteinculture liquid
in accordance with the above-mentioned ordinary lactose protein culture concentrate three times the preparation, divided into Duhan's tube test tube, each tube 5mL.
1, maelin sodium sulphate culture base (for flat
)10g, Lactose 10g, K2HPO4 3.5g,
agar
15 to 20g, distilled water 1000mL, waterless sodium sulfate 5g, 5% alkaline mammoth ethanol solution 20mL, pH 7.2 to 7.4.
4, EMB culture (EMB culture base) (for fermentation plate separation, 3 and 4 optional)
protein10g, lactose 10g, K2HPO4 2.0g, Agar 20g, distilled water 1000mL, 2% Yihong (Twilight) aqueous solution 20mL, 5% melanin (m8 blue) aqueous solution 13mL, pH 7.2 to 7.4.
(ii) Sterilized pipetting tubes, sterilized diluted water, test tubes, Duhan's tubes, Terrain chromosomals, sterilized petri dishes.the experimental process
(a) water sample collection
water samples for bacteriological testing, must be collected in accordance with the basic requirements of general sterile operation, and ensure that the re-delivery, storage process is not contaminated. Water samples should not exceed 4h from collection to inspection, should not exceed 24h at 0 to 4 degrees C, if not analyzed within 4h, the preservation time and conditions should be indicated on the inspection report.
1, tap water sampling: should be in the tap open water 5min, and then use sterile containers to take water samples, to be analyzed. If the water sample contains residual chlorine, the sample bottle is sterilized and 3% Na2S2O3.5H2O solution 1mL is added to each 500mL water sample.
2, river, river, lake, pool natural water body sampling: available sampler, sampling bottle should be sterilized first. After sampling, there should be a gap in the bottle. If samples are taken jointly with other laboratory items, bacteriologically analyzed water samples should be taken before other samples.
(ii) primary fermentation test
1, water sample dilution: preparation of water sample 10-1, 10-2 dilution, method for using sterile pipe to absorb water sample 10mL put in 90 In the conical bottle of mL sterile water and several glass beads, the mixture is fully oscillated so that the bacteria in it are as present as possible in a single presence, i.e. 10-1 diluent; And so on, dilute to the desired multiply.
2, inoculation and culture: sterile operation in 5 triple concentrated medium (before vaccination must first check whether there are bubbles in the Duhan's tube) each add water sample 10mL, 5 ordinary medium test tubes to add water sample 1mL, 5 ordinary medium test tube to add 10-1 dilution 1mL, carefully mixed into the 37 degrees C culture tank culture 24h. This is the 5-tube method.
3, the results of observation: 37 degrees C culture 24h after removal observation, observation of the availability of gas (Duhan's tube gas) and acid production (culture medium color change). Between 48h, any amount of gas accumulation in the inverted Duhan tube in the culture tube, or the change of the culture base color from purple to yellow, can be initially determined to be positive.
if all 15 tubes tested positive indicate that the thick water sample is seriously polluted, the sample can be further diluted before being inoculated, cultured and observed according to the above method.