The detection of coliform bacteria in water can be applied to (1) the examination of various water samples (2) the identification of indicators of water pollution.
Operation method
Detection of coliform bacteria in water
Principle
The so-called coliforms is a general term for partially anaerobic Gram-negative non-bacteria that can ferment lactose to produce acid and gas within 24h of incubation at 37°C. The bacteria are mainly composed of four genera of Enterobacteriaceae, namely Escherichiaceae, Citrobacter, Klebsiella and Enterobacter. It is mainly composed of bacteria within four genera in the family Enterobacteriaceae, namely Escherichiaceae, Citrobacter, Klebsiella and Enterobacter. The coliform count of water is the actual value of coliform bacteria contained in a 100 mL water sample, expressed as the most proximate number of coliform bacteria (MPN). Under normal conditions, the intestinal tract is dominated by a variety of bacteria including coliforms, Streptococcus faecalis, and Bacillus anaerobicus. All these bacteria can enter the water source with human and animal excreta. Because of the large number of coliform bacteria in the intestinal tract, the number of coliform bacteria in the water source is an important indicator that directly reflects the contamination of the water source by human and animal excreta. At present, it is internationally recognized that coliform is an indicator of fecal contamination. Therefore, drinking water must be checked for coliform bacteria.
Materials and Instruments
Water Sample Move (-) Culture medium 1、Ordinary lactose peptone culture medium Peptone 10g, beef paste 3g, lactose 5g, sodium chloride 5g, 1.6% bromocresol purple ethanol solution 1.0mL, distilled water 1000mL, pH 7.2~7.4. Peptone, beef paste, lactose, sodium chloride heated dissolved in 1000mL distilled water, adjust the pH to 7.2~7.4, then add 1.6% bromocresol violet ethanol solution 1.0mL, mix well, divided into test tubes with Duhamel's tubules, 10mL per tube, 115 ℃ sterilized for 20min. 2、Triple concentrated lactose peptone culture solution Concentrate lactose peptone culture solution three times according to the above, divided into test tubes with Dukhan's tubules, 5mL per tube. 1、 Sodium Magenta Sulfite Medium (for plate separation) Peptone 10g, lactose 10g, K2HPO4 3.5g, agar 15-20g, distilled water 1000mL, anhydrous sodium sulfite 5g, 5% alkaline magenta ethanol solution 20mL, pH 7.2 ~ 7.4. 4, Erythromycin blue medium (EMB medium) (for fermentation method of plate separation, 3 and 4 can choose one) Peptone 10g, lactose 10g, K2HPO4 2.0g, agar 20g, distilled water 1000mL, 2% Eosin (eosin) aqueous solution 20mL, 5% Meilan (methylene blue) aqueous solution 13mL, pH 7.2~7.4. (ii) Sterilized pipettes, sterilized dilution water, test tubes, Doohan's tubules, Gram staining solution, sterilized Petri dishes. Experimental process (I) Collection of water samples For bacteriological testing of water samples, must be collected in accordance with the basic requirements of general aseptic operation, and to ensure that the transportation and storage process is not contaminated. Water samples from the collection to the test should not exceed 4h, in 0 ~ 4 ℃ should not exceed 24h, such as can not be analyzed within 4h, should be indicated in the test report on the preservation of time and conditions. 1, tap water sampling: the tap should be opened to drain 5min, and then use a sterile container to receive water samples to be analyzed. If the water sample contains residual chlorine, the sampling bottle after sterilization of each 500mL water sample plus 3% Na2S2O3.5H2O solution 1mL. 2, rivers, lakes, pools, natural water sampling: available sampler, sampling bottle should be sterilized. After sampling, the bottle should be left empty. If joint sampling with other laboratory projects, bacteriological analysis of water samples should be taken before other samples. (B) primary fermentation test 1, water dilution: the preparation of water samples 10-1, 10-2 dilution, the method is to use a sterile pipette to suck water samples 10mL put in a conical flask containing 90mL of sterile water and a number of glass beads, vibrate fully to make the mixture homogeneous, and so that the bacteria as far as possible to be the presence of a single, that is, for the 10-1 dilution; and then 10-2 dilution of the 10-1 preparation. And so on, diluted to the required times. 2, inoculation and culture: aseptically operated in 5 triple concentrated medium (before inoculation must first check the Dukhan's tube with or without air bubbles) in each of the water samples added 10mL, 5 ordinary medium tube to add water samples 1mL, 5 ordinary medium tube to add 10-1 dilution of 1mL, carefully mixed into the incubator at 37 ℃ incubator culture for 24h. This is the 5-tube method. 3、Observation of results: After 24h incubation at 37℃, take out and observe whether there is gas (whether there is gas in the Doohan's tube) and acid production (whether the medium is discolored). In the 48h period, if there is any amount of gas accumulation in the inverted Duhamel's tubules, or the color of the medium changes from purple to yellow, it can be initially concluded as a positive reaction. Caveat 1. the experiment needs to be averaged over multiple groups for the data to be convincing, in addition, a blank control group is also needed. 2. the experiment should be recorded in the experimental time and place. 3. water samples need to be taken out of the sampler, sampling bottles need to be sterilized before use. Common Problems 1. For the detection of coliform bacteria in water, the multi-tube fermentation method and the filter membrane method are commonly used. Multi-tube fermentation method can be applied to a variety of water samples, but the operation is cumbersome and takes a long time. Filter membrane method is only applicable to tap water and deep well water, the operation is simple and fast, but not applicable to the water samples with more impurities and easy to block the filter holes. 2. If there are positive reactions in all 15 tubes measured in the experiment, it means that the concentrated water samples are seriously contaminated, and the samples can be further diluted and then inoculated, cultured and observed according to the above methods. For more product details, please visit Aladdin Scientific website.
Plain Lactose Peptone Solution Triple Concentrated Lactose Peptone Solution Sodium Magenta Sulfite Medium Erythromycin Blue Medium
Pipettes Conical flasks Test tubes Incubators