EC blue for detection of Coliforms and Escherichia coli in waters
Both the Ministry of Health, Labor and Welfare (MHLW) in 2003 and the Japan Waterworks Association (JWWA) in 2001 officially approved EC Blue as one of the official methods for the detection of Escherichia coli and Coliforms in water. EC Blue was developed on the basis of the specific enzyme substrate culture medium method which is used as a specific enzyme substrate for the detection of E. coli and coliforms in water.
Basis of the method.
The substrate of the colorimetric enzyme, Xgal (5-bromo-4-chloro-3-indolyl-β-D galactopyranoside) in the medium is decomposed to highlight blue/blue-green color by β-galactosidase specifically generated by coliforms. Also MUG (4-Methyl-Umbelliferyl-β-Ｄ-Glucuronide) in the medium is decomposed by β-Glucuronidase which generates. E. coli specifically, to isolate a 4-Methyl-Umbelliferone fluorescence substrate. The EC Blue series comes in a special pressurized package (EC Blue 100P), a disposable bottle (EC Blue 100) or a disposable tube (EC Blue 10). These packages are sterilized by electron irradiation; therefore, no preparation of the medium is necessary. The EC Blue 100P test procedure is very simple and just adds it to the test sample. In addition, the EC Blue 100 and 10 test procedure is simple and adds sample water into these bottles (tubes).
Composition of the medium
Per 1 liter ...............................................................................17,4 g
Peptone ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥..... 5.0 g
Sodium chloride ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥.... 5.0 g
Monopotassium phosphate ‥‥‥‥‥‥‥‥‥‥‥‥‥‥..‥‥ 1.0 g
Dipotassium phosphate ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥......‥ 4.0 g
Sodium lauryl sulfate ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥.......‥‥‥‥ 0.1 g
Sodium pyruvate ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥....‥.‥ 1.0 g
Potassium nitrate ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥....‥.‥ 1.0 g
Isopropyl-β-D-thiogalactopyranoside (IPTG) ‥‥‥‥‥‥‥‥‥. 0.1 g
5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal) ‥ 0.1 g
4-Methyl-Umbelliferyl-β-D-glucuronide (MUG) ‥‥‥‥‥‥‥‥ 0.1 g
pH 7,1 ±
Operating Procedure EC Blue 100P "Nissui" for 100 ml qualitative test
1) Separate one piece of the Snap package from the 5 coupling packages.
2) Prepare a sterilized culture tube (bottle). Dispense 100 ml of the clean captured sample from the collection bottle into a sterilized culture tube (bottle).
3) Refer to the figures below and add one packet of EC Blue 100P into the culture tube (bottle). Tighten the cap and shake well to dissolve the medium in the water sample.
4) Judge the color change (blue - blue green) of the medium after 24 hours of incubation at 35 - 37 ℃. When confirming the blue-blue-green color change, check the fluorescence additionally (for detection of E. coli).
Operating procedure EC Blue 100 "Nissui" for qualitative test of 100 ml
1) Prepare EC Blue 100 (bottle) and dispense 100 ml of test sample into the bottle under sterile conditions. The bottle is graduated with a 100 ml mark on the circumference.
2) Squeeze the cap and shake well to dissolve the medium in the water sample.
3) Evaluate the color change (blue - blue green) of the medium after 24 hours of incubation at 35 - 37 ℃. When confirming the blue-blue-green color change, check the fluorescence additionally (for detection of E. coli).
Operating procedure. EC Blue 10 "Nissui" For 10 ml quantitative test (MPN)
1) Prepare a 10-fold dilution series aseptically (refer to the MPN method in the official documents issued).
2) Dispense 10 mL of diluted sample into each tube. The tube is graduated at a 10 mL mark on the circumference.
3) Tighten the cap and shake well to dissolve the medium in the water sample.
Interpretation of results:
1) For coliforms: If the color of the culture medium (blue - blue-green) is darker or the same as the EC Blue color comparator under natural light, determine the test result as "Coliform positive".
2) For E. coliWhen confirming the blue-blue-green color change; check the fluorescence additionally under a 365 nm UV lamp. If the fluorescence of the sample is stronger than or equal to that of the EC Blue comparator with a 365 nm UV lamp, determine the test result as "E. coli positive "
Storage and shelf life:
Keep at room temperature (1-30 ℃), in the dark.
* EC Blue 100P "Nissui" Two (2) years after manufacture
* EC Blue 100 "Nissui" Two (2) years after manufacture
* EC Blue 10 "Nissui" One (1) year after manufacture