E. coli measurement kit
＞ 【ニュースリリース】簡易・迅速な大腸菌数測定技術の実用化 2020.9.23
Measurement method: Enzyme activity assay
Excitation wavelength: 360 nm
Measurement wavelength: 460 nm
Samples: Sewage, industrial wastewater, river water, groundwater, drinking water
Species reactivity: E.coli
Storage temperature: Store at 2-8 ℃
Expiration date: 6 months after production
Measuring equipment: Fluorescence microplate reader
The plate with E. coli selective freeze-dried medium 96 well × 4
The culture medium for E. coli contains organic and inorganic compounds that promote E. coli growth, as well as a specific fluorogenic enzyme substrate in which a fluorescent molecule is bound to β-D-glucuronide. The specific fluorogenic enzyme substrate dose not emit fluorescence until being degraded by β-D-glucuronidase which is specifically produced by E. coli. Because the fluorescence intensity is proportional to the amount of degradation of the specific fluorogenic enzyme substrate, the change of fluorescence intensity over time (slope) represents the enzyme activity. The enzyme activity is equal to the amount of enzyme, that is, the number of E. coli. The number of coliform bacteria can be measured by using a specific fluorogenic enzyme substrate in which a fluorescent molecule is bound to β-D-galactopyranoside.
Significance of measurement
This kit is for measuring the number of Escherichia coli (E. coli). Currently, the number of E. coli is measured by incubating E. coli for 24 hours on an agar plate or in agar medium. A clean environment is required to prepare the culture medium, and the analyst's subjective may affect the measurement. The kit provides a simple, rapid, and low-cost method for measuring E. coli that overcomes all the shortcomings of existing technologies. In addition, 96 samples can be analyzed simultaneously. This kit is able to measure the number of E. coli in all kinds of liquids including sewage, industrial wastewater, river water, groundwater, and drinking water, etc., and quickly detect fecal contamination that causes waterborne diseases and food poisoning.
(1) Set the plate reader at 37 °C
(2) Make holes in the seal attached to the plate (or remove the seal.)
(3) Dispense 200μL of the test sample into each well of medium-containing 96-well plate.
(4) Set the excitation wavelength at 360 nm and the measurement
wavelength at 460 nm. Measure every 10 minutes for 3 hrs.
J.-B. Burnet 2019.
“Autonomous online measurement of beta-D-glucuronidase activity in surface water: is it suitable for rapid E. coli monitoring?”
Water Research. Vol.152 pp.241-250.
F.J. Ferrero 2016.
“A Novel Handheld Fluorimeter for Rapid Detection of Escherichia coli in Drinking Water.”
IEEE Sensors Journal. Vol.16, pp.5136–5144.
I. George 2000.
“Use of enzymatic methods for rapid enumeration of coliforms in freshwaters.”
Journal of Applied Microbiology. Vol.88, pp.404–413.
N.S.K. Gunda 2016.
“A hydrogel based rapid test method for detection of Escherichia coli (E. coli) in contaminated water samples.”
Analyst. Vol.141, pp.2920–2929.
B. Heery 2016.
“ColiSense, today’s sample today: A rapid on-site detection of β-D-Glucuronidase activity in surface water as a surrogate for E. coli.”
Talanta. Vol.148, pp.75–83.
N. Hesari 2016.
“A biosensor platform for rapid detection of E. coli in drinking water.”
Enzyme and Microbial Technology. Vol.83, pp.22–28
G. Magro 2014.
“Synthesis and Application of Resorufin β-D-Glucuronide, a Low-Cost Chromogenic Substrate for Detecting Escherichia coli in Drinking Water.”
Environmental Science & Technology. Vol.48, pp.9624–9631.
D. Wildeboer 2010.
“Rapid detection of Escherichia coli in water using a hand-held fluorescence detector.”
Water Research. Vol.44, pp.2621–2628.
1.) Hisashi Satoh, Kai Kikuchi, Yutaka Katayose, Shu Tsuda, Reiko Hirano, Yuga Hirakata, Masaaki Kitajima, Satoshi Ishii, Mamoru Oshiki, Masashi Hatamoto, Masahiro Takahashi, Satoshi Okabe (2020) Simple and Reliable Enumeration of Escherichia coli Concentrations in Wastewater Samples by Measuring β-D-glucuronidase (GUS) Activities via a Microplate Reader. Science of The Total Environment. In press
2.) Hisashi SATOH, Shu TSUDA Shu, Kai KIKUCHI Kai, Reiko HIRANO Reiko (2019) “Development of a simple and rapid method for determining coliforms in wastewater based on fluorogenic enzymatic substrate” Journal of Japan Sewage Works Association. 56(684), 110-117.