Coliform bacteria produce the enzyme β-D-galactosidase which splits the
enzyme substrate 5- bromo-4-chloro-3-indolyl-β-D-galactoside (X-GAL). A colored
end product is produced; if coliforms are present the liquid sample turns a
E. coli produces the enzyme β3 -D-glucuronidase, which breaks down the enzyme
substrate 4- methylumbelliferyl-β-D-g1ucuronide (MUG) to generate an end
product. The sample is illuminated with a UV light at about 365nm; if E. coli is
present the sample will glow a bright blue, fluorescent, color.
- Wash your hands thoroughly with soap and water
- Carefully remove the bottle cap; do not touch the inside of the cap or
bottle opening. Fill with l00mls of the water sample to be tested (about
½" below the neck of the bottle). Do not overfill. Securely recap the
- Shake the bottle vigorously until all the media is dissolved. The
solution should be clear yellow in color (a bit less clear with turbid
- Incubate the sample for:
- After the 24 hour or 48 hour incubation time (based on temperature)
observe the color of the sample:
- Clear yellow negative for coliforms
- Blue-green = positive for coliforms
E. coil Confirmation:
- Shine a UV light (approximately 365nm) from the bottom of the sample.
(Avoid looking directly at the light).
- No fluorescence negative for E. coli
- Bright blue fluorescence = positive for E. coli .
If further confirmation of E. coli is desired add 1 ml of Kovacs
Indole reagent (sold separately) to the sample, recap the bottle, and wait 30
seconds. A red ring will form if E. coli is present.