Isolation of E. coli from Water or Sewage

!! Safety !!

1. Follow universal lab safety precautions: wear a lab coat and gloves when working in the lab, no open-toed shoes, no eating and drinking.
2. Wash all surfaces every day with freshly made 1% bleach followed by 70% isopropanol.
3. Immediately clean any spills or drips with 1% bleach. Surfaces must stay wet for at least 1 minute.
4. Wash your hands after handling infectious material and before leaving the lab.
5. Minimize generation of aerosols (e.g. uncap and pour slowly, minimize splashing, etc.).
6. Dispose of liquid waste by adding bleach to a final concentration of 10% bleach. Let sit for at least 30 minutes and then decant down the drain while running the tap.
7. Dispose of solid waste by adding 5 ml of 10% bleach to agar plates. Let sit for 30 minutes and then decant any liquid waste down the drain while running the tap. Dispose of solid waste in the trash.
8. For consumables (e.g. tips, loops), soak in 10% bleach in a disposable plastic bottle for at least 30 minutes. When the bottle is full, drain the bleach solution down the drain while running the tap, then cap the bottle and discard in the regular trash.

Protocol Overview

1. (Day 1) Collect water and plate on E. coli & Coliform MC Media Pads. Alternatively, dilute sewage in sterile saline and plate 1 ml on MC Media Pads.
2. (Day 2) Count the total number of coliform (light blue) and E. coli (dark blue) colonies on the MC Media Pads, targeting 25-250 colonies/pad. Subculture E. coli (dark blue) on MacConkey II agar plates for purity testing.
3. (Day 3a) Start the disk diffusion assay on isolates from the MacConkey II agar plates (Disk Diffusion Assay).
4. (Day 3b) Create subcultures in LB broth for banking of isolates (glycerol stocks and DNA).
5. (Day 4a) Record results of the disk diffusion assay (photos and measurements) for later analysis.
6. (Day 4b) Extract DNA and create glycerol stocks to freeze at -20C.

Reagents

Equipment and Consumables

Protocol

Inoculate MC-Media Pads

1. Open the aluminum bag, and remove a MC-Media Pad. You’ll need one pad per sample.
2. Write sample information and the date around the edge of the pad.
3. Open the cover film diagonally.
4. Inoculate with 1 ml of sample (e.g. ocean water, diluted sewage). Sample will diffuse automatically across the surface.
5. Close the cover film and lightly press the edges of the film to seal.
6. Incubate 24 ± 2 hours at 35°C or 48 ±2 hours at room temperature.
7. Re-seal the bag and store at 2–15 °C for up to 4 weeks.
8. Count colonies and record numbers. Target 25-250 colonies per pad. E. coli will be dark blue and coliforms will be light blue.
9. Photograph pads using your phone for documentation.
10. Store the used MC-Media Pads in the fridge for weekly batch processing of E. coli isolates.
11. See the interpretation guide for troubleshooting and more information.

Subculture E. coli Isolates from MC-Media Pads to MacConkey II Agar Plates

1. Decide what E. coli colonies to select for antibiotic susceptibility testing, targeting up to 5 colonies per pad. Assign each an IsolateID and record sample information on the metadata document. On the MC-Media Pad film, circle the colonies and label with the IsolateID. Take a photograph.
2. Divide a McConkey II agar plate into two even sections with a sharpie. Label each section with the date and isolateID.
3. Using a sterile toothpick, touch one circled colony of E. coli and then rhythmically streak the plate in one section, starting at the edge of the plate and moving inward. Do not cross over sector lines or over any streaks.
4. Repeat with other colonies.
5. Incubate plate upside down at 35°C overnight.
6. Inspect your plates the next day and immediately proceed to the Kirby Bauer Disk Diffusion and Banking Protocols.
7. E. coli colonies will be pink to rose-red on MacConkey II agar. This is an example of a mix of E. coli (dark pink colonies) and non-E. coli (light pink colonies).