Methods: E. coli Isolation

For more information on ChromAgar ECC: Alonso JL, et al. Comparison and Recovery of Escherichia coli and Thermotolerant Coliforms in Water with a Chromogenic Medium Incubated at 41 and 44.5°C Appl. Envir. Microbiol 65: 3746-3749

It is essential that the bacteria isolated for DNA fingerprinting be verified as E. coli. Our E. coli isolation and verification procedure is outlined in the flow chart, and described below.

Materials

Initial isolation

Fecal samples: Collect fresh fecal material from the animal using sterile Whirl-Pac bag and tongue depressor or Culturette transport swab system. Record animal information. Process fecal samples within 24 hours, keep at 4°C until processed. Streak fecal material onto mFC agar plates (100x15 mm) for isolation. Incubate at 44.5°C for 24 hours.

Water samples (two day process): Collect water samples in sterile Whirl-Pac bags. Keep at 4°C until processed. Process samples within 6 hours. Analyze sample for fecal coliform on mFC agar by membrane filtration method (Standard Methods 9222 D). We have this done by a certified laboratory.

Pick isolated colonies from mFC plate and re-streak to 100x15 mm mFC agar plates using sterile toothpicks. Incubate at 44.5°C for 24 hours.

Purification

Pick isolated colonies from mFC plates with a sterile toothpick. Streak for isolation onto MacConkey agar plate. Patch onto a ChromAgar ECC plate. Incubate MacConkey and ChromAgar plates at 37°C for 24 hours.

Read MacConkey and ChromAgar plates. Only colonies yielding typical results for E. coli on MacConkey agar (pink to red color with or without a zone of precipitated bile) and ChromAgar ECC (light to dark blue) are further tested by IMViC and EC-MUG tests. [A word of caution about interpreting ChromAgar ECC results: Some E. coli appear white on ChromAgar ECC. We test these isolates further by using API 20E test kits to determine if they are E. coli. Follow package directions.] ChromAgar ECC works well for differentiating between E. coli (blue) and Klebsiella (pink), the two organisms typically selected for by mFC agar.

Prep for culture preservation and IMViC and EC-MUG tests

Pick a well-isolated colony from the MacConkey agar plate using a sterile swab. Swirl the swab into 1 ml of 0.85% sterile saline to create a cell suspension. Use the same swab to completely cover the surface of a PCA plate (for confluent growth), and to patch onto a Simmons citrate agar plate. Inoculate each of the following with a drop of the saline cell suspension:

• 1 tube 1% tryptone broth
• 2 tubes MR-VP broth (we have discontinued the VP test, and use only 1 tube)
• 1 tube EC-MUG broth

Incubate the PCA plate overnight at 37°C. Incubate the tryptone broth, MR-VP broth and citrate plates at 37°C for 48 hours. Incubate the EC-MUG broth at 44.5°C for 48 hours.

Run positive (E. coli) and negative (Klebsiella) controls for the IMViC and EC-MUG tests.

Preserve cultures

Scrape up growth from the 24-hour PCA plate with a sterile swab and swirl in 2-3 ml 50% glycerol to create a heavy cell suspension. Dispense into sterile cryovials and/or 96-well plates. Store at -80°C.

Read IMViC and EC-MUG tests

Read IMViC and EC-MUG tests after 48 hours of incubation.

Consult a bacteriology methods text (e.g. Methods for General and Molecular Bacteriology, ASM Press, 1993; or Standard Methods for the Examination of Water and Wastewater, APHA-AWWA-WEF, 20th ed., 1998) for reagent recipes, specifics about growth media and reagent volumes, and test reactions.

• Indole - Add Kovac's reagent. Positive response red ring on top of culture.
• Methyl red - Add methyl red indicator. Positive response is red color.
• Voges-Proskauer - Add Voges-Proskauer reagents. Positive response red color.
• Citrate - Positive response is blue growth on the plate.

EC-MUG - View EC-MUG tube under long-wavelength (366 nm) UV light. [Our 312 nm transilluminator seems to give better results than a 366 nm source.] Compare fluorescence to positive and negative controls. Examine fermentation tube for gas.

Typical responses for E. coli are:

• Indole +
• Methyl red +
• Voges-Proskauer -
• Citrate -
• EC-MUG
  • fluorescence +
  • gas +

Isolates giving atypical responses to these tests should be further screened using an API 20E test kit or similar system.