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.
- mFC agar plates
- Culturette collection and transport swabs
- MacConkey agar plates
- Whirl-Pac bags
- ChromAgar ECC plates
- Sterile tongue depressors
- Simmons Citrate agar plates
- Sterile toothpicks
- Plate count agar (PCA) plates
- Kovac's reagent
- 1% tryptone broth
- Voges-Proskauer reagents
- MR-VP broth
- UV light source
- EC-MUG broth with fermentation tube
- 0.85% saline solution
- 50% glycerol
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.
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
- 1 tube 1% tryptone broth
- 2 tubes MR-VP broth (we have discontinued the VP test, and use only
- 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.
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
- Voges-Proskauer - Add Voges-Proskauer reagents. Positive response
- 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 -
Isolates giving atypical responses to these tests should be further screened
using an API 20E test kit or similar system.