SOP: E. coli Detection by MPN Method

Finding E. coli means fecal contamination found its way into the product, which is why water, produce, and dairy labs test for it constantly. The most probable number method gives a statistical estimate from tube patterns when counts are too low for plating.

Microbiological testing protects consumers from pathogens and tracks hygiene across the plant. Because living organisms are the analyte, aseptic technique and precise incubation conditions are not optional extras; they are the measurement itself. A single lapse in sterility or temperature control invalidates the result.

Purpose

To detect and enumerate E. coli in foods and water by the multiple-tube MPN technique with confirmation.

Scope

Applies to water, produce wash water, dairy products, and foods where low-level fecal indicator enumeration is required.

Equipment and Materials

• Lauryl sulfate tryptose (LST) broth tubes with Durham tubes
• Brilliant green lactose bile (BGLB) broth tubes
• EC broth tubes with Durham tubes
• Eosin methylene blue (EMB) agar plates
• Incubators at 35 C and 44.5 C (water bath for EC)
• Sterile diluent blanks and pipettes
• MPN tables (3-tube or 5-tube series)

Procedure

1. Prepare the 1:10 sample homogenate and decimal dilutions as for total plate count.
2. Inoculate 3 (or 5) tubes of LST broth per dilution with 1 mL each. Incubate at 35 C for 24 to 48 hours.
3. Tubes showing gas in the Durham tube are presumptive positive. Transfer a loopful from each positive LST tube to BGLB broth.
4. Incubate BGLB at 35 C for 48 hours. Gas-positive tubes are confirmed coliforms.
5. From positive BGLB tubes, inoculate EC broth and incubate in a 44.5 C water bath for 24 hours. Gas production indicates fecal coliforms.
6. Streak gas-positive EC tubes onto EMB agar. Incubate at 35 C for 24 hours.
7. Pick typical nucleated colonies with metallic sheen and confirm as E. coli by IMViC or a validated rapid method.
8. Read the MPN from the table using the pattern of confirmed positive tubes.

Recording Results

Report as MPN of E. coli per g or per mL with the tube series used (for example “MPN 43 per g, 3-tube series”). Record incubation temperatures, which must be within 0.5 C of target for the EC step.

Quality Control

Verify each new lot of culture media with reference strains before use: the target organism must show typical growth and morphology, and a non-target strain must be inhibited or show atypical growth as expected. Include a positive control (reference strain) and a negative control (sterile diluent) with every test batch. Incubate an uninoculated plate from each poured batch as a sterility check. Monitor incubator temperatures twice daily with a calibrated thermometer; do not use incubators found outside tolerance until corrected and re-verified. Track contamination rates in blanks; a rising blank contamination rate signals a systemic problem requiring investigation. Participate in proficiency testing at least annually and investigate any unsatisfactory scores with documented corrective action.

Troubleshooting

• Weak gas production: old Durham tubes trap air. Use fresh tubes and check for cracks.
• EC water bath temperature drift: the 44.5 C step is selective; even 1 C low lets non-fecal coliforms grow. Calibrate the bath and use a calibrated thermometer.
• Atypical colonies on EMB: confirm with IMViC; some Enterobacter give sheen-like colonies.
• All tubes positive at high dilutions: extend the dilution series next time.

Safety Notes

• E. coli includes pathogenic strains. Work aseptically and treat all cultures as infectious.
• Autoclave all tubes and plates before disposal.
• Disinfect the water bath regularly to prevent biofilm.