Testing Coliforms in Food: MPN & Plate Count Methods

Coliforms are not classified bacteriologically but are a term used in the field of hygiene microbiology. They do not refer to a specific bacterium or genus, but rather to a group of bacteria associated with fecal contamination that share certain characteristics, although they are not completely identical biochemically or serologically. Generally, coliform bacteria may include Escherichia coli, Citrobacter species, Enterobacter species, and Klebsiella species. Excessive levels of coliforms can cause diarrhea, gastrointestinal infections, and other related diseases.

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Experimental Principle

Terms and Definitions

• Coliforms: Gram-negative, non-spore-forming rods that are aerobic or facultatively anaerobic and can ferment lactose to produce acid and gas under certain culture conditions.
• Most Probable Number (MPN): An indirect counting method based on the Poisson distribution.

MPN Method

The MPN method is a quantitative detection technique that combines statistics and microbiology. The test sample is subjected to a series of dilutions and cultured. Based on the lowest dilution showing no growth and the highest dilution showing growth, the most probable number of coliforms in the sample is estimated using statistical probability theory.

Plate Count Method

Coliforms ferment lactose and produce acid on solid media. With the action of an indicator, colonies appear in red or purple, with or without precipitate rings, making them countable.

Scope of Application

The MPN (Most Probable Number) method is suitable for counting coliform bacteria in foods with relatively low coliform content; the plate count method is suitable for counting coliform bacteria in foods with relatively high coliform content.

Main Reagents for the Experiment

Lauryl sulfate tryptose (LST) broth, brilliant green lactose bile (BGLB) broth, violet red bile agar (VRBA), sterile phosphate-buffered saline, sterile physiological saline, 1 mol/L NaOH solution, and 1 mol/L HCl solution.

Main Instruments and Equipment for the Experiment

In addition to the conventional sterilization and culture equipment of a microbiology laboratory, the following instruments and materials are required:

• Incubator [(36 ± 1) °C]
• Refrigerator [(2–5) °C]
• Constant temperature water bath [(46 ± 1) °C]
• Balance (sensitivity 0.1 g)
• Homogenizer
• Shaker
• 1 mL sterile pipettes (with 0.01 mL graduations)
• 10 mL or micropipettes (with 0.1 mL graduations) with tips
• 500 mL sterile conical flasks
• Sterile Petri dishes (90 mm diameter)
• pH meter, pH colorimetric tubes, or precise pH test paper
• Colony counter

Coliform MPN Counting Method

The test procedure for coliform MPN counting is shown in Figure 1.

Figure 1 Inspection Procedure for Coliform MPN Enumeration

Experimental Procedure

1. Sample Dilution

1) Solid and Semi-solid Samples: Weigh 25 g of the sample and place it in a sterile homogenization cup containing 225 mL of phosphate-buffered saline (PBS) or physiological saline. Homogenize at 8,000–10,000 r/min for 1–2 min, or place the sample in a sterile homogenization bag containing 225 mL of PBS or physiological saline and homogenize by a stomacher for 1–2 min to prepare a 1:10 sample homogenate.

2) Liquid Samples: Using a sterile pipette, transfer 25 mL of the sample into a sterile conical flask containing 225 mL of PBS or physiological saline (preloaded with an appropriate number of sterile glass beads) or another sterile container. Shake thoroughly by hand or in a mechanical shaker to fully mix, preparing a 1:10 sample homogenate.

3) pH Adjustment: The pH of the sample homogenate should be 6.5–7.5. If necessary, adjust using 1 mol/L NaOH or 1 mol/L HCl.

4) Serial Dilution to 1:100: Using a 1 mL sterile pipette or micropipette, take 1 mL of the 1:10 sample homogenate and slowly dispense it along the inner wall of a sterile test tube containing 9 mL of PBS or physiological saline (avoid touching the liquid surface with the pipette tip). Shake the tube or use a new 1 mL sterile pipette to repeatedly pipette up and down to mix evenly, preparing a 1:100 sample homogenate.

5) Further Serial Dilutions: Based on the estimated contamination level of the sample, prepare a series of 10-fold serial dilutions as described above. Use a new 1 mL sterile pipette or tip for each dilution step. The entire process, from preparing the sample homogenate to inoculation, must not exceed 15 min.

2. Initial Fermentation Test

For each sample, select three appropriately consecutive dilutions of the sample homogenate (liquid samples may use the original sample). Inoculate 1 mL of each dilution into 3 tubes of LST broth per dilution (if the inoculum exceeds 1 mL, use double-strength LST broth). Incubate at (36 ± 1)°C for (24 ± 2) h. Observe whether gas is produced in the inverted tubes. Samples producing gas at (24 ± 2) h undergo a secondary fermentation test (confirmation test). Samples that do not produce gas continue incubation up to (48 ± 2) h; those that produce gas at this point also undergo the confirmation test. Samples that do not produce gas are considered negative for coliforms.

3. Secondary Fermentation Test (Confirmation Test)

Using an inoculating loop, transfer one loopful of culture from the gas-producing LST broth tube into a BGLB tube. Incubate at (36 ± 1) °C for (48 ± 2) h and observe gas production. Tubes producing gas are considered positive for coliforms.

Calculation of Experimental Results

Reporting of Coliforms Most Probable Number (MPN):

Based on the number of confirmed coliform-positive BGLB tubes, refer to the MPN table to report the MPN value of coliforms per gram (g) or milliliter (mL) of the sample.

MPN Table for Coliforms:

The maximum probable number (MPN) of coliforms in 100 g (or mL) of the test sample can be found in Table 1.

Table 1. MPN Table for Coliforms

Precautions for the Experiment

Maintain a sterile environment throughout the operation to avoid contamination that could affect the test results.

Coliform Plate Count Method

The procedure for the coliform plate count method is shown in Figure 2.

Figure 2. Test Procedure for Coliform Plate Count Method

Experimental Procedure

1. Sample Dilution

Sample dilution is performed as described in "1. MPN Method for Coliform Enumeration."

2. Plate Count

Select 2–3 appropriate consecutive dilutions. Inoculate 1 mL of each dilution into 2 sterile Petri dishes per dilution. Also, add 1 mL of physiological saline into a sterile Petri dish as a blank control. Immediately pour 15–20 mL of VRBA, melted and equilibrated to 46 °C, into each dish. Carefully rotate the dishes to mix the medium with the sample thoroughly. After the agar solidifies, overlay each plate with 3–4 mL of VRBA, invert the plates, and incubate at (36 ± 1)°C for 18–24 h.

3. Selection of Plate Colonies

Select plates with 15–150 colonies. Count both typical and suspected coliform colonies (e.g., colonies smaller in diameter than typical colonies). Typical colonies appear purple-red, with a red bile salt precipitate ring around the colony, and a diameter of 0.5 mm or larger. For plates at the lowest dilution with fewer than 15 colonies, record the exact number of colonies.

4. Confirmation Test

Pick 10 different typical and suspected colonies from the VRBA plates. If fewer than 10 colonies are present, pick all typical and suspected colonies. Transfer each colony into a BGLB broth tube. Incubate at (36 ± 1) °C for 24–48 h and observe gas production. Any BGLB tube producing gas is considered positive for coliforms.

Calculation of Results

The number of coliforms per gram (or mL) of sample is calculated by multiplying the proportion of tubes confirmed positive in the final confirmation test by the number of colonies counted on the plate in "(3) Selection of Plate Colonies," and then by the dilution factor.

Example: For a 10^-4 dilution, 1 mL is plated on VRBA, yielding 100 typical and suspected colonies. Ten colonies are selected and inoculated into BGLB broth, and 6 tubes are confirmed positive. The coliform count is calculated as:

100×6/10×10⁴/g(mL)=6.0×10⁵cfu/g(mL)

If no colonies grow on plates at all dilutions (including the original liquid sample), the count is reported as<1 multiplied by the lowest dilution factor.

Precautions

Maintain aseptic conditions throughout the procedure to avoid contamination, which could affect the accuracy of the results.