Overview: How to perform MPN tests

Before starting the MPN test, sterile single- and double-strength Phenol Red Lactose broth should be freshly made (MacFaddin)². A 10 times dilution of the water sample should also be prepared with phosphate buffer (0.5 mM K₂HPO₄, pH 7.2). 

The protocol is divided into two dispensing programs, MPN-1 and MPN-2 (Figure 1).

Overview of the steps and corresponding programs:

• Program 1: Dispensing the broth (MPN-1)
• Program 2: Dispensing the samples (MPN-2).

The detailed programs are given in the appendix (Tables 2 and 3, respectively). Both programs need to be manually set up on the DOSE IT peristaltic pump.

Downloads: Protocols for the Most Probable Number method

Here you can download the two programs used in this application in a ZIP file and use them on your DOSE IT. 

Experimental set-up

Step-by-step procedure: Program 1 - Dispensing the broth

1. DOSE IT set-up

Tubing adjustment, priming and calibration for dispensing the broth

Open the pumphead, adjust the tubing clamps and insert the 4.0 mm tubing in the DOSE IT. Put the aspiration tube with the tube collar into the single-strength Phenol Red Lactose broth and the dispensing tube into an empty bottle in order to fill the empty tubing with liquid. Choose MPN-1 from the APPLICATIONS menu and press START to access the program window (Figure 2).

Perform the priming and calibration steps according to the Operating instructions.

2. Dispensing the single-strength broth

Filling 10 tubes with 10 ml single-strength Phenol Red Lactose broth

Put the dispensing tube into the first test tube. Press START. DOSE IT directly dispenses 10 x 10 ml of single strength broth with 2 second intervals between tubes (Step 1). After each dispense, move the dispensing tube into the next empty test tube. 'Waiting time: manual' is displayed on the screen (Step 2) when tube filling has finished. Remove the aspiration tube with the tube collar from the single-strength broth and put the dispensing tube into an empty bottle to drain. Press NEXT STEP. The single-strength broth will be removed from the tubing (Step 3). Press PAUSE once all liquid is drained from the tubing.

3. Dispensing the double-strength broth

Filling five tubes with 10 ml double-strength Phenol Red Lactose broth

Put the aspiration tube with the tube collar into the double-strength broth and the dispensing tube into an empty bottle to refill the empty tubing. Press NEXT STEP (Step 4). Press PAUSE once the tubing is filled with double-strength broth. Put the dispensing tube into the first test tube. Press NEXT STEP (Step 5). DOSE IT dispenses 5 x 10 ml of broth into the tubes with a 2 second break between tubes. During each pause, move the dispensing tube into the next empty test tube. At the end, 'Custom application complete.' will appear on the screen. Press MAIN MENU to continue with program MPN-2.

Tip:

• As the less concentrated broth (single-strength broth) is dispensed before the more concentrated broth (double-strength broth), cleaning of the tubing is not required between the dispensing steps.

Step-by-step procedure: Program 2 - Dispensing the samples

1. DOSE IT set-up

Tubing adjustment, priming and calibration for dispensing the sample

Open the pumphead and remove the 4.0 mm tubing. Adjust the tubing clamps and insert the 2.0 mm tubing into the DOSE IT. Put the aspiration tube with the tube collar into the diluted water sample and the dispensing tube into an empty bottle. Select MPN-2 from the APPLICATIONS menu and press START to access the program window. Priming and calibration should be carried out as described in the Operating instructions.

2. Dispensing the diluted water sample

Addition of 1 ml of diluted water sample to 10 ml of single-strength broth (corresponds to 0.1 ml of original water sample volume – see Figure 1)

Put the dispensing tube into the first test tube containing single-strength broth. Press START. DOSE IT directly dispenses 5 x 1 ml of the diluted water sample with a 2 second interval between tubes (Step 1). During each pause, move the dispensing tube into the next empty test tube. Make sure the dispensing tube doesn't touch the surface of the broth in the tube. When complete, 'Waiting time: manual' will appear on the screen (Step 2). Remove the aspiration tube with the tube collar from the diluted water sample and put the dispensing tube into an empty bottle to drain. Press NEXT STEP. The diluted water sample will be pumped out of the tubing (Step 3). Press PAUSE when it is done.

3. Dispensing the original water sample to the single-strength broth

Addition of 1 ml original water sample to 10 ml single-strength broth (1 ml sample – see Figure 1)

Put the aspiration tube with the tube collar into the undiluted water sample and the dispensing tube into an empty bottle. Press NEXT STEP (Step 4). Press PAUSE when the tubing is filled with the water sample. Put the dispensing tube into the first test tube containing single-strength broth. Press NEXT STEP (Step 5). DOSE IT dispenses 5 x 1 ml of water sample to the tubes with a 2 seconds interval between tubes. During this pause, move the dispensing tube into the next empty test tube. Make sure the dispensing tube doesn't touch the surface of the broth in the tube. Once finished, 'Waiting time: manual' will appear on the screen (Step 6).

4. Dispensing the original water sample to the double-strength broth

Addition of 10 ml original water sample to 10 ml double-strength broth (10 ml sample – see Figure 1)

Put the dispensing tube into the first test tube containing double-strength broth. Press NEXT STEP (Step 7). DOSE IT directly dispenses 5 x 10 ml of water sample to the tubes with 2 seconds between each tube. During this pause, move the dispensing tube into the next empty test tube. Once finished, 'Custom application complete.' will appear on the screen.

Tip:

• As the diluted water sample is dispensed prior to the original water sample, cleaning of the tubing is not required between the dispensing steps.

After adding broth and sample to the tubes, mix and incubate at 37 °C for 24 hours. 

If you have further samples, repeat programs MPN-1 and MPN-2, or adapt the MPN-1 program to fill more tubes in one go, to speed up your process even more.

Remarks

The MPN method can be used to estimate the number of several types of microorganism. For example, it is commonly used in the food industry for estimating the number of Clostridium tyrobutyricum in cheese or Listeria monocytogenes (causing listeriosis) in meat and dairy products. 

How to interpret the results of the MPN test of water samples

In this specific test, Phenol Red Lactose broth is used to detect coliforms in drinking water. It contains phenol red indicator, which is red at pH 7.4 ± 0.2, changing to yellow at acidic pH. Coliforms ferment lactose and produce an acid byproduct. Therefore, when they are present in drinking water, the Phenol Red Lactose broth turns yellow. After incubation, the number of positive test tubes per dilution step should be recorded.

How to use the MPN table

The number of positive (yellow) tubes from the five tubes in the first, second and third dilutions should be noted, then matched to the MPN table (Table 1). Based on the result, an estimation is made on how many bacteria are found in 100 ml of sample.

In the example above (Figure 3), the pattern of positive (yellow) tubes is: 5-2-0, which means that on average approximately 49 bacteria (with the confidence interval: 17-130 bacteria) were in 100 ml original water sample.

¹M. H. McCrady (1918). Tables for Rapid Interpretation of Fermentation-tube Results. The Public Health Journal 9(5): 201-220.

²J. MacFaddin (1985): Cultivation-Identification-Maintenance of Medical Bacteria. Vol.1. Williams and Wilkins, Baltimore, MD.