Posted on September 30th, 2021

Why MPN works

The most probable number method of enumeration estimation works because the distribution of microorganism enumeration data is based on Poisson statistics. Poisson is a distribution of probabilities, meaning that each interval of time represents the distribution of probabilities of an event occurring.

Note that the MPN method is an estimate of microorganism enumeration. However, given the variables affecting the direct enumeration of microorganisms, the direct count is also an estimation of the microbial count. It is only by gathering many data points that bioburden can begin to be defined. Nonetheless, MPN bioburden is a valuable and flexible method.

Uses of MPN

There are four uses for the MPN method(Sutton,S, 2010.):

• Extensive use in QC microbiology (e.g., media qualification).

• It is used as the source for D-value determination using a fraction negative-based calculation.

• Used for validation of rapid microbiological methods

• Used for the evaluation of environmental monitoring data.

Use of Fraction Negative in determining D-value

The terms decimal reduction time, log reduction, and d-value represent a means of measuring microorganism resistance through logarithmic reduction of the microorganism population, typically by using a sterilization process. The decimal reduction time (D10) may be determined by any of several methods, including the limited Spearman-Karber procedure (LSKP), the Stumbo-Murphy-Cochran procedure (SMCP), and the survival-curve method (EN).

The most popular method of determining the D-value using MPN is the Stumbo Murphy and Cochran procedure (SCMP). In employing the SCMP method, Halvorsen and Ziegler utilized the MPN method to estimate the concentration of spores surviving replicate sets of samples (Anellis, A and Werkowski, S.,1968). The MPN method involves taking the natural log of the samples tested and dividing it by the number of negative samples. If a SIP is used, the equation is multiplied by the inverse of the decimal value of the SIP.

The Limited Spearman Karber Method (LSKM) is considered statistically sound and preferable to other calculations. SCMP was criticized for its inaccuracy in a presentation delivered at an ISO meeting, but this was not published. This encouraged Japanese delegates to the ISO meeting to compare the accuracies of D10 values obtained with these methods by simulation and in experimental procedures using a biological indicator (BI) and a biological indicator evaluator resistometer (BIER) with ethylene oxide gas sterilization. The results indicated that the SMCP was not consistently less accurate than the LSKP in determining D10 (Takahashi,1995).

While there has been plenty of discussion on the accuracy of fraction negative methods, it is essential to evaluate such practices within the framework of their intended use. The use of the Takahashi, overkill method often allows for such a conservative approach to process definition that the accuracy that may be sacrificed in choosing fraction negative over survivor kill curve becomes somewhat irrelevant.

Discussion

The use of MPN bioburden is critical to many necessary microbiological and sterilization analyses in modern industrial sterilization. Its utility in various applications of QC microbiology, sterilization process development, and process evaluation makes it a key application of microbiological statistics in everyday industrial microbiology.

References:

Anellis, A and Werkowski, S. (1968).  Estimation of Radiation Resistance Values of

Microorganisms in Food Products.  Applied Microbiology.  16(9):1301

Sutton, S. (2010).  The Most Probable Number Method and its use in QC Microbiology.  Journal of GXP Compliance.  14(4):29

Masaaki Takahashi, Kohki Hatayeyama, Hideharu Shin Tani, Kuranosuke Ishii, Hideo Hayashi

PDA Journal of Pharmaceutical Science and Technology Sep 1995, 49 (5) 220-225.