INTRODUCTION

The "most probable number" (MPN) method is a useful, if under utilized, tool for the microbiologist. It is part of the harmonized compendial chapter on bacterial enumeration. The test is a method to estimate the concentration of viable microorganisms in a sample by means of replicate liquid broth growth in ten-fold dilutions and is particularly useful with samples that contain particulate material that interferes with plate count enumeration methods. The basic concept to the MPN method is similar to the fraction negative method of the value determination. Nutrient broth will support growth of organisms and turn turbid. The basic pattern of growth vs. no growth can provide information as it is a reflection of sampling error. For example, if one replicate tube of media receives a dilution of the sample that contains a bacterial cell, the tube will turn turbid. Its neighbor, an "identical" replicate, may not receive any bacteria in its sample due to pipetting or sampling and would not turn turbid. This information is particularly useful at low numbers of organisms. However, this accuracy can be greatly increased by diluting the inoculums and then comparing the recoveries of all tubes in the dilution series. This is the basis of the MPN method. The most probable number (MPN) technique is an important technique in estimating microbial populations in soils, waters, and agricultural products. Many soils are

heterogeneous, therefore exact cell numbers of an individual organism can be impossible to determine. The MPN technique is used to estimate microbial population sizes in situations like this. The technique does not rely on quantitative assessment of individual cells, instead it relies on specific qualitative attributes of the microorganism being counted. The important aspect of MPN methodology is the ability to estimate a microbial population size based on a process-related attribute.

DISCUSSION
A. The Methods of Most Probable Number ( MPN ) The MPN technique estimates microbial population sizes in a liquid substrate. The methodology for the MPN technique is dilution and incubation of replicated cultures across several serial dilution steps. This technique relies on the pattern of positive and negative test results following inoculation of a suitable test medium (usually with a pH sensitive indicator dye) such as tubes (upper left photo) and microwell plates (upper right photo). The results are used to derive a population estimate based on the mathematics of Halvorson and Ziegler. Here is the general equation for determining the MPN of organisms in a substrate after it is serially diluted and several units are inoculated of each dilution. Here is the general equation for determining the MPN of organisms in a substrate after it is serially diluted and several units are inoculated of each dilution (Halvorson & Ziegler, 1933). [a1p1/(1-e^(-a) 1^x)] + ... + [akpk/(1-e^(-a) k^x)] = a1n1 + ... + aknk where a = the dilution level of each dilution, n = the number of inoculated units at each dilutions level, p = the number of positive units within each dilution level, k = the highest dilution level of the series, e = the base of the natural logarithm. There are two assumptions underlying the mathematical solution. First, it is assumed that organisms in the initial and all subsequent dilutions are randomly distributed. Secondly, it is assumed that one or more organisms contained within an inoculant volume are capable of producing a positive result. If the second assumption is not satisfied, unusual patterns of positive and negative occurs. The MPN techniques also assume that all test organisms occupy a similar volume.

OVERVIEW OF MPN PROCEDURES The are four main design features of a MPN determination that must be chosen before using the MPN procedure. The first one is to select a base dilution ratio, and then to select the number of units at each dilution level. The other two design features are to determine an initial dilution volume and its value and to select the inoculation volume. Next the required materials must be gathered. If you choose to use a procedure with six fivefold dilution steps and four units per dilution level, as the experiment in Paul L. Woomer?s "Most Probable Number Counts" article, you will need the following materials: 100 grams of soil, one sterile 1 L wide-neck Erlenmeyer flask and stopper containing 400 mL of sterile-N mineral nutrient diluent, one sterile 5 mL wide-mouth pipette, five sterile 5 mL pipettes, one sterile 1 mL pipette, one growth pouch rack, and twenty-four growth pouches containing nodulation legume seedlings previously selected for uniformity of root and shoot development. Below is a dilution scheme for a most probable number count experiment.

To record your data, prepare a data sheet that assigns either a positive or a negative value for each experimental unit. Figure 2 is an example of a data sheet where a (+) represents root nodulation.

After the data is recorded go the appropriate MPN table to assign a population estimate. Tables can be generated using MPNES software (Woomer et al., 1990).

Table 1. Manual.

MPN table for a three-replicate design from FDA's Bacterial Analytical

The method offers real opportunities as an enumeration tool. It can also be employed for semiquantitative estimation of growth-promotion capability of liquidmedia and in estimation of precision for alternate microbiological methods with a simple modification. In the compendial version ofthe MPN test, the sample to be tested is prepared in 10-fold dilution series, and then 1mL samples of each dilution are inoculated into triplicate broth culture tubes for incubation. As the dilutions increase, the possibility that the broth tubes will fail to be inoculated with any microorganism increases. At some point therefore, very few of the replicate tubes will be inoculated with viable microorganisms. Following incubation, all tubes are examined for turbidity and the pattern of growth in the tubes is scored againstatable of such values (3). TheMPNtablefromthe US Food and Drug Administration's Bacterial Analytical Manual (BAM) is provided above. A typical design uses three replicates with a three-loglO unit dilution series (although varying numbers of replicates and different dilution series may also be used). In this design, if all tubes showed growth, then the results will be noted as 333. If only one tube in each replicates how growth it would be denoted as 111. The pattern of growth is then read from the table to provide the

most probable number and 95% confidence interval. By this, the result of 210 would reflect an MPN of 21, and a result of 322 would be interpreted as an MPN of 21O. The MPN table normally only presents results for three dilutions in sequence (e.g.,10',10', 10 ),but the dilution series tested might have been from the 10
3 2

to 10- tubes

(see the FDA BAM discussion on how to select appropriate tubes to read). The worker will need to take the dilution factors in the table and in the actual experiment into account to derive the most probable number from this study. The results of this test should be expressed as "MPN" rather than CFU (colony forming unit) to reflect the capabilities of the method.

This method, with the associated table, only works if there is a succession of 1/10 dilutions being tested (in an appropriate medium) for growth, and the tubes chosen to compare with the table are in consecutive order of increasing dilution. Some things to keep in mind:

Other amounts than 1 mL can be inoculated into the broth tubes. For example, inoculating 0.1 mL of a 10-3 dilution is equivalent to inoculating 1 mL of a 10-4 dilution; the "plated dilution" (10-4) is the same in each case.

It doesn't matter what amount of medium there is in the tubes being inoculated. For example, the same growth response should be evident if we doubled the amount of broth in each tube.

Also, we don't have to inoculate our tules from dilutions. For example, one can set up a series of tubes where 10 grams are inoculated into each tube in the first set, 1 gram is inoculated into each tube in the second set, and 0.1 gram is inoculated into each tube in the third set. The sequence of decimally-decreasing amounts is maintained

The method assumes a random distribution of microorganisms in the sample and an accurate dilution of the sample through the dilution series. It also assumes that the microorganisms are separate and do not affect each other (i.e., attract or repel). In addition, it must be assumed that every tube (or plate, etc.) whose inoculum has a single viable organism will result in visible growth.

Figure 3: The bacterial visible growth of MPN Although the compendial version utilizes three replicates and a ten-fold dilution series, there is no theoretical reason for these parameters. In fact, it is well known that the accuracy of the method increases dramatically when increasing the number of replicates and decreasing the interval ofthe dilution series (five-fold or two-fold) (4, 5). The FDA BAM website referenced provides an Excel spreadsheet to assist in creating different MPN tables as needed.

Applications Of MPN In Environmental Monitoring Data Environmental monitoring data is a problem for microbiology. We are urged to "qualify" our control levels and our sample sites. However, we are using a technology (plate count) that is exceedingly imprecise at numbers of CFUbelow25. The aseptic core of a modern facility will commonly yield counts of zero, with concern expressed if the count is approaching three. These control levels are in truth of little value despite their popularity in regulatory circles (6-8). One approach suggested to deal with this mismatch between regulatory expectations and plate count capabilities has been to explore the possibility of looking at a frequency distribution models to establish control levels in these areas (9) or incident models (10). A recent publication by Sun et al. (11) pointed out the possibilities in using MPN methods for evaluation of clean room monitoring data. The basic idea is to use the fundamental statistics as if only a single dilution were being considered. In this approach, the application is not dissimilar to fraction negative studies of biological indicators for sterilization studies. Preliminary studies presented by this group look promising, and this is an approach that could be pursued with existent data for evaluation. MPN In Qualification Of Alternate (Rapid) Microbiological Methods It should be obvious to the reader that the previous discussion on the use of MPN in growth promotion studies has immediate application for determination of the relative limit of detection for two microbiological methods, for example a "traditional" method and an "alternate" method. The USP chapter recommends this approach even for quantitative methods. Although this might at first seem counter-intuitive, the MPN method (when used with a dilution series) can actually be more accurate than plate counts at low numbers. The only modification that needs to be made is to ignore the counts and treat every plate or membrane as a separate "tube"-the MPN method fits right into the experimental design.

Measurement of Fecal Coliform Bacteria are single-celled organisms that can only be seen with the aid of a very powerful microscope. However, coliform bacteria form colonies as they multiply, which may grow large enough to be seen. By growing and counting colonies of coliform bacteria from a sample of water, it is possible to determine approximately how many bacteria were originally present. There are several ways coliform bacteria are grown and measured. Methods commonly used include the most probable number (MPN) method and the membrane filter (MF) method. In the MPN method, a "presumptive test" is performed first. A series of fermentation tubes that contain lauryl tryptose broth are inoculated with the water sample and incubated for 24 hours at 35 C. Fermentation tubes are arranged in 3 or more rows, with 5 or 10 tubes per row, with varying dilutions of the samples in the tubes. The fermentation tube contains an inverted tube to trap gases that are produced by the coliform bacteria. After 24 hours, the fermentation tube is examined for gas production. If there is no gas production, the samples are incubated for another 24 hours and reexamined. If gas production is observed by the end of 48 hours, the presumptive test is positive; coliform bacteria are present in the sample. A "confirmed test" is then performed to determine if fecal coliform bacteria are present. For the confirmed test, some of the content of the fermentation tube is transferred with a sterile loop to a fermentation tube containing another broth. The sample is incubated in a water bath at 44.5 C for 24 hours. Gas production in the fermentation tube after 24 hours is considered a positive reaction, indicating fecal coliform. Based on which dilutions showed positive for coliform and/or fecal coliform, a table of most probable numbers is used to estimate the coliform content of the sample. The results are reported as most probable number (MPN) of coliform per 100 ml (American Public Health Association, 1998).

The MF method is more rapid than the MPN method, but the results are not as reliable for samples that contain many non-coliform bacteria, high turbidity, and/or toxic substances such as metals or phenols. The water sample is filtered through a sterile membrane filter. The filter is transferred to a sterile petri dish and placed on a nutrient pad saturated with broth. The plates are inverted, placed in watertight plastic bags, and incubated in a water bath at 44.5 degrees C for 24 hours. Colonies produced by fecal coliform bacteria are blue, and are counted using a microscope or magnifying lens. The fecal coliform density is recorded as the number of organisms per 100 ml. Sometimes the unit of colony producing units per 100 milliliters of water (CPU/100 ml) is used; this is equal to the number of organisms per 100 ml. Most Probable Number (MPN) Presumptive Test The MPN test is a statistics-based test which estimates the number of fecal coliforms in a water sample based on the degree of lactose fermentation by organisms in the sample. In this test, a series of tubes of phenol red lactose broth are inoculated with measured amounts of water to determine if the water contains any lactosefermenting bacteria that produce gas. If, after incubation, fermentation plus gas production is demonstrated, it is presumed that coliforms are present in the water sample. By counting the number of positive tubes positive at each dilution, the most probable number (MPN) of coliforms is statistically determined using a standardized chart.

Explanation :

Tube 1: Positive for fecal coliforms. Note (1) the gas present in the Durham tube and (2) the color change from red to yellow as acid end-products react with the pH indicator. Tube 2: Negative for fecal coliforms. Note the absence of gas in the Durham tube. Even though the pH indicator has changed from red to yellow, gas must be produced for a positive result. Tube 3: Negative for fecal coliforms. Advantage and Disadvantages There are many clear advantages of using the MPN technique. MPN methodology results in more uniform recovery of a microbial population across different soil types than filter counts. Furthermore, the detection of organisms through process-related attributes often results in the recovery of mixed populations with similar functional roles in soils. For a more detailed study, the mixed populations can be separated into individual colonies. Another advantage of MPN techniques is that, unlike direct quantitative procedures, it measures only live and active organisms. Microscopic techniques sometimes confuse live and dead cells. Also, MPN methodologies provide more realistic estimates of infective propagules of VA mycorrhizae in field soils than does the more conventional method of spore counts (Porter, 1979). Despite the numerous advantages of using MPN methodology there are a few disadvantages to the method. MPN procedures tend to require more labor and materials than microscopic procedures. Also, MPN estimates often have a lower order of precision than do well-replicated direct counts.

SUMMARY
The basic concept for the MPN method is to dilute the sample to such a degree that inocula in the tubes will sometimes (but not always) contain viable organisms. By replicates, and dilution series, this will result in a fairly accurate estimate ofthe mostprobable number ofcells in the sample. While this method is most commonly used in the pharmaceutical industry for water testing or the compendial bioburden test, it has significant potential to the quality control microbiology lab. These possible applications of MPN include environmental monitoring, media growth promotion studies and aspects ofthe validation ofrapid microbiological methods. To envision the theoretical basis for the Most Probable Number (MPN) Method, think of a ten-fold dilution series being made of a water sample with one mL of each dilution being inoculated into a separate tube of an all-purpose broth medium. To increase the statistical accuracy of this type of test, more than one broth tube can be inoculated from each dilution. Standard MPN procedures use a minimum of 3 dilutions and 3, 5 or 10 tubes per dilution. The statistical variability of bacterial distribution is better estimated by using as many tubes as possible or practical.