1. Lab 6: Most Probable Number Method (MPN)

2. Most Probable Number Method (MPN)
Objective
What is the MPN method?
How to determine the amount of bacteria from the MPN method?

3. Most Probable Number Method (MPN)
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.
It is particularly useful with samples that contain particulate material that interferes with plate count enumeration methods and useful for low concentrations of organisms (<100/g), especially in milk and water.
Most probable number (MPN) analysis is
a statistical method based on the random
dispersion of microorganisms per volume in
a given sample. 

4. Most Probable Number Method (MPN)
In this method, measured volumes of water is added to a series of tube containing a liquid indicator growth medium.
The media receiving one or more indicator bacteria show growth and a characteristic color change.
Color change is absent in those receiving an inoculums of water without indicator bacteria.

5. Most Probable Number Method (MPN)
Only viable organisms are enumerated by the MPN determination.
If, in the microbiologist's experience, the bacteria in the prepared sample in question can be found attached in chains that are not separated by the preparation and dilution, the MPN should be judged as an estimate of growth units (GUs) or colony-forming units (CFUs) instead of individual bacteria.

6. Most Probable Number Method (MPN)
From the number and distribution of positive and negative reactions, the MPN of indicator organisms in the sample may be estimated by reference to statistical tables.
MPN test is completed in three steps :
Presumptive test
Confirmed test
Completed test

7. Presumptive test
It is used for detection and estimation of coliform in water sample(Screening).
For estimation of coliforms, lactose containing broth medium is used.
Commonly used medium is MacConkey broth that contains the indicator bromocresol purple or lauryl tryptose (lactose) broth.
An inverted Durham’s tube is placed.
The color of media changes into yellow and on collection of gas in Durham's tube, bacteria are assumed to be coliform.
Number of positive tubes are counted and referred to the standard chart to find MPN of total 100 ml water sample.

8. Confirmed test
Some spore forming bacteria give false positive test in presumptive test.
Confirmed test is done to determine that the coliforms are of fecal origin or not. And they are E. coli or not.
For this positive presumptive test are inoculated in selective media like Eosine Methylene Blue (EMB) agar or Endo agar and incubated at 44.5°C and 37°C.
Presence of typical colonies at 37°C confirms positive coliform test and those at 44.5°C confirms the presence of E. coli.

9. Completed test
Subculture typical colonies in lactose containing medium and incubated at 37° C and 44.5 °C.
Presence of E. coli is confirmed by the production of gas at 44.5 °C.
From each of the solid-medium plates used for the confirmed test, transfer selected colonies each to:
3 ml  lactose-broth or brilliant green lactose fermentation tube
an agar slant (Gram stain)
3 ml tryptone water.

10. Most Probable Number Method (MPN)
For simplicity, however, here we will speak of these GUs or CFUs as individual bacteria.
The following assumptions are necessary to support the MPN method.
The sample is prepared in such a way that the bacteria are distributed randomly within it.
The bacteria are separate, not clustered together, and they do not repel each other.

12. Most Probable Number Method (MPN)
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 pippetting or sampling and would not turn turbid.
This information is particularly useful at low numbers of organisms.

13. Confidence Intervals
The 95 percent confidence intervals in the tables have the following meaning.
Before the tubes are inoculated, the chance is at least 95 percent that the confidence interval associated with the eventual result will enclose the actual concentration.

15. Most Probable Number Advantages Disadvantages
Relatively simple and sensitive
Can count a specific type in the presence of others
Can use large sample volumes
Time consuming and labor intensive
Requires large volumes of glassware
Doesn’t give the “real” value
Doesn’t give isolated colonies

16. 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 of the most probable number of cells in the sample.
The "outcome", i.e., the numbers of inoculate producing growth at each dilution, will imply an estimate of the original, undiluted concentration of bacteria in the sample.
While this method is most commonly used in the personal products, medical device, and pharmaceutical QC microbiology labs for water testing , it has significant potential for other applications.

17. END OF LECTURE