Microbiology Guide: Most Probable Number (MPN) Method

Document ID

Document ID TE8844

Version

Version 3.0

Status

Status Published

Published Date

Published Date 09/06/2018
Question
Microbiology Guide: Most Probable Number (MPN) Method
Summary
Microbiology Guide: Media and Equipment Selection; Most Probable Number (MPN) Method
Answer
From: Microbiology Guide: Media and Equipment Selection


Sample Dilution and Interpretation
  • Sample Size Potable Water
    • Potable water should contain no coliforms per 100 mL, so testing should be done on undiluted samples. Use either the 5- or 10-tube MPN test. The 5-tube MPN test is not accepted by the U.S. Environmental Protection Agency (USEPA).
 
  • Sample Size Non Potable Water
    • Testing nonpotable water and wastewater samples requires inoculating a series of tubes with appropriate decimal dilutions of the original sample, based on probable coliform density. Three different dilutions should be inoculated into test tubes. For example, when examining swimming pool water: add undiluted sample to 5 tubes, sample with a 1:10 dilution to 5 tubes, and sample with a 1:100 dilution to 5 tubes. See specific procedures for dilution instructions.
 
  • Procedure
    • Wash hands.
    • Open a bottle of sterile Buffered Dilution Water.
    • Invert the sample container in a waist-to-ear motion, approximately 25 times (for 30 seconds).
    • Use a sterile pipet to add 11 mL of sample into the dilution water bottle.
    • Add 11 mL of the 10x dilution to another dilution bottle and mix well (100x dilution).
    • Add 11 mL of the 100x dilution to a third bottle and mix well (1000x dilution).
    • Continue to make dilutions until the necessary dilution level has been reached.
 
  • Five-Tube MPN Test
    • Use Table 1 to determine MPN/100 mL for 5-tube MPN tests. Example: if 4 of the 5 tubes showed a positive response, the MPN/100 mL would be 16.0.
    • Table 3, illustrates typical decimal dilutions for various sample types.

  • Calculation
    • Using Table 4 MPN Index, it is possible to estimate the number of organisms from any combination of positive and negative test results.
    • The MPN Table (Table 4) is based on 3 dilutions of decreasing decimal increments.
      Example: Five tubes of undiluted sample, 5 tubes of 10X dilution, and 5 tubes of 100X dilution are inoculated initially. Positive results are obtained from 5 of the undiluted samples, 5 of the 10X dilutions, and 3 of the 100X dilutions.
    • Calculation Equation= MPN Index per 100mL value x the Lowest dilution factor


Inoculation Step
  • Sample Size depends on the media.  See Hach method
 
  • Pipets and Tips
    • Tensette® Pipet, 1.0-10.0 mL (Product # 1970010)
    • Pipet Tips, for Tensette® Pipet 1970010, Sterile, 1.0-10.0 mL, pk/50 (Product # 2558996)
 
  • Inoculating Loop
    • Loop, Inoculating, Disposable, PS 10UL pk/25 (Product # 2749125)
    • Loop, Inoculating, Wire (Product # 2112100) - Reusable

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