Connecting the Dots

Article by Regina Simmons, QC Chemist

Microbial growth is characterized by the number of cells or colony forming units per volume of sample.   This concept is a mass/volume ratio that is roughly proportional to the growing conditions as well as the initial number of different organisms found in the sample.  By better understanding how the range of the sample is related to the optimum colony forming units per volume tested we can better determine the dilution factors in which to use for the most accurate count.  The most prevalent bacteriological standard used for monitoring water for contamination is Escherichia coli (E.coli) since it is easily enumerated and a member of the total, fecal, and thermotolerant coliform groups.  In some methods it is also monitored for use as an indication of other parasitic pathogens. 

Conventional methods for verification of E.coli are very laborious and time-consuming especially when dealing with fecal contaminated water samples.  To better compensate the industry has had to adapt and modify the techniques used for detection thus allowing testing to become faster and more reliable through better methods and selective culture medium.  Methods employed by most testing facilities include membrane filtration (MF), standard plate count (SPC), enzyme substrate technology (EST), and most probable number (MPN) or multiple tube fermentation (MTF) techniques.  Although the methods themselves are straight forward in the way they are done the big deciding factor for accurate detection is the choice of the culture medium, the incubation temperature, and the volume of sample used to get a desired plate count.  Laboratories are limited by the guidelines set down by the Environmental Protection Agency (EPA) for the criteria of culture medium and incubation temperature used per method but the volume of sample tested is dependant upon the final concentration range expected for the sample and the optimum plate count.

A final concentration range of 20-2400 CFU/100mL of sample can be broken down in the following suggested dilutions to use for the MF technique.

The optimum plate count for a 47mm plate is 20—60 CFU/plate.  By using the above table as a guide for testing on a WP Microbiological PT sample you can see that some portions of the PT concentration range to be reported overlap for the sample volume dilution used for testing.  For example, if two of the sample volume dilutions used for testing is 10mL and 20mL of a 100mL reconstituted sample that the PT concentration range to be reported is between 100 and 200 CFU/100mL the value is covered by both sample volume dilutions.  So a possible strategy for deciding what sample volume dilution to use would be to perform the MF technique on each of the listed sample volume dilutions.  This would likely provide at least one plate within the 20-60CFU/plate requirement and cover the PT concentration range of 20-2400CFU/100mL.  Using this strategy will consume 88mL of the 100mL reconstituted PT sample. 

For the MPN/MTF technique testing on a WP Microbiological PT a ten fold dilution series of 10mL, 1mL, 0.1mL, and 0.01mL will cover the entire PT sample concentration range.  If you are using a five tube per dilution arrangement you will consume 55.55mL of the 100mL of reconstituted sample.  Therefore, if you need to report both MPN/MTF and MF values for your WP certification you will need to order more than one PT sample. 

Using the MF technique for the SPC PT with a final concentration range of 5-500CFU/mL and keeping in mind the 20-60CFU/plate guideline, analysis of a PT sample can be broken down in the following dilutions.