What do we do with your samples once they have arrived?
Amy C. Jordan, Laboratory Scientist V
Water Analysis Lab Microbiology Unit Supervisor
Public Health Laboratories
What do we do with your samples once they have arrived?
You’ve collected a river sample, and it made its way to the Public Health Laboratory – Water Analysis Laboratory (PHL-WAL). You have a result, but how did we get it? What happens between the time a sample is dropped off and you get your result? In the summary below, we explain what happens at the laboratory after your sample arrives.
The Upper Merrimack Monitoring Program (UMMP) river samples received at the laboratory are logged in to our Laboratory Information Management (LIMS) system for E.coli analysis using an IDEXX method called Colilert. This LIMS system is where we enter and review data and is also where your report is generated. Once logged in, samples are delivered into the Water Microbiology lab where they are analyzed.
How does the Colilert MPN test work?
Since rivers are considered freshwater swim samples we analyze them for E.coli. The Colilert test utilizes defined substrate technology, which basically means that it utilizes nutrients and enzymatic indicators to detect total coliform and E.coli bacteria. E.coli is a coliform bacteria and is used as an indicator organism to determine water quality, as its presence in a sample is indicative of fecal contamination from warm-blooded animals, including humans. Not all E.coli strains are harmful (some are harmful to human health), but the presence of E.coli in a water sample can also be indicative of the potential for other organisms present that may also be harmful to your health (viruses/parasites/fungus/other harmful bacteria). It is also super easy to grow in a laboratory setting, which is another benefit to using it as an indicator organism.
The Colilert test consists of prepackaged reagent containing the nutrients and indicators to detect E.coli in individual “snap-pack” containers. One snap-pack is used per sample.
Image 1: Colilert Reagent
River samples first undergo a 1/10 dilution in sterile water, the reagent is added to this dilution, and the sample is placed on a shaker to dissolve the reagent. Once the reagent is dissolved, the sample is then aseptically transferred into a Quanti-Tray (Q-tray), put through a sealer to seal the sample and reagent into the individual wells, and then incubated at 35C for 18-28 hours, depending on the Colilert analysis chosen.
Image 2: Quanti-tray with sealer
We use both an 18- and 24-hour Colilert test throughout the day. (See images 3 and 4 for the two Colilert analyses used at PHL-WAL.)
Image 3: Colilert 24-hour Image 4: Colilert 18-hour
After incubation, the sample is then checked for both color change and fluorescence. In order for the sample to be positive for E.coli, the individual wells must be both yellow AND fluoresce under UV light at 365nm. (See the following pictures below. Image 5 shows yellow wells indicating the sample is positive for total coliform bacteria. Image 6 shows that the yellow wells also fluoresce under UV light, indicating presence of E.coli in the sample).
Image 5: Total coliform positive result Image 6: E.coli positive result
The Q-tray contains 49 large wells and 48 small wells. Each positive well is counted, and a corresponding Most Probable Number (MPN) is documented off the IDEXX Q-tray MPN table. Any positive large and/or small wells of the Q-tray provide two numbers that correspond to an MPN/100mL result using the probability table below that will estimate the MPN based on those positive wells. For example, in image 6 above, we have 49 positive large wells and 18 positive small wells. We can then go to the MPN table and look at where the numbers for 49 large and 18 small meet to obtain a result of 307.6 MPN/100mL (See an example of the MPN table below).
What is MPN?
Most Probable Number (MPN) is an estimation of the concentration or number of bacteria in liquid media (in this case, river water). The MPN is a statistical method of quantitation, which assumes even distribution of the microbes across the sample as well as random separation of the organisms across the wells of the Q-tray. The Colilert analysis gives a result in MPN/100mL because that is the standard amount of water used to obtain a result with this test.
Image 7: MPN Table for Quanti-Tray 2000 /100mL