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April 30, 2014

In The Lab: Water Quality Testing — Part 1

  • Food Safety

By InstantLabs

By Lauren Bambusch

We’ve had several people ask about the possibility for adapting our AOAC-approved Salmonella species and E. coli O157 Food Safety Kits for use with water samples.  It’s a concept we’ve been thinking about, so it was time to fully develop the modifications needed to the sample collection and  DNA extraction protocols and test the effectiveness of the assays on water samples.

First Things First:

What do the testers want to know?  Are they interested in the presence or absence of even one cell of Salmonella or E. coli O157?  Is it a question of being above a specific threshold of bacterial load?  Or is quantification important?  The form the question needs to inform the design of the protocol.

After doing my research into industry standards and regulations from food production to industrial processing to environmental water quality, it turns out all three question are valid.  Environmental water quality has standards set by the Environmental Protection Agency that require levels of bacteria to be below a certain threshold, while food producers want to know if there’s even a single cell of bacteria in the water source for their hydroponic farms.  Industrial processors just want to keep an eye on bacteria levels in their water to make sure they don’t get out of hand.  Each industry needs a different sample processing technique to answer their unique questions.

Method One – Threshold Testing:

I come from a long line of civil engineers, and environmental water quality has always been close to my heart.  For that reason, I started with developing a protocol to satisfy the EPA’s 235 cells per 100 mL of water threshold.  First, I grew up pure cultures of Salmonella enterica Sbsp. Enteritidis and E. coli O157:H7, which I used to seed Baltimore’s finest tap water. We ended up with approximately 300 cells per 100 mL per organism, verified by plating on appropriate media.  I left one sample as it came out of the tap, then one sample was seeded with only Salmonella, one was seeded with only E. coli, and one was seeded with both organisms.  These samples were then mixed thoroughly, and the fun began.

 

I “borrowed” a vacuum-powered water filtration system which we are using extensively for another project, plugged in a new disposable filtration cup, and fired her up.  After pouring 100 mL of the sample into the filtration cup and vacuum-filtering it, the filter was removed and processed with a bead beater to extract the DNA. This method is similar to the one specified by the EPA’s Method A.  In this method, the filter—along with any organisms on it—are decimated by violently shaking it in a tube filled with a stabilization buffer and small beads.  This process releases the DNA from the cells into the buffer, giving me the DNA needed to run real-time PCR.  This was repeated for all 4 samples in replicate.

These samples were then tested using our AOAC-approved Salmonella species and E. coli O157 Food Safety Kits on the Hunter® Real-Time PCR instrument.  All results matched expectation; the samples seeded to create positive results tested positive, while those that should have been negative remained so.  The chart below shows the results; the amplification of the sampled seeded with E. coli started amplification around cycle 33.  Because late amplification, like in the graph seen below, indicates a bacterial load that is approaching the test’s limit of detection (the minimum number of bacterial cells required to obtain a positive result), the results suggest that a test could be developed to positively determine if a sample meets a specific threshold, such as the EPA’s threshold mentioned above.

The Verdict:

Our simple protocol passed step one of the proof of principle testing.  There’s much more to do, but this is an encouraging start to developing a set of protocols to answer water testing questions.  Next up: testing for the presence/absence of a single cell of Salmonella and E. coli  in a water sample using a modified enrichment protocol.

Lauren Bambusch is a microbiologist by trade as well as a writer and baker by hobby. She lives in Baltimore with her husband, two cats, a super-sized mutt, and a school of fish, all of whom root for her Alma mater, Michigan State. Go Green!