EPA Method 1603 “Spike the Ball” Study

Oral Presentation

Prepared by M. Citriglia, C. Lannan
Northeast Ohio Regional Sewer District, 4747 East 49th Street, Cuyahoga Height, OH, 44125, United States

Contact Information: CitrigliaM@neorsd.org; 216-641-6000


EPA Method 1603 (“Escherichia coli [E. coli] in Water by Membrane Filtration using Modified membrane-Thermotolerant Escherichia coli Agar [Modified mTEC]") requires the use of a spike of a known value to test “Ongoing Precision and Recovery” (OPR), and “Matrix Spikes” (MS). The method proposes the use of a manufactured E.coli strain, called a “BioBallTM,” of known concentration, or the use of a lab-prepared E.coli strain. The lab-prepared E. coli strain has an optimal incubation temperature of 35-37°C and the manufactured BioBallTM has an optimal temperature incubation of 35-37°C. Previous studies in the lab demonstrated a decrease in recovery when a laboratory E.coli strain was incubated at method temperatures, 2 hours at 35°C, and 22-24 hours at 44.5°C (the temperature at which thermo-tolerant E. coli grow). The increased temperature drastically reduced the count and viability of the colonies. Additional studies in the lab using the BioBallTM spike showed a decrease in recovery when the BioBallTM was plated onto Modified mTEC Agar. The decrease was a result of the inhibitory properties of the modified mTEC agar and the increased incubation temperature at 44.5OC. The Analytical Services department of the Northeast Ohio Regional Sewer District initiated a study for alternative spiking procedures for Method 1603 in order to resolve the limitations of the current bioball procedure. The spiking procedure was developed using an environmental thermo-tolerant strain of E. coli. Percent recoveries and RSD were assessed. A percent recovery of 83% to 110%, and an RSD of 9.6% validates the spikes and confirms repeatability. The recovery range of the thermo-tolerant spikes approached 100% recovery, whereas the BioBallTM was at 26%. This alternative approach to spiking demonstrated higher efficiency by eliminating the analysis of lab-prepared suspensions for every single spiking requirement and lowered costs of testing by the use of a laboratory isolated strain rather than a manufactured strain, such as the BioBallsTM. A further advantage of our method is its ability to indicate any matrix interference as compared to the other two protocols which provide wide and unreliable recovery ranges by using E. coli strains that cannot grow favorably at 44.5 oC and/or on a selective media such as modified mTEC agar.