2.3. Bacterial Culture and Trial Preparation

The wild-type E. ictaluri isolate S97-773 (recovered from diseased channel catfish at the Thad Cochran National Warmwater Aquaculture Center in Stoneville, Mississippi; accession number: {"type":"entrez-nucleotide","attrs":{"text":"JX867005","term_id":"575524040"}}JX867005) was utilized for this study [14,69]. Cryopreserved S97-773 stocks were revived on brain-heart infusion (BHI) agar and incubated for 48 h at 28 °C. Next, a pure E. ictaluri colony was placed in 1 L of BHI broth and incubated at 28 °C and 115 revolutions per minute (RPM) for approximately 48 h. The broth culture was centrifuged at 4000× g for 10 min in a 5810 R benchtop centrifuge (Eppendorf North America Inc., Enfield, CT, USA), washed in cold 1X phosphate-buffered saline solution (PBS) with an adjusted pH of 7.4. Bacterial cells were resuspended and adjusted to an optical density of 0.200 ± 0.005 at 550 nm using an Eppendorf Biospectrometer^® Basic (Eppendorf North America Inc.), resulting in an average inoculum concentration of 8.33 × 10⁷ colony forming units (CFU) mL⁻¹.

Preparation of the F. covae inoculum, using isolate ALG-00-530 (recovered from a diseased channel catfish at the Alabama Fish Farming Center in Greensboro, Alabama; accession number: {"type":"entrez-nucleotide","attrs":{"text":"MG516971","term_id":"1277442650"}}MG516971), followed a similar procedure [70,71]. However, the culture media was modified by Shieh [58] containing the antibiotic tobramycin at a concentration of 1 mg L⁻¹ of media (MST) resulting in a more selective media [72]. The F. covae was passed over the selective MST agar five times to ensure the bacterium had grown accustomed to the antibiotic. After the fifth pass, a pure colony of F. covae was placed into 1 L of Modified Shieh broth and incubated for 24 h at 28 °C and 115 RPM. Once the broth culture had grown, the bacterial cells were spun down, as mentioned previously, and instead washed with a 0.1X PBS solution with an adjusted pH of 7.0. Bacterial cells were resuspended and adjusted to an optical density of 0.200 ± 0.005 at 550 nm using a DR3900 visible spectrophotometer (Hach Company, Loveland, CO, USA) to determine bacterial concentration [73]. The final F. covae inoculum concentration in PBS was 1.78 × 10⁷ CFU mL⁻¹.

A randomized block design was used for each PT to assign the four sediment types to the 12 total chambers. In each chamber, 20 mL of either E. ictaluri or F. covae optically adjusted bacterial inoculum was added to 200 g of sterilized sediment and 500 mL of disinfected dechlorinated city water. The sediment mixture was vigorously stirred with a sterile stainless-steel spatula for 1 min durations every 5 min over 1 h. After the mixing period, water volume within each chamber was increased to a total of 8 L. To simulate production pond aeration, a 3.5 cm × 1 cm × 1 cm cuboid Pawfly air stone at a fixed location within each chamber would expel air supplied via a Whitewater Silent Air Pump™ v201 (Pentair Aquatic Eco-Systems™, Apopka, FL, USA) for 12 h beginning at 18:00 h and stopping at 06:00 h the following day.