Agar gsp

In total, 18 fish were sampled from each fish farm monthly and during the additional visits. The studied population was selected based on the maximum probability of isolating the Aeromonas bacteria from fish farms that were recently infected with furunculosis. The fish sample size for each pond (≤22,500 fish per raceway) was determined by considering that the expected frequency of furunculosis was 30% in the studied farms based on the analysis of Cannon and Roe [33 ,34 (link)]. Fish samples were autopsied and clinical lesions were recorded for each case. The samples of spleen, mucus from the posterior digestive tract, gills and skin mucus were dissected and cultured on Agar GSP (Merck KGaA, Darmstadt, Germany), the selective medium for detecting Aeromonas spp. [35 ].

One litre of pond water was collected from each study pond using a sterile water bottle. Each water sample was filtered in 10 parts (10 × 100 mL) using the filtration manifold system (Millipore, Darmstadt, Germany) through a cellulose nitrate membrane filter, 47 mm diameter, 0.22 μm pore size (Sartorius, Goettingen, Germany). The filter membrane then was placed in a Petri dish into which 1 mL of sterile normal saline solution was added. The bacteria were detached from the filter membrane via pipetting the sterile water on the membrane [36 (link)]. The solution then was diluted at 10⁻¹ and 10⁻². Then, 100 µL of each dilution was inoculated and thinly spread on Agar GSP (Merck KGaA, Darmstadt, Germany).

Prior to the start of the study, biofilm experimental surfaces were created on a plastic structure and installed in each study pond. Each month, two biofilms were removed from each pond and taken for analysis. One biofilm surface was taken for the bacteriological analysis of the cumulative effects of previous months. Another biofilm surface was also collected from the previous month and then replaced with the biofilm surface of the following month. Each biofilm plate was placed in a sterile bottle filled with the corresponding pond water. The plastic biofilm surface (5 × 5 cm) was detached from the plate and put into the filtered sterile stomacher bag (177 × 302 mm) into which 10 mL of sterile normal saline solution was added. After being put in a mini-mixer (stomacher) (Lab-Blender 400, Leicestershire, UK), which operated at a speed of 230 rpm for 15 min, attached cells were removed from the biofilm surface into the stomacher bag. Using a sterile pipette, the filtered cells were aspirated from the stomacher bag and placed in a sterile tube [37 ]. The samples were diluted at 10⁻² and 10⁻³, and then 100 µL of solution was inoculated and thinly spread on Agar GSP (Merck KGaA, Darmstadt, Germany).