Bx40 clinical microscope

Total white and red blood cell counts were measured using a Coulter Z1 (Coulter, Miami, FL, USA). One hundred-cell differential counts were performed using a microscope (Olympus BX40 clinical microscope, Center Valley, PA, USA).

The fish were euthanatized on the farm and sent refrigerated to the Fish Diseases Laboratory of the Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta (Turin) within 24 h, where necropsy, parasitological, and bacteriological tests were carried out. For the anatomopathological examination, the fish were macroscopically inspected by means of sterile tools and the lesions noted. Internal organs (liver, spleen, kidney) were removed for culture to detect NTM.
The gill lamellae, the cutaneous, and the gill mucus were inspected for parasites using an optical microscope (BX40 Clinical Microscope; Olympus, Tokyo, Japan) at increasing magnification (10×, 20×, 40×) The coelomic cavity and the intestinal package were examined macroscopically and microscopically for endoparasites.
For bacteriological culture, brain and kidney samples were aseptically removed using 10-μL sterile loops, inoculated on Columbia blood agar (Liofilchem, Roseto degli Abruzzi (TE), Italy), marine agar, and thiosulfate citrate bile sucrose (TCBS) agar and incubated at 22 ± 2 °C for 72 h. Isolates were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a VITEK MS system (bioMérieux S.A., Marcy-l’Étoile, France).

Stained slides were evaluated by a trainee (N.H.I.) and a pathologist (A.Y.) on an Olympus BX40 Clinical Microscope and imaging software MicroSuite™ system (Olympus America Inc.). Both assessors were blinded to the treatment groups to minimize operator biasness. A minimum of ten fields were examined and photographed for each tissue section using Olympus DP72 Microscope Digital Camera (Olympus America Inc.). The localisation of the four proteins (Aqp 1, Aqp 5, CFTR and Muc 1) was based on a combinative semi-quantitative H-scoring method (52 (link)). The percentage area of positively labelled cells (value A) and intensity of IHC reaction (value B) were quantified by the operators, and the product of the two values will give a final score (AxB). In the event of discrepancy, slides were re-evaluated to achieve consensus. The scoring for value A is based on percentage area of positively labelled cells (0 = 0%; 1 = below 30%; 2 = 30-60%; 4 = more than 60%). The scoring for value B is based on intensity of IHC reaction (0 = no reaction; 1 = weak reaction; 2 = mild reaction; 3 = strong reaction).