The isolation and detection of pathogenic bacteria (E. coli, Salmonella spp and Vibrio spp) were performed using the membrane filter technique as described in Standard Methods [17 ]. MacConkey agar (Merck, SA), XLD agar (Merck, SA) and TCBS agar (Merck, SA) were used as a culture medium and the plates were incubated at 37°C overnight. Colonies were enumerated and recorded as CFU/100 mL for the intake raw water. The counts of attached pathogenic bacteria were calculated using the equation mentioned above and they were reported as CFU/cm2.
Tcbs agar
Manufactured by Merck Group
Sourced in Saudi Arabia, Germany, United States
TCBS agar is a selective and differential culture medium used for the isolation and identification of Vibrio species, particularly Vibrio cholerae, the causative agent of cholera. It contains sucrose, sodium citrate, sodium thiosulfate, and bromothymol blue as key ingredients. TCBS agar inhibits the growth of most other bacteria while allowing Vibrio species to grow and produce colonies with distinctive color and appearance.
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4 protocols using tcbs agar
1
Isolation and Detection of Pathogenic Bacteria
2
Isolation and Identification of Vibrio parahaemolyticus
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V. parahaemolyticus was isolated as described by Mok et al. [13 (link)]. Briefly, 5 mL of seawater was added to a flask containing 95 mL of peptone water with 1.5% NaCl and then incubated at 28 °C for 24 hours. Subsequently, 100 μL aliquots were spread on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Merck) and incubated for 48 hours at 28 °C. Colonies that grew on TCBS were characterized by their color, shape, and size. The blue colonies were subsequently inoculated onto CHROMagar medium (Titan). Purple presumptive colonies were further confirmed through biochemical and polymerase chain reaction (PCR) assays.
3
Isolation of Vibrio spp. from Seafood
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Bacteria were isolated from gilthead sea bream (Sparus aurata L.) and shellfish (Penaeus indicus H. Milne-Edwards). These samples were obtained from a local market in Hail region-Saudi on 25 February 2022. Fish with red spots on their skin were targeted, as this is an indication of microbial infection. Upon arrival, gilthead sea bream and prawns were immediately washed using sterile seawater, gutted, headed, and shucked with a sterile knife. Twenty-five grams from prawn abdomen meat, and the intestines, gills, and muscle meat from S. aurata were enriched in 225 mL of alkaline peptone water supplemented with 1% NaCl [18 (link)]. The inoculated broth media was incubated overnight at 37 °C. After incubation, a loopful from each enrichment culture was steaked onto thiosulfate–citrate–bile salt–sucrose agar (TCBS) (Agar; Sigma Aldrich, Darmstadt, Germany) and onto Vibrio ChromoSelect agar (Sigma Aldrich, Germany), before incubating for 18 to 24 h at 37 °C.
4
Tissue-mimicking Phantom for Blood Flow
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To model biological tissue, 10 phantoms were made by heating 1% TCBS agar (Sigma-Aldrich, Missouri) and 2% intralipid diluted from 20% stock (Sigma-Aldrich, St. Louis, Missouri) in water. intralipid mimics the scattering properties of biological tissue, 22, (link)23 (link) and agar solidifies the resultant suspension upon cooling. The heated mixture was cooled in chambers containing two microtubes to simulate blood vessels. Five prepared phantom chambers contained microtubes with internal diameters of 600 μm, and five more with internal diameters of 300 μm. A double-syringe high-precision pump (Model NE-4002X, New Era Pump Systems, Farmingdale, New York) was used to *Address all correspondence to: Homa Assadi, E-mail: homa.assadi@ryerson .ca fill the two microtubes simultaneously; OCT measurements under both no-flow and flow conditions were then possible. The average blood flow velocities in both microtubes were 0, 0.5, 1, 2, 3, 5, 8, and 12 mm∕s corresponding to venules, capillaries, and arterioles. [24] [25] (link)[26] One syringe was filled with blood, the other with the mixture of blood and MBs. Rotating magnets inside both syringes were used to avoid red blood cells (RBC) settling and MBs floating during all experiments.
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