The microplates were incubated at 37 [degrees]C for 24 h; then, 30 [micro]L 0.02% resazurin
(Sigma-Aldrich) aqueous solution was added to every well (Sarker et al., 2007).
Kumarasamy, Microtitre plate-based antibacterial assay incorporating resazurin
as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals, Methods, 42, 321 (2007).
Ten microliters of the resazurin
dye (0.1% solution) was added in each well as a visual indicator of the MIC.
The metabolic conversion of resazurin
to resorufin was used as a measure for viability, which was estimated 1 h and 24 h post-treatment with AAPH and UV radiation, respectively.
The viability of cells in individual wells was assessed by adding 20 [micro]L of resazurin
toxicology reagent (Sigma-Aldrich) per well and measuring fluorescence intensity (exc.
For enrichment culture and isolation, the medium was prepared based on the modification by Sowers and Schreier  and contained the following compounds (per liter distilled water): K[H.sub.2]P[O.sub.4], 0.51 g; [K.sub.2]HP[O.sub.4], 0.24 g; MgS[O.sub.4] x 7[H.sub.2]O, 0.204 g; [(N[H.sub.4]).sub.2]S[O.sub.4], 0.255 g; NaCl, 1.02 g; Ca[Cl.sub.2] x 2[H.sub.2]O, 0.134 g; vitamin solution, 10.0 mL; yeast extract, 1.0 g; sodium acetate, 50 mM; methanol, 50 mM; sodium formate, 50 mM; rumen fluid, 300 mL; NaHC[O.sub.3], 5.0 g; resazurin
, 0.001 g; 0.2 M cysteine hydrochloride, 15 mL; and 0.2 M [Na.sub.2]S x 9[H.sub.2]O, 4.0 mL.
Then, 30 [micro]L of resazurin
0.1% (Sigma-Aldrich, St.
Several approaches are possible to determine the cytotoxicity of material like LDH release, MTT formation, XTT formation, neutral red uptake, kenacid blue binding, acid phosphatase activity, sulforhodamine B binding and resazurin
test to verify pathogenic efficiency of pasteurization.
The medium used for isolation and cultivation of strains grown on Thioglycolate Agar (TGA) medium and was prepared as follows; 15 g/L pancreatic digest of casein, 5.5 g/L dextrose, 5 g/L yeast extract, 2.5 g/L sodium chloride, 0.5 g/L sodium thioglycolate, 0.5 g/L L-cystine, 1 mg/L resazurin
, 75 g/L agar with final pH 7 (29).
Compound Quantity (ml) for 100 ml of medium Distilled water 52.9 Clarified rumen liquid (1) 30.0 Mineral solution I (2) 5.0 Mineral solution II (3) 5.0 Sodium carbonate 5.0 ([Na.sub.2][CO.sub.3]), 8% (4) Sulphide-cysteine solution (5) 2.0 Resazurin
solution 0.1% (6) 0.1 (1)Clarified filtered rumen liquid filtered centrifuged at 17664 g for 15 min and sterilized 20 min at 21[degrees]C at 15 psi.
After the incubation, 20 [micro]L of resazurin
dye for each well was added and, then, the colorimetric reaction was observed.