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Out of eighteen Bacillus spp. screened for aflatoxin M1 (AFM1) sensitivity Bacillus brevis MTCC 3136 was found most sensitive followed by B. megaterium ATCC 9885 at 0.5 ppb (Codex limit). Bacillus brevis 3136 was found weakly positive for lipase activity while B. megaterium 9885 showed significant acetyl esterase activity. Maximum sporulation was achieved in Arret and Krishbaum sporulation agar (90.63%) followed by tryptone yeast extract broth, tryptone glucose yeast extract broth, minimal media and sucrose salt medium. A field level chromogenic assay for AFM1 in milk has been developed using spores of B. megaterium 9885 grown in Arret and Krishbaum sporulation agar. Spores were added with milk containing germinants (dextrose, sucrose, tryptone) and chromogenic substrate followed by incubation at 37˚C for 75 ± 5 min. The colour change from colourless to sky blue colour within 75 ± 5 min is indication of spore germination followed by release of enzyme and its action on chromogenic substrate. However, no colour change indicates that milk is positive for AFM1 at 0.5 ppb. The developed assay was validated with raw, pasteurized and dried milks with a 96% correlation by comparing with radioimmunoassay i.e., Charm 6602 assay. The reliability of assay has also been checked by spiking AFM1 in milk. The developed assay was also validated by certified reference materials of heavy metals and pesticides for checking the cross reactivity of these with our developed assay. The developed chromogenic assay is cost effective, portable, having no interference of antibiotics,
detergents, sanitizers, heavy metals and pesticides. The developed assay can be used in dairy farm and laboratories with fewer resources for AFM1 detection in milk.