title: Short communication: Improved method for centrifugal recovery of bacteria from raw milk applied to sensitive real-time quantitative PCR detection of Salmonella spp.
abstract: Centrifugation is widely used to isolate and concentrate bacteria from dairy products before assay. We found that more than 98% of common pathogenic bacteria added to pasteurized, homogenized, or pasteurized homogenized milk were recovered in the pellet after centrifugation, whereas less than 7% were recovered from raw milk. The remaining bacteria partitioned into the cream layer of raw milk within 5 min, and half-saturation of the cream layer required a bacterial load of approximately 5×10(8) cfu/mL. Known treatments (e.g., heat, enzymes or solvents) can disrupt cream layer binding and improve recovery from raw milk, but can also damage bacteria and compromise detection. We developed a simple, rapid agitation treatment that disrupted bacteria binding to the cream layer and provided more than 95% recovery without affecting bacteria viability. Combining this simple agitation treatment with a previously developed real-time quantitative PCR assay allowed the detection of Salmonella spp. in raw milk at 4 cfu/mL within 3 h. To our knowledge, this is the first report of an effective method for achieving high centrifugal recovery of bacteria from raw milk without impairing bacterial viability.
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, USDA, Agricultural Research Service, North Atlantic Area, Eastern Regional Research Center, Wyndmoor, PA 19038. Electronic address: jeffrey.brewster@ars.usda.gov.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Brewster
authors.affiliation: Dairy and Functional Foods Research Unit, USDA, Agricultural Research Service, North Atlantic Area, Eastern Regional Research Center, Wyndmoor, PA 19038.
authors.forename: Moushumi
authors.initials: M
authors.lastname: Paul
mesh_terms: D000818:Animals; D058491:Bacterial Load / Q000379:methods; D002498:Centrifugation / Q000379:methods*; D005516:Food Microbiology / Q000379:methods; D008892:Milk / Q000382:microbiology*; D061352:Pasteurization; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000302:isolation & purification*
keywords: bacteria; partitioning; raw milk; recovery