Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2015
Publication Date: 5/1/2016
Publication URL: http://handle.nal.usda.gov/10113/62624
Citation: Brewster, J.D., Paul, M. 2016. Short communication: Improved method for centrifugal recovery of bacteria from raw milk applied to sensitive real-time quantitative PCR detection of Salmonella spp. Journal of Dairy Science. 99(5):1-5.
Interpretive Summary: Raw milk and other raw (unpasteurized) dairy products are a significant source of foodborne illness. Rapid, sensitive tests for pathogenic bacteria in raw milk can help reduce consumer exposure and improve the safety of these products. Many rapid tests use centrifugation to separate milk into a cream layer, a liquid layer, and a small solid layer containing bacteria. The liquid and cream are removed to give a concentrated bacteria suspension that can be assayed with high sensitivity. We found that this step fails with raw milk because the majority of bacteria are bound to the cream layer and discarded before assay, leading to inaccurate test results. We studied this phenomena with three pathogenic bacteria under a wide variety of conditions. We developed a simple modification to existing assays that allows pathogens to be efficiently recovered from raw milk and detected accurately.
Technical Abstract: Centrifugation of milk is widely used as a separation/concentration step in assays for pathogenic microorganisms. Separation of the cream and liquid supernate from the pellet containing sedimented solids, somatic cells and microorganisms eliminates many interfering substances, and resuspension of the pellet in a small volume of liquid affords substantial concentration of the sedimented target. Centrifugal recovery of bacteria from pasteurized, homogenized milk is very efficient, as expected based on the relatively high density of bacteria relative to cream and milk serum. However, recovery of microorganisms from untreated raw milk was very low under a wide variety of centrifugation conditions, with the vast majority of bacteria partitioning into the cream layer rather than the pellet. The effect of homogenization, heat treatment, detergents, calcium chelators, sugars, lipid extraction reagents and agitation on centrifugal recovery was studied for three model pathogens. Intense vortexing in the presence of EDTA allowed efficient centrifugal concentration of bacteria from raw milk, as demonstrated with a rapid assay for Salmonella spp.