Culture-independent bacterial cell extraction from fluid milk and oat-based beverage for basic qualitative microscopy

Abstract

Butterfat and protein complicate attempts to extract bacterial cells from milk by centrifugation for use in basic microscopy. Some types of bacteria preferentially separate into the butterfat layer upon centrifugation and are lost when this layer is discarded, and the action of bacterial protease enzymes can cause milk proteins to precipitate and partition into the centrifugal pellet. Butterfat and precipitated protein remaining in the centrifugal pellet along with the desired bacterial cells can confound the results of differential staining and microscopy. Oat- and other plant-based beverages, which are often manufactured by dairy processors on shared equipment, present similar hurdles to bacterial extraction and microscopic visualization because of the presence of oils, starch granules, and dietary fiber particles in these products. Herein we describe methods for centrifugal separation of bacterial cells for microscopy from unflavored milk, chocolate milk, and oat-based beverage. Cell suspensions prepared through these methods were used for phase-contrast microscopy, Gram staining, and viability staining. These techniques can be used to provide rapid, culture-independent diagnostic information when bacterial cells are expected to be present in high concentrations, as in the event of sporadic product spoilage or mass product spoilage incidents.