Selective Cultivation 171 Selective Enrichment

Microorganisms of public health significance associated with foods are usually present as a small minority of the total microbial population. For both detection and quantitation of such microorganisms, selective enrichment cultivation is usually undertaken. Selective enrichment involves conditions of cultivation that favor the development of the target organism over that of the usual majority of extraneous microorganisms. Selective agents are often derived from the environment in which such organisms of public health significance are found. Most culture media for selective enrichment of E. coli make use of the fact that the organism is a common inhabitant of the intestinal tract of mammals and hence is normally in contact with surface-active agents such as bile salts, which are frequently used as selective agents for the organism. Bile salts are notably inhibitory to many Grampositive organisms. Gram-negative bacteria, in general, are significantly more tolerant of various dyes compared to Gram-positive bacteria, resulting in the incorporation of a variety of dyes for the enrichment cultivation of numerous Gram-negative organisms. This difference in sensitivity to bile salts and dyes is due to the presence of lipopolysaccharide in the external membrane of Gram-negative bacteria that is absent in Gram-positives.

Incubation temperature has also been used successfully with selective chemical agents for the selective cultivation and enumeration of certain bacteria. The use of 45.5°C for the most probable number (MPN) enumeration of E. coli using the culture broth known as EC functions selectively in conjunction with the content of 0.15% bile salts no. 3. Selective enrichment of pathogenic vibrios such as Vibrio vulnificus and Vibrio parahaemolyticus makes use of the fact that they are marine organisms and hence are quite tolerant of the somewhat alkaline pH of seawater, which is usually about 7.8. This tolerance toward an alkaline growth environment extends to a pH of 8.5 to 8.7, which is the usual initial pH range used for enrichment cultivation of these organisms. The natural habitat for Staphylococcus aureus is the human skin. The level of sodium chloride on the skin surface during physical exertion under warm weather conditions is often at or near saturation, when one considers the process of evaporation. S. aureus, as would be expected, exhibits a significant level of salt tolerance, resulting in the use of enrichment and isolation media for the organism containing 7.5-10% NaCl. NaCl at a level of 7.5% is notably inhibitory to most Gram-positive and Gram-negative bacteria.

A wide variety of antibiotics is now added to media for selective isolation of bacterial pathogens from foods. Yeasts and molds are closely associated with acid fruits. The use of acidified culture media (pH 3.5-5.5) is a long established and highly effective method of inhibiting most bacteria, while allowing unrestricted growth of yeasts and molds.

The successful use of selective enrichment media is predicated on cultivation and enumeration of undamaged cells. Cells of a target organism that have undergone membrane damage through freezing and thawing or heating will often not survive the stress imposed by a selective culture medium originally developed using undamaged cells. The enumeration of damaged cells therefore usually requires initial growth in a nonselective repair medium prior to transfer to a selective medium.

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