Significance of the presumptive coliform test as applied to orange juice.

The presumptive test for the determination of coliforms in food products is based on the inoculation of tubes of lactose broth with specified amounts of the material (A.P.H.A., 1946). Fermentation of the sugar with formation of gas indicates a positive presumptive test for coliforms. This test was originally developed for the examination of water and milk samples, but in recent years it has been used for the examination of many food products regardless of differences in their chemical composition. With the phenomenal development of frozen concentrated orange juice, the presumptive test has been widely employed by regulatory and industry laboratories in the examination of these products for coliforms. Orange juice is a complex mixture of chemicals including carbohydrates, free amino acids, vitamins, enzymes, and other compounds. For example, approximately 76 per cent or higher of the soluble solids of orange juice is carbohydrate. McCready et al. (1950), using chromatographic techniques, found that most of this carbohydrate is sucrose, dextrose, and levulose in the ratio of 2:1:1. Therefore, an orange juice containing 10 per cent, or more, of these fermentable sugars is not uncommon. When 10 ml of lactose broth is inoculated with 1 ml of orange juice, two-thirds of the sugars in the inoculated broth may be other than lactose. It is not improbable that gas produced in such tubes could result from the fermentation of the sugars from the juice by microorganisms which are unable to attack lactose. It is a common experience for the citrus products microbiologist to find high presumptive coliform indices in orange samples which are either negative for the completed coliform test or have a low coliform index. Martinez and Appleman (1949) noted that a large number of positive presumptive tests resulted when coliform determinations were run on 62 samples of frozen orange juice from which no coliforms were isolated. Although they did not discount the possibility that fermentation of the orange juice sugars might have caused some of their spurious results, they attributed many of their positive presumptives to lactose-fermenting yeasts which produced a sheen on eosin-methylene blue agar. 1'Western Utilization Research Branch, Agricultural Research Service, United States Department of Agriculture. Wolford (1950) found that the presumptive coliform index in 78 out of 79 samples tested was more than ten times the completed index obtained. In a later study, Wolford (1954) obtained 2,232 positive presumptives out of a total of 3,422 lactose broth tubes inoculated with orange juice or surface rinses of oranges. False positives accounted for 1,297 of the 2,232 positives. Fermentation of the orange juice sugars was postulated as playing a part in the production of the many false positive presumptive tests. The present paper reports on an investigation undertaken to determine the extent to which fermentation of the sugars and/or other substances in the juice may play a part in the presumptive test for coliform organisms in orange juice.