Microbiological Methods

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Tests for microbiological quality and safety of frozen desserts and their ingredients are described in Standard Methods for the Examination of Dairy Products (Marshall, 1993), the Compendium of Methods for the Microbiological Examination of Foods (Vanderzant and Splittstoesser, 1992), and the Official Methods of Analysis (Cuniff, 1999). Tests most relied on to reflect overall microbiological quality have been the standard plate count (aerobic plate count) and the coliform count. Petrifilm (3M Health Care, St. Paul, MN) methods of performing each of these counts are given official status in standard methods and can be substituted for the plating methods. Furthermore, the spiral plating method for determination of the total aerobic plate count is an officially recognized method in standard methods.

Methods for enumeration of microorganisms are classified in Standard Methods for the Examination of Dairy Products (SMEDP) and in the official methods manual of AOAC International. Classification is based on three criteria: (a) research that thoroughly evaluates the method, (b) collaborative testing in qualified laboratories, and (c) demonstration of applicability based on extensive use. The AOAC decides whether these qualifications have been met and awards a method first action status when it has been thoroughly evaluated and collaboratively tested; final action status is assigned when those methods have been proven in extensive use. The SMEDP classification terminology for these methods is A2 and A1, respectively. Recently, two additional classifications have been added. Class A3 applies to methods approved after meeting criteria set by the United States Conference on Interstate Milk Shipments for milk produced and shipped under provisions of the U.S. Pasteurized Milk Ordinance. Class A4 apples to methods granted Performance Tested status after evaluation by AOAC International Research Institute (Wehr, 2001).

As given in Standard Methods for the Examination ofDairy Products (Marshall, 1993), the agar method for enumerating coliform bacteria in ice cream products calls for making a 1:2 or 1: 10 dilution and distributing 10 mL of this dilution equally into three Petri dishes to which is added Violet Red Bile Lactose Agar. Matushek et al. (1992) showed that dilution of ice cream produced more accurate results than did direct plating. The major reason for inaccuracies with the direct plating method was atypical colonies produced with the directly plated samples. Non-lactose-fermenting bacteria can ferment sugar contained in plating media to which ice cream or frozen desserts are added, producing false-positive tests. The lower the dilution of the sample, the greater the concentration of sugar in the medium and the greater the chance for false-positive results (red colonies arise when acid is produced from fermentable sugar in the medium). Confirmation of suspect colonies as coliforms can be done by incubation in brilliant green bile lactosebroth in which coliformbacteriaproduce gas when incubated at 32°C. False-negative results can occur when ingredients of frozen desserts inhibit growth. Inaccuracies may occur when excess product on a plate causes overcrowding (more than approximately 150 colonies), resulting in colonies that are less than 0.5 mm in diameter. Finally, pipeting undiluted sample cannot be done with precision because of the high and variable viscosities of frozen dessert mixes.

The official procedure (Marshall, 1993) for enumerating coliform bacteria with the Petrifilm method calls for making a 2:3 dilution of ice cream and plating

0.5 mL of this dilution onto one or each of three prehydrated coliform count plates. Experiments by Matushek et al. (1992) demonstrated that the Petrifilm coliform count method was highly satisfactory with higher confirmation rates (94-100%) than any of the other methods tested.

Freezing of desserts produces dead, injured, and fully viable cells. Many factors interact to determine the fate of microorganisms on freezing. The common practice used to differentiate injured from uninjured cells is to plate a sample on an inhibitory medium such as Violet Red Bile Agar (VRBA) (used to enumerate coliform bacteria) as well as a productive but nonselective medium such as Tryp-ticase Soy Agar. The injured cells among the survivors of freezing will grow on the nonselective medium but not on the selective agar, whereas noninjured cells will grow on both. This principle has been used in the Modified VRBA procedure of SMEDP in which the sample is plated in 10 mL of Tryptic Soy Agar (TSA). After solidification, the base medium is overlaid with an equal amount of double-strength VRB agar. The remainder of the test is unchanged from the VRBA procedure. The bile salts, neutral red, and crystal violet in the double-strength VRB agar diffuse into the TSA providing the needed inhibition of noncoliform bacteria.

Testing of 353 environmental samples taken at four ice cream and six liquid milk plants by Cotton and White (1990) failed to show a relationship between standard plate count, coliform count, or psychrotrophic bacteria count and the presence of L. monocytogenes, but high counts by these methods were associated with the presence of Yersinia enterocolitica (P < .01).

New tests for small numbers of pathogens in frozen desserts are being made possible by technological developments. For example, E. coli 0157:H7 was recovered and identified within 10 h at concentrations as low as 1 cfu/g of ice cream (Gooding and Choudary, 1997). Samples were enriched in Tryptic Soy Broth for 4 h, captured by immunomagnetic separation, amplified by polymerase chain reaction of parts of the verotoxin genes (SLT-I and SLT-II), and detected by agarose gel electrophoresis.

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