U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
December 9, 1999

This method, developed for the next edition of the FDA Bacteriological Analytical Manual (BAM), should be read in conjunction with the current BAM (Edition 8, Revision A, 1998) and/or the Official Methods of Analysis of AOAC International (Edition 16, Revision 5) to which it refers for further information on food sample preparation, the appearance of typical, as well as atypical, Salmonella colonies, and reactions on triple sugar iron agar (TSI) and lysine iron agar (LIA) slants. As in all testing, Salmonella positive and negative culture controls should be performed alongside test samples. In addition to the positive control cultures (typical Salmonella), 3 additional Salmonella cultures are recommended to assist in the selection of atypical Salmonella colony morphology on selective agars. These cultures are a lactose-positive, H₂S-positive S. arizonae (ATCC 12325) and a lactose-negative, H₂S negative S. abortus equi (ATCC 9842); OR a lactose-positive, H₂S-negative S. diarizonae (ATCC 29934). These cultures may be obtained from the American Type Culture Collection, P.O. Box 3605, Manassas, VA 20108-0971, USA.

Sample preparation

As a food for consumption by the immunocompetent adult population, orange juice is sampled as a Food Category II commodity; a total of thirty 25mL analytical units is examined. If intended for consumption by immunodeficient individuals, particularly the aged, the infirm, and infants, orange juice is sampled as a Food Category I commodity; a total of sixty 25mL analytical units is examined. (See BAM, Chapter 1.)

Pre-enrichment

Add a 25 mL analytical unit of orange juice to 225 mL Universal Pre-enrichment (UP) broth (1:9 sample-to-broth ratio) in a sterile, wide-mouth, screw-capped jar (500 mL) or other appropriate container. The UP broth may be made from its individual ingredients, or the commercially available medium may be used. If a composite is analyzed, then the composite should be added to UP broth at a 1:9 sample-to-broth ratio (e.g., for 15 analytical units, 375 mL orange juice should be added to 3375 mL UP broth) in a sterile appropriate container as described above. Swirl the flask contents thoroughly. Do not adjust the pH. Incubate for 24 ± 2 h at 35 ± 2°C.

Selective enrichment

After incubation of the pre-enrichment mixture, remove a 1 mL aliquot to a 10 mL volume of tetrathionate (TT) broth, vortex, and incubate for 24 ± 2 h at 35 ± 2°C. Also, remove a 0.1 mL aliquot to a 10 mL volume of Rappaport-Vassiliadis (RV) medium, vortex, and incubate for 24 ± 2 h at 42 ± 0.2°C. The RV medium should be incubated in a circulating, thermostatically-controlled water bath.

Selective plating

Streak a 3 mm (10 mL) loopful from each incubated selective enrichment onto bismuth sulfite (BS), Hektoen enteric (HE), and xylose lysine desoxycholate (XLD) agar plates. Incubate the selective agar plates for 24 ± 2 h at 35 ± 2°C.

After incubation, pick a minimum of 2 typical presumptive-positive Salmonella colonies from each of the 3 plating agars to TSI and LIA slants.

If there are no typical colonies present on the HE and XLD plates, then pick a minimum of 2 atypical colonies to the TSI and LIA slants. Incubate the TSI and LIA slants for 24 ± 2 h at 35 ± 2°C. If typical or suspicious colonies are not present on BS agar after 24 ± 2 h, then do not pick any colonies.

Irrespective of whether or not BS agar plates are picked at 24 ± 2 h, re-incubate BS agar plates an additional 24 ± 2 h. If no colonies were picked from BS agar after 24 ± 2 h or if the TSI and LIA reactions from colonies picked from BS agar at 24 ± 2 h give atypical reactions, then pick a minimum of 2 typical colonies (or a minimum of 2 atypical colonies, if typical colonies are not present) from the BS agar plates after 48 ± 2 h incubation.

(See BAM, Chapter 5, or the AOAC sec. 967.26 for the appearance of colonies and the interpretation of TSI and LIA reactions).

Confirmation of presumptive positive isolates

Growth from presumptive-positive TSI slants should be screened biochemically as recommended by the BAM Salmonella culture method (ch. 5) or by the AOAC Salmonella culture method (sec. 967.27). An AOAC-approved biochemical identification kit may be used in place of the conventional biochemical tube system recommended by the AOAC/BAM. The AOAC-approved test kits include the following: API 20E^®, Enterotube II^®, Enterobacteriaceae II Set (sec. 978.24); MICRO ID^® (sec. 989.12); Vitek GNI (sec. 991.13). Isolates must be confirmed serologically as recommended by the AOAC/BAM method (sec. 967.28).

Preparation of UP broth

Formula per liter:

Add the above ingredients to one liter distilled or deionized water and heat gently to dissolve completely. Sterilize at 121-124°C for 15 min. Final pH 6.3 ± 0.2 at 25°C.