APPLIED AND ENVIRONMENTAL , Feb. 1977, p. 485-487 Vol. 33, No. 2 Copyright © 1977 American Society for Microbiology Printed in U.S.A. Plating Medium for Differentiating arizonae from Other Salmonellae ALLAN M. LITTELL Food and Drug Administration, Brooklyn, New York 11232 Received for publication 18 June 1976

A differential plating medium developed for isolation of Salmonella arizonae produces a uniform reaction for both lactose-negative and -positive S. arizonae and differentiates S. arizonae from other salmonellae. Salmonella arizonae, capable of causing salmonellae exhibited H.S production on this food-borne infections in man (3), can be easily medium. Some Citrobacter strains are also ca- overlooked on plating media used for detecting pable of H,S production and incapable of dulci- Salmonella. This is because 61.3% (2) of S. tol utilization. Such strains will be confused arizonae strains utilize lactose within 24 to 48 with S. arizonae. H,S-positive Proteus can also h, whereas almost all of the other Salmonella be confused, since none use dulcitol. Therefore, strains are incapable of using this carbon sucrose was added to the plating medium to source. This large percentage of S. arizonae make it more selective for S. arizonae, since producing acid from lactose can be easily con- most strains do not use this disaccharide. The fused with lactose-fermenting microorganisms strains of Citrobacter and Proteus that utilize such as Escherichia coli and Citrobacter sp. this sugar would give an acid reaction similar Most plating media used routinely in Salmo- to that of salmonellae. nella analysis use lactose utilization as an im- In preliminary studies, it was found that the portant differential characteristic. This prop- addition of 0.1% glucose gave faster colonial erty alone, however, is of limited value for the growth, thus allowing better differentiation at analysis of S. arizonae. 24 h. This small amount of glucose is rapidly A plating medium was developed that pro- utilized by both S. arizonae and other salmo- duces a uniform reaction for both lactose-nega- nellae without observable acid production. tive and positive S. arizonae strains and also The organisms used in evaluation of the me- differentiates S. arizonae from many other dium were obtained from the culture collections gram-negative organisms, including most other of the Food and Drug Administration's Brook- salmonellae. This medium was patterned after lyn laboratory and the National Center for Dis- that used for Hektoen enteric agar (4). Salicin ease Control, Atlanta, Ga. The cultures were and lactose were deleted from the formulation, grown in brain heart infusion broth 24 h before and malonate, dulcitol, and glucose were testing. The medium was initially tested by added. All other ingredients, including sucrose, streaking 24-h cultures of S. arizonae, salmo- were the same as in Hektoen enteric agar, ex- nellae, and also strains of H,S-positive Citro- cept that the indicator phenol red was substi- bacter and Proteus on plates, which were then tuted for bromothynol blue and acid fuchsin. incubated for 24 to 30 h at 35°C. The formulation ofS. arizonae agar is shown in The appearance of typical colonies is de- Table 1. scribed in Table 2. The medium readily distin- The rationale for these modifications is based guishes S. arizonae from other salmonellae. on differences in the ability of the two groups to Some gram-negative organisms can be mis- utilize dulcitol, malonate, and lactose. The ma- taken for S. arizonae on the new medium. Of jority of S. arizonae utilize lactose and malo- particular concern are those organisms that nate but not dulcitol, whereas the majority of produce H-,S and fail to use dulcitol or sucrose. salmonellae use dulcitol but not lactose or mal- Examples are certain strains of Citrobacter, onate. The utilization of malonate by S. ari- Proteus, and Pseudomonas putrefaciens, which zonae caused an alkaline reaction on the new is the only H.,S-producing pseudomonad. plating medium, turning the indicator pinkish- The ability of the medium to recover S. ari- red. Salmonellae produced acid from dulcitol, zonae from foods and feeds was tested with forming a yellow precipitate due to precipita- samples inoculated with known strains of S. tion of the bile salts. Both S. arizonae and other arizonae both as a pure culture and as mixed 485 486 NOTES APPL. ENVIRON. MICROBIOL. cultures (combined with salmonellae or with is shown in Table 3. With the media used rou- salmonellae and Citrobacter). Twenty-five- tinely, colonies are normally picked only if they gram samples of mushroom powder, oregano, conform to that described for salmonellae or pumpkin seeds, white pepper, fish meal, and lactose-negative S. arizonae. With bismuth flour were placed in 225 ml of lactose broth and sulfite agar, lactose-positive S. arizonae colo- inoculated with approximately 100 cells from nies could possibly be picked; however, they an appropriate dilution of a 24-h brain heart cannot be distinguished from other Salmonella infusion broth culture. The inoculated products and can be missed if high numbers of other were incubated and analyzed for Salmonella Salmonella are also present. The new medium according to procedures of the Association of was formulated specifically for S. arizonae, and Official Analytical Chemists (AOAC) (1), ex- only colonies exhibiting alkaline (red) reactions cept that the S. arizonae agar, instead of the with H.2S production should be picked. The prescribed plating media, was streaked. The other four media should be used for the isola- isolates were confirmed as S. arizonae by tion of other potential Salmonella. AOAC biochemical and serological procedures. A total of 94 food and feed samples were In every case, the inoculated strains of S. ari- tested (Table 4). S. arizonae were detected in zonae were easily recovered in the presence of five samples using the new medium, whereas other inoculated organisms and the natural the routine agars detected S. arizonae from flora in the product. Uninoculated controls only two of the samples. The S. arizonae that were all negative for both S. arizonae and other were missed from the other three samples using salmonellae. the routine agars were all lactose positive. Of The S. arizonae agar medium has been in- the two samples from which S. arizonae were cluded as an additional plating medium along detected on the routine agars, one isolate was with bismuth sulfite, brilliant green, salmo- lactose negative; the other was lactose positive nella-, and Hektoen enteric agars, and was picked only from bismuth sulfite which are normally used for Salmonella analy- plates. sis in this laboratory. A comparison of colonial characteristics for salmonellae and S. arizonae TABLE 2. Colonial appearance on S. arizonae agar at 24 h Group of organisms Colonial appearance TABLE 1. Formulation of S. arizonae agar, S. arizonae (lactose Pinkish-red, black centered Ingredient Amt (g) + and lactose - colonies, 2-3 mm in diame- strains) ter. The medium surround- Proteose peptone (Difco) ...... 12 ing the colony is pinkish- Yeast extract ...... 3 red. Bile salts no. 3 (Difco) ...... 9 Sucrose ...... 12 Other salmonellae Dark-centered, yellow colo- Malonic acid disodium salt ...... 6 nies, surrounded by a yel- Dulcitol ...... 20 low precipitate. Slow dulci- Glucose ...... 1 tol-utilizing strains give Sodium chloride ...... 5 weak acid reactions at 24 h Sodium thiosulfate ...... 5 and typical reactions at 30 Ferric ammonium citrate ...... 1.5 h. Agar ...... 14 Phenol red ...... 0.04 Citrobacter and Resembles salmonellae if Distilled water ...... 1,000 ml Proteus (H2S + dulcitol or sucrose is fer- strains) mented. Resembles S. ari- a Do not autoclave. Add ingredients, heat to boil- zonae if neither substrate ing, adjust pH to 7.1 to 7.2 (pinkish-red color), and is utilized. pour plates as desired.

TABLE 3. Comparison of colonial descriptions on various plating media Colonial appearance Medium Salmonellae S. arizonae Lac- S. arizonae Lac+ Brilliant green Pinkish-red Pinkish-red Yellow-green Salmonella-shigella Uncolored, black centered Uncolored, black centered Red, black centered Bismuth sulfite Black Black Black Hektoen enteric Green, black centered Green, black centered Salmon, dark centered S. arizonae Yellow, dark centered Red, black centered Red, black centered VOL. 33, 1977 NOTES 487 TABLE 4. Results ofSalmonella analysis offood and used routinely for isolation of Salmonella. It feed samples using S. arizonae agar along with will, however, serve to differentiate S. arizonae routinely used plating media from the other salmonellae in those cases No. positive for S. ari- where this information is desired. zonae (This paper was presented at the 76th An- Product Total nual Meeting of the American Society for Mi- tested S. ar- Routine zonae crobiology, 2-7 May 1976, Atlantic City, New agar agars Jersey.) Frozen frog legs 40 4 2 ACKNOWLEDGMENTS Nut meats 14 1 0 Frozen foods 5 0 0 I thank James D. Macmillan, of the Food and Drug Animal feeds 9 0 0 Administration and Rutgers University, for his invaluable Chocolate products 7 0 0 assistance in the preparation of this manuscript. The com- Spices 4 0 0 ments and suggestions of Leonard W. Mestrandrea and Noodle products 8 0 0 Leonard D. Fantasia of the Food and Drug Administration Soup mixes 7 0 0 are also gratefully acknowledged. Total 94 5 2 LITERATURE CITED a Numbers refer to samples containing S. arizonae as 1. Association of Official Analytical Chemists. 1975. Offi- confirmed biochemically and serologically by AOAC meth- cial methods of analysis, 12th ed. Association of Offi- ods. cial Analytical Chemists, Washington, D.C. 2. Edwards, P. R., and W. H. Ewing, 1972. Identification of . Burgess Publishing Co., Min- On the basis of the work reported here, S. neapolis. arizonae agar proved superior to the four agars 3. Edwards, P. R., M. A. Fife, and C. H. Ramsey. 1959. routinely used in a Salmonella analysis for the Studies on the Arizona group of Enterobacteriaceae. Bacteriol. Rev. 23:155-174. isolation of S. arizonae from foods and feeds. It 4. King, S., and W. I. Metzger. 1968. A new plating me- should be emphasized that the new medium dium for the isolation of enteric pathogens 1. Hektoen will not serve as a replacement for the media enteric agar. Appl. Microbiol. 16:577-578.