Evaluation of direct plating methods to enumerate Alicyclobacillus in beverages

Ten agar media were evaluated for their suitability to support spore germination and colony development by six strains of Alicyclobacillus acidoterrestris, three strains of Alicyclobacillus acidocaldarius, and one strain of Alicyclobacillus cycloheptanicus. The influence of plating method (pour versus spread), incubation temperature (43 °C and 50 °C), and incubation time (up to 10 days) on colony development were determined. K agar, Alicyclobacillus medium (ALI agar), and Bacillus acidoterrestris thermophilic (BAT) agar recovered the highest numbers of spores. Orange serum agar and Hiraishi glucose yeast extract agar were the least suitable. Overall, surface plating was superior to pour plating and, with the exception of one strain of A. acidocaldarius which grew better at 50 °C, incubation of K agar, ALI agar, and BAT agar plates at 43 °C or 50 °C resulted in recovery of equivalent numbers of spores. Essentially all viable spores were detected on media incubated for 3 days at 43 °C. The ability of one strain of each Alicyclobacillus species to grow in ten non-carbonated commercially manufactured beverages at 30 °C and 43 °C was markedly affected by the composition of the beverages. Results show that surface plating samples on BAT agar, followed by incubating plates at 43 °C for 3 days provide the most suitable conditions to enumerate ten strains of three species of Alicyclobacillus most commonly responsible for spoilage of beverages.