Colonies produced by enrichment or direct plating on low pH media must be subjected to appropriate analytical techniques to confirm their identification as alicyclobacillus. Deciding which analytical techniques are considered appropriate is the basis of controversy that has yet to be adequately addressed. A microscopic examination is necessary to confirm that the isolate is not fermentative yeast or another type of acidophilic organism, and that it is a Gram-positive spore-forming bacterium. The sporogenous nature of the isolate may require further plating and incubation on other media followed by spore staining and microscopic observations.
Confirmation activities include growth on low pH media at elevated temperatures (45 to 55°C) with concurrent lack of growth on media of neutral pH, such as traditional plate count agar or nonacidified minimal media, and lack of growth at reduced temperatures. One possible protocol is to streak suspect colonies onto duplicate plates of low pH and neutral pH media. One plate of each is incubated at 45 to 50°C is incubated and the remaining plates at 20 to 25°C for appropriate time periods. Observation of growth at 45°C and little or no growth at 25°C on low pH agar with no growth on the remaining plates at either temperature is consistent with the genus alicyclobacillus. Further testing is suggested for final confirmation.
The most solid proof of identity is obtained from sequence analysis of species-specific DNA or RNA segments, such as for the 16S ribosomal subunit, coupled with observations of basic phenotypic characteristics (ther-moacidophilic, Gram-positive bacterial spore-former). Ribotyping has been investigated by the National Food Processors Association for identification of the alicyclobacilli with limited success. A protocol for use of randomly amplified polymorphic DNA was developed by Yamazaki et al.  to identify A. acidoterrestris. Further research is required by other laboratories to verify the use of these technologies.
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