species do not grow well on the non-selective solid media typically used to determine total viable counts in food and beverages. As acidophiles, they require a reduced pH environment (pH ≤4.0). A number of different media have been adapted, or developed specifically to detect and/or enumerate Alicyclobacillus
, including orange serum agar (OSA), acidified potato dextrose agar, K agar, YSG agar and Bacillus acidoterrestris
medium (BAT). Various methods based on these have been developed, mainly by the beverage industry, for quality assurance purposes and suitable media are commercially available. Most of these are designed primarily to detect A. acidoterrestris
. Attempts have also been made to develop standard method protocols, and although there is as yet no universally accepted method, the International Federation of Fruit Juice Producers (IFU) Method No. 12 is widely used.
IFU method 12 includes procedures for the examination of raw materials, including process water, end products sampled immediately after processing and end products taken from the market to investigate possible spoilage.
Spores are of most concern in raw materials and the IFU method includes a heat shock treatment (typically equivalent to 80o
C for 10 min) of the sample to promote spore germination. The sample can then be plated directly onto BAT agar or another suitable medium and incubated for 3-5 days at 45o
C. Any visible colonies are presumptive Alicyclobacillus
species. If low numbers of spores are a concern the sample can be enriched in BAT broth for 2-4 days at 45o
C, or in the case of filterable samples, concentrated by membrane filtration before plating out.
Finished products after processing
Again the method is designed to detect spores, but a heat shock is not considered necessary immediately after pasteurisation. Since low numbers of spores are likely to be present, membrane filtration, pre-incubation of the finished product in its final packaging for seven days at 45o
C, or enrichment in BAT broth is recommended before the samples are plated onto solid media.
Finished products from the distribution chain
Samples can be pre-incubated for seven days at 45o
C and optional heat shock treatment and enrichment procedures may also be applied. But where spoilage is suspected, viable Alicyclobacillus
vegetative cells may be detectable by direct plating.
Confirmation and identification
Growth of selective media can be taken as a presumptive identification of Alicyclobacillus
, but conventional confirmation testing can be unreliable. One commonly used approach is to test colonies for guaiacol production, since the compound is characteristic of tainting spoilage by A. terrestris
. The method is based on the production of guaiacol from vanillin after incubation in a liquid medium and detection by the peroxidase enzyme colourimetric assay (PECA) test, which produces a brown coloured complex in the medium.
can be differentiated from A. acidocaldarius
by incubating cultures at 30, 45 and 65o
C. A. acidoterrestris
does not grow at 65o
C and A. acidocaldarius
does not grow at 30o
Conventional culture methods can take 10-12 days to detect the presence of Alicyclobacillus
species in samples and a further 24 hours to confirm guaiacol production by isolates. Identification of isolates is likely to require even longer. It is not surprising therefore that research effort has been directed at reducing the time needed to achieve a result. A number of rapid technologies have been investigated and a few tests have been developed into commercial products for the beverage industry.
Flow cytometry has been widely applied to beverages for determining total viable counts and the detection of yeasts, but its use for Alicyclobacillus
detection has also been investigated. The technique depends on the fluorescent labelling of viable cells, which are then detected by a laser. Flow cytometry is not particularly sensitive and requires at least 103
cells per ml for detection to occur. This means that a time consuming pre-incubation or enrichment step will still be required, but the 3-5 days needed to culture the bacteria can be cut to about 10 hours.
Molecular biology methods have been developed for rapid detection of Alicyclobacillus
. PCR-based detection methods have been widely investigated and at least one commercial test kit is available. Merck Millipore has developed the foodproof® Alicyclobacillus Detection Kit, which can be used with foodproof Sample Preparation Kits for real-time PCR detection of Alicyclobacillus
species. The lower limit of detection is around 100 cells per ml, and the method is not able to detect spores, so that pre-incubation or enrichment is still needed to allow germination of spores and multiplication of vegetative cells, but only for 48 hours instead of 5-7 days. Detection then takes a matter of a few hours and the test kit is also able to identify A. acidoterrestris
to species level.
Gene probe technology has also been employed to detect Alicyclobacillus
and has been developed into a commercial product in the shape of the Vermicon VIT-Alicyclobacillus
detection test. The test can be applied directly to enrichment cultures and to samples after a short (48 hours) pre-incubation, but without the need for DNA extraction. It uses fluorescent-labelled specific gene probes, which bind to rRNA within the bacterial cells during a short incubation period. Samples are then examined by fluorescence microscopy to detect and differentiate viable cells of Alicyclobacillus
species and of A. acidoterrestris
. The test produces results in three hours.
The difficulty of confirming presumptive Alicyclobacillus
detection in samples and of identifying isolates by conventional techniques has lead to the investigation of alternative methods.
Fourier transform infrared spectroscopy (FT-IR) has been used to analyse the components of the bacterial cell wall and membrane and has been shown to be capable of distinguishing between Alicyclobacillus
species and other bacteria in cultures. FT-IR is also able to distinguish between different strains of A. acidoterrestris
and to identify guaiacol-producing strains.
Molecular methods have shown promise in the identification of Alicyclobacillus
species. For example, DNA ribotyping of the genes coding for 16S and 23S rRNA has been investigated and found to be capable of identifying many isolates. DNA sequencing has also been used successfully and the rapid advances in sequencing technology may soon make this a more practical and less expensive identification technique. Micro arrays designed to analyse the complete bacterial genome have been developed and are able not only to identify different species and strains, but also to identify markers for specific characteristics, such as guaiacol production.