Table

Composition of Minimal Mineral Media for Isolation of the Alicyclobacillia

Ingredient

Ref. 3

Ref. 5

Ref. 1

Refs. 13, 23

Ref. 40

Ref. 44

Refs. 45, 54

CaCl2-2H2O

0.1g

0.25 g

0.25 g

0.25 g

0.25 g

0.25 g

0.25 g

FeSO4-7H2O

0.025 g

0.28 mg

KH2PO4

1.0g

3.0g

3.0g

3.0 g

0.6g

3.0g

3.0g

MgSO4-7H2O

0.5g

0.5g

0.5g

0.5 g

0.5g

0.5g

0.5g

MnCl2-4H2O

1.25 mg

MnSO44H2O

0.025 g

NaCl

1.0g

Na2HPO412H2O

2.5g

(NH4)2SO4

0.2g

0.2g

0.2 g

0.2g

0.2g

0.2g

NH4Cl

1.0g

ZnSO47H2O

0.4mg

Glucose

5.0g

0.1-1.0g

1.0g

5.0 g

1.0g

1.0g

5.0g

Glycerol

0.1-1.0g

Peptone

Ribose

0.1-1.0g

Soluble starch

2.0 g

2.0 g

Tween 80

Yeast extract

0.1 g

2.0 g

1.0g

1.0g

2.0 g

2.0 g

Deionized water

1000 ml

1000 ml

1000 ml

1000ml

1000 ml

1000 ml

1000 ml

Trace elementsb

1 ml

1 ml

Biotin

10 ^g

Agar

15g

15-20 g

15-20 g

a Media are typically acidified with organic or mineral acids to pH 3.5 to 4.0 after autoclaving.

b Solution of 0.66 g/l CaCl2-2H2O, 0.30 g/l Na2MoO4-2H2O, 0.18 g/l ZnSO47H2O, 0.18 g/l CoCl26H2O, 0.16 g/l CuSO45H2O, 0.15 g/l MnSO4-4H2O, 0.10 g/l H3BO3.

is diluted, heat shocked for 10 minutes at 80°C, incubated at 45°C for 7 days, and plated onto an acidic, low-nutrient medium (BAT agar reported by Eguchi et al. [45]; see Table 7.3). Growth at 45°C after 5 days is microscopically examined to exclude yeasts, followed by restreaking onto BAT and PCA. After incubation at 45°C, strains that grow on BAT but not PCA are considered probable alicyclobacillus although "further bio- and genotyping of suspect alicyclobacillus" is encouraged to ensure the identification.

The most recent edition of the Compendium of Methods for the Microbiological Examination of Foods describes a direct plating and enrichment procedure [57]. While the protocols are fundamentally sound, the use of a mineral-containing medium in addition to, or in place of, K agar is suggested. Additionally, direct plating of concentrate is not recommended since higher Brix levels may inhibit colony formation. In the enrichment protocol, the heat shock at 90° C is too severe and should be reduced to a range of 70 to 80°C.

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