Technical Data Sheet

GranuCult®
BAT broth acc. IFU Method No. 12

Ordering number: 1.07993.0500

For the enrichment of spore-forming thermo-acidophilic spoilage bacteria (Alicyclobacillus spp.) from
juice and juice-related products and their ingredients, environmental samples including process water
and other materials in the area of food production and food handling.

This culture medium complies with the specifications given by IFU Method No. 12.

BAT broth is named from Bacillus acidoterrestris (BAT), the former name of Alicyclobacillus
acitoterrestris.

Mode of Action

BAT broth contains glucose as a carbon and energy source. Yeast extract is source for vitamins,
particularly the B-group. Potassium dihydrogen phosphate acts as a buffering system. The low pH
value and the high incubation temperature inhibit the contaminating flora. This medium contains many
trace elements which supply the specific requirements of the spore-forming thermo-acidophilic
spoilage bacteria (Alicyclobacillus spp.).

Following the latest revision of IFU method No. 12 of 2019, the toxic components cobalt(II) chloride
hexahydrate and boric acid have been removed from the formulation.

Typical Composition
GranuCult®
Specified by IFU Method No. 12
BAT broth acc. IFU Method No. 12
Yeast extract 2 g/l Yeast extract 2 g/l
Glucose anhydrous 5 g/l D(+) glucose 5 g/l
Calcium chloride dihydrate 0,25066 g/l Calcium chloride 0.25066 g/l
Magnesium sulfate
0,5 g/l Magnesium sulfate 0.5 g/l
heptahydrate
Ammonium sulfate 0,2 g/l Ammonium sulfate 0.2 g/l
Potassium dihydrogen Potassium dihydrogen
3,0 g/l 3 g/l
phosphate phosphate
Zinc sulfate monohydrate 0,00018 g/l Zinc sulfate 0.00018 g/l
Copper sulfate
0,00016 g/l Copper sulfate 0.00016 g/l
pentahydrate
Manganese sulfate hydrate 0,00015 g/l Manganese sulfate 0.00015 g/l
Sodium molybdate
0,00030 g/l Sodium molybdate 0.00030 g/l
dihydrate
Water 1000 ml/l Water n/a
pH at 25 °C 4.0 ± 0.2 pH at 25 °C 4.0 ± 0.2
Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Version 2019-12-02 Page 1 of 4

Preparation

Dissolve 11.0 g in liter of purified water, adjust the pH to 4.0 ± 0.2 by adding 1N H2SO4 and autoclave
(15 min. at 121 °C).

The prepared medium is clear and yellowish.

Experimental Procedure and Evaluation

Depend on the purpose for which the medium is used.

Following the procedure for detection given by IFU Method No. 12, prepare the sample and dilute it
using BAT broth, followed by the heat treatment at 80 °C for 10 min.

Incubate for 5 d at 45 °C ± 1 °C aerobically.

Disperse 0,1 ml of the incubated enrichment onto the surface of a plate containing GranuCult® BAT
agar (article number 1.070094.0500) or alternatively using 1 ml of the enrichment with poured plate
method using BAT agar.

Incubate 48 h ± 4 h at 45 °C ± 1 °C aerobically. If growth occurs, proceed with confirmation. If there

is no growth after 48 h ± 4 h, return the plate for further incubation at 45 °C ± 1 °C aerobically for
additional 72 ± 4 h.

Examine the plates for the presence of colonies.

BAT media support the growth of all currently known species of Alicyclobacillus spp. It is therefore
likely that a wider range of colony types will be visible on these media.

For the confirmation of presumptive Alicyclobacillus spp. from the detection procedure take at least
one colony. One confirmed isolate per sample is sufficient for the detection procedure. If the first
colony is negative, take further colonies up to a maximum of five colonies.

From each selected colony, streak a portion onto one plate containing a culture medium with neutral
pH, e.g. GranuCult® Plate Count agar (article number 1.05463.0500), and one plate containing
GranuCult® BAT agar (article number 1.070093.0500).

Incubate both plates for 72 h ± 4 h at 45°C ± 1 °C aerobically.

Spore-forming thermo-acidophilic bacteria (Alicyclobacillus spp.) negative isolates will show growth
on both culture media.

Spore-forming thermo-acidophilic bacteria (Alicyclobacillus spp.) positive isolates will show growth
only on the BAT agar and no growth on the culture medium with the neutral pH, e.g. Plate Count agar.

Storage

Store at +15 °C to +25 °C, dry and tightly closed. Do not use clumped or discolored
medium. Protect from UV light (including sun light). For in vitro use only.

According IFU Method No. 12, self-prepared medium can be stored in closed containers at 5 °C ± 3 °C
in the dark for up to 4 weeks.
Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Version 2019-12-02 Page 2 of 4

Quality Control

Expected
Function Control strains Incubation Method of control
results

Alicyclobacillus
acidoterrestris
DSM 2498
Alicyclobacillus
acidoterrestris up to 5 days Growth
DSM 3922 Qualitative single tube
Productivity at 45 ± 1 °C, good to very
method (turbidity)
Alicyclobacillus aerobic good
cycloheptanicus
DSM 4006
Alicyclobacillus
hesperidium
DSM 12766

Please refer to the actual batch related Certificate of Analysis.

The performance test is in accordance with the current version of IFU Method No. 12 and EN ISO 11133.

Literature

IFU International Fruit and Vegetable Juice Association. Method on the detection and enumeration of
spore-forming thermo-acidophilic spoilage bacteria (Alicyclobacillus spp.). Method of Analysis No.
12:2019.

ISO International Standardisation Organisation. Microbiology of food, animal feed and

water - Preparation, production, storage and performance testing of culture media. EN ISO
11133:2014+Amd1:2018.

Deinhard, D., Blanz P., Pooralla K. and Alatan, E. (1987) Bacillus acidoterrrestris sp. nov., a new
thermotolerant acidophile isolated from different soils. system. Appl. Microbiol. 10 (1), 47-53.

Fritze, D. and Pukall, R. (2012): Culture media for Bacillus spp. and related Genera
Relevant to Foods. In: Handbook of Culture Media for Food and Water Microbiology. (Corry,
J.E.L., Curtis, G.D.W. and Baird, R.M. eds). pp. 90 – 114. Royal Society of Chemistry,
Cambridge, UK.

Smit, Y., Cameron, M., Venter, P. and Witthuhn, R.C. (2011) Alicyclobacillus spoilage and isolation — A
review. Food Microbiol. 28 (3), 331-349.

Steyn, C.E., Cameron, M. and Witthuhn, R.C. (2011) Occurrence of Alicyclobacillus in the fruit
processing environment — A review. Int. J. Food Microbiol. 147 (1), 1-11.

Yokota, A., Fuji T., and Goto K. (eds) Alicyclobacillus: Thermophilic Acidophilic Bacilli. Springer, Japan,
First Edition, 2007.

Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Version 2019-12-02 Page 3 of 4

Ordering Information

Product Cat. No. Pack size

GranuCult® BAT broth acc. IFU Method No. 12 1079930500 500 g

GranuCult® BAT agar acc. IFU Method No. 12 1079940500 500 g

GranuCult® Plate Count agar acc. ISO 4833, ISO 17410 and
1054630500 500 g
FDA-BAM

20 x 55 mm
ReadyPlate™ 55 Plate Count Agar 1467630020
plates

600
Cellulose mixed ester filter: individually
HAWG047S6
S-Pak® filters 0,45 μm, 47 mm, white gridded sealed filters,
sterile

4 bands of
Cellulose mixed ester filter:
EZHAWG474 150 sterile
EZ-Pak® filters 0,45 μm, 47 mm, white gridded
filters

EZ-Stream® Vacuum Pump EZSTREAM1

EZFITBASE 1,
EZ-Fit® Manifold
3, 6

EZ-Pak® Dispenser Curve EZCURVE01

Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Version 2019-12-02 Page 4 of 4