17.6.03 H2O, and mix thoroughly.

At time agar is added to tube, place clean iron

AOAC Official Method 972.45 strip or nail in each tube. No adjustment of reaction is necessary.
Thermophilic Bacterial Spores in Sugars Prepare medium and Na2SO3 solution, if used in place of solid Na2SO3,
Microbiological Method fresh weekly. Autoclave medium 20 min at 121°C and cool to 55°C.
First Action 1972
D. Culture Technique
Final Action 1989
(a) Flat sour spores.—Into 5 separate Petri dishes, pipet 2 mL
(Sugar, both beet and cane, may carry spores of all 3 groups of
boiled sugar solution. Cover and mix inoculum with glucose
thermophilic bacteria that are important as spoilage agents in
tryptone agar. Incubate plates 35–48 h at 55°C and, to prevent
low-acid canned foods, i.e., flat sour bac te ria [Ba cil lus
drying of agar, humidify incubator. Combined count from 5 plates
stearothermophilus], thermophilic anaerobes not producing H2S
represents number of spores in 2 g original sugar. Multiply this count
[Clostridium thermosaccharolyticum], and sulfide spoilage bacteria
by 5 to express results in terms of number of spores/1 0 g sugar.
or thermophilic anaerobes producing H2S [C. nigrificans]. These
Characteristic colonies are round, 1–5 mm in diameter, with
bacteria are not of health significance, but excessive numbers may
typical opaque central “spot,’’ and, usually, surrounded by yellow
survive commercial heat processes.)
halo in field of purple. This halo may be insignificant or missing
A. Sampling with certain low acid-producing types or if plate is so thickly seeded
Take 225 g (0.5 lb) laboratory samples from 5 separate bags or that entire plate has yellow tinge. Typical subsurface colonies are
barrels of shipment or lot, place in clean containers, and seal. compact and may approach “pin point” conditions.
Sample liquid sugar by drawing 5 separate 200–250 mL (6–8 oz) If identity of subsurface colonies is doubtful, observe nature of
portions during pumping transfer from tank trucks to storage tanks surface colonies. If they show reasonable purity of formed flora,
or at refinery during filling of tank trucks. assume that subsurface colonies have been formed by similar
Number of laboratory samples will vary in relation to size of bacterial groups. If plate is heavily seeded, counts may not be
shipment or lot. If there is significant variability in lot, this fact will accurate and colony structure and size may be atypical. If plates are
become evident, in majority of cases, through individual tests on the so heavily seeded that counting is impractical, dilute original
5 laboratory samples. solution and repeat procedure.
B. Preparation of Test Portion To determine if typ ical subsurface colonies are flat sour
organisms, apply streak from colonies to agar plates to determine
(a) Dry sugar.—Place 20 g test portion in sterile 150–250 mL
surface characteristics.
Erlenmeyer marked to indicate 100 mL. Add sterile H2O to 100 mL
mark. Bring rapidly to bp, and boil 5 min. Replace liquid evaporated (b) Thermophilic anaerobes not pro duc ing hy dro gen
with sterile H2O. sulfide.—Divide 20 mL boiled sugar solution equally among 6 liver
broth tubes and stratify liquid medium with plain nutrient agar. After
(b) Liquid sugar.—Add test portion containing 20 g dry sugar,
agar has solidified, preheat to 55°C and incubate 72 h at that
determined on basis of °Brix (e.g., 29.41 g 68° Brix [%] liquid sugar
temperature.
is equivalent to 20 g dry sugar), to sterile 250 mL flask and proceed
as in D(a). Thermophilic anaerobes not producing H2S are identified by
splitting of agar, presence of acid, and, occasionally, cheesy odor.
C. Culture Media Method is suitable as qualitative test but provides only rough
(a) Glucose tryptone agar.—For detection of flat sour bacteria. estimation; results cannot be expressed as number of spores/unit
Use commercially standardized dehydrated medium (Dextrose weight sugar.
Tryptone agar) preferably, or prepare as follows: Suspend 10.0 g (c) Sulfide spoilage bacteria.—Divide 20 mL boiled sugar
tryptone, 5.0 g glucose, 15.0 g agar, and 0.04 g bromocresol purple solution equally among 6 freshly exhausted tubes containing
in 1 L H2O, and mix thoroughly. Final pH should be 6.7 ± 0.1. modified sulfite agar. Incubate 48 h at 55°C.
Autoclave 30 min at 121°C and cool to 55°C. In sulfite agar, sulfide spoilage bacteria form characteristic
(b) Liver broth.—For detection of thermophilic anaerobes not blackened spherical areas. Due to solubility of H2S and its fixation
producing H2S (C. thermosaccharolyticum). Mix 500 g chopped by Fe, no gas is noted. Some thermophilic anaerobes not producing
beef liver with 1 L H2O. Slowly boil mixture 1 h, adjust to ca pH 7.0, H2S generate relatively large amounts of H2, which splits agar and
and boil ad di tional 10 min. Press boiled ma te rial through reduces sulfite, thereby causing general blackening of medium. This
cheesecloth and dilute liquid to 1 L. To broth, add 10.0 g peptone and condition, however, is readily distinguishable from restricted
1.0 g K2HPO4, and adjust to pH 7.0. To test tube, add 1–2 cm blackened area mentioned above. Count blackened areas to obtain
previously boiled ground beef liver and 10–12 mL broth. Sterilize quantitative results.
20 min at 121°C. Before using medium, unless freshly prepared,
E. Reporting Results
exhaust by subjecting to flowing steam ≥20 min, and, after
Report flat sour and sulfide spoilage results as number of
inoculation, stratify with 5–6 cm layer of plain nutrient agar
spores/10 g sugar. Report thermophilic anaerobes not producing
(common formula) that has been cooled to 50°C.
(c) Sulfite agar, modified.—For detection of sulfide spoilage H2S as number of tubes positive or negative (+ or −).
bacteria. Suspend 10.0 g tryptone, 1.0 g Na2SO3, and 20.0 g agar in 1 L References: JAOAC 19, 438(1936); 21, 457(1938); 55, 445(1972).

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