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17.3.07 (b) SSD.

—5 mm diameter polyethylene discs containing
AOAC Official Method 992.30 5-bromo-4-chloro-3-indolyl-β- D -galactopyranoside and
Confirmed Total Coliform and Escherichia coli 4-methylumbelliferyl-β- D -glucuronide (MUG). Available as
in All Foods ColiComplete Substrate Supporting Disc from BioControl Systems, Inc.,
Substrate Supporting Disc Method (ColiComplete®) 12822 SE 32nd St, Bellevue, WA 98005, USA.
First Action 1992 (c) Butterfield’s buffered phosphate diluent.—(1) Stock solu-
Final Action 1996 tion.—Dissolve 34.0 g KH2PO4 in 500 mL H2O, adjust to pH 7.2
(Applicable to simultaneous enumeration of confirmed total with ca 175 mL 1M NaOH, and dilute to 1 L. Store in refrigerator.
coliform and E. coli in all foods. Not applicable to E. coli strain (2) Diluent.—Dilute 1.25 mL stock solution to 1 L with H2O. Pre-
O157:H7.) pare dilution blanks with this solution, dispensing enough to allow
for losses during autoclaving. Autoclave 15 min at 121°C.
See Table 992.30 for the results of the interlaboratory study sup- D. Test Sample Preparation
porting the acceptance of the method. (Prepare all decimal dilutions with 90 mL sterile diluent plus
A. Principle 10 mL previous dilution unless otherwise specified. Shake all dilu-
Substrate supporting discs (SSD), containing tions 25 times in 30 cm arc. Pipets must accurately deliver required
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and volume; do not use to deliver <10% of their total volume.)
4-methylumbelliferyl-β-D-glucuronide (MUG), are used to determine lev- (a) Frozen and/or prepared foods and raw ingredients.—Use
els of total coliforms and E. coli. Discs are added to lauryl sulfate tryptose balance with capacity of ≥2 kg and sensitivity of 0.1 g to aseptically
broth (LST) inoculated with selected dilutions of food samples. Test sam- weigh 50 g unthawed (if frozen) test sample into sterile blender jar.
ples are incubated at 35°C and examined after 24 and 48 h for confirmed Add 450 mL diluent, (c)(2), and blend 2 min at high speed. (If neces-
total coliform count and after 30 h for confirmed E. coli presence. sary to temper frozen test sample to remove 50 g portion, hold ≤18 h
β-Galactosidase, from coliforms present in food samples, cleaves at 2–5°C.) Not more than 15 min should elapse from time test sample
X-Gal into 5-bromo-4-chloro-indoxyl intermediate which under- is blended until all dilutions are made in appropriate media.
goes oxidation to yield water-insoluble blue dimer, visually detect- If entire test sample consists of <50 g, weigh portion equivalent to
1 test sample and add volume of sterile diluent required to make 1:10
able on disc or in surrounding medium as confirmed positive result 2

of total coliform activity. dilution. Total volume in blender jar must completely cover blades.
β-Glucuronidase, from E. coli present in food samples, cleaves (b) Tree nut meat halves and larger pieces.—Aseptically weigh
MUG into glucuronide and methylumbelliferone which fluoresces 50 g test portion into sterile jar. Add 50 mL diluent, (c)(2), and
under longwave UV light (366 nm) as confirmed positive result of E. shake vigorously (50 times through 30 cm arc) to obtain 100 dilu-
tion. Let stand 3–5 min and shake just before making serial dilu-
coli presence. β-Glucuronidase has been found specific to genus
tions and inoculations.
Escherichia (Escherichia and Shigella) and some Salmonella. Prac-
(c) Nut meal.—Aseptically weigh 10 g test portion into sterile jar.
tically, from most food samples, only E. coli yields positive results.
Add 90 mL diluent, (c)(2), and shake vigorously (50 times through
This method will not detect E. coli O157:H7.
30 cm arc) to obtain 101 dilution. Let stand 3–5 min and shake to re-
B. Apparatus suspend just before making serial dilutions and inoculations.
(a) Pipets.—1.0 and 10.0 mL, with 0.1 mL graduations. E. Analysis
(b) Blender.—2-speed model with explosion-proof motor, capa-
Inoculate LST tubes with appropriate test portion dilution series se-
ble of high-speed operation 16 000–18 000 rpm. One glass or metal
lected to determine levels or presence/absence of total coliforms and E.
blender jar (1 L capacity) with cover for each analytical unit.
coli in test sample. Aseptically add 1 SSD/tube. Incubate tubes at 35°C.
(c) Vortex mixer.
(d) Incubator.—Capable of maintaining 35–37°C. (1) For total coliforms.—After ≥ 24 h incubation, examine tubes
(e) UV light source.—366 nm, handheld, 6 watts, is recom- for visually detectable blue color on disc or in surrounding medium.
mended. Wear safety glasses when light is on. Presence of detectable blue color indicates confirmed positive total
coliform result. (Note: A wide range of blue color intensity may be
C. Media and Reagents expected, depending on test sample composition and microflora. All
Ingredients and reagents used to prepare the following media may be blue reactions are positive regardless of intensity of color.)
obtained from any manufacturer. For convenience, commercial dehy- Reincubate at 35°C for additional 24 h; reexamine. Continued ab-
drated media equivalent to formulation may be used. Commercial me- sence of blue indicates negative result; presence of blue color indi-
dia should be tested with known coliforms (Enterobacter, Klebsiella cates confirmed positive result for total coliforms.
spp.) and E. coli prior to use to verify growth promotion properties. (2) For E. coli.—Examine tubes from step 1 under UV light
Tubes used for media preparations should be examined under UV light (366 nm) after 30 ± 2 h from start of initial incubation. (Caution: Do
(366 nm) to confirm lack of autofluorescence prior to use. not look directly into light source.) Fluorescent tubes indicate con-
Before autoclaving, adjust media pH, as necessary, by adding 1M firmed positive result for E. coli.
NaOH or 1M HCl, to attain stated post-autoclaving pH. Use pH me- (3) Controls.—Known positive E. coli and negative control tubes
ter standardized against standard buffers, 964.24 (see A.1.04). must be included to facilitate interpretation of MUG fluorescent reac-
(a) LST.—Dissolve 20.0 g trypticase or tryptose (pancreatic di- tion. Negative controls must include both non-E. coli/coliform tube
gest of casein), 5.0 g NaCl, 5.0 g lactose, 2.75 g K2HPO4, 2.75 g (Enterobacter or Klebsiella spp.) and uninoculated media tube.
KH2PO4, and 0.1 g sodium lauryl sulfate in l L H2O, mixing over low
Reference: J. AOAC Int. 76, 988(1993).
heat, if necessary. Dispense 10 mL portions into 20 ×150 mm tubes.
Autoclave 15 min at 121°C. Final pH must be 6.8 ± 0.1. Revised: June 2000

 2000 AOAC INTERNATIONAL

0 Frozen vegetablesa Low 1.568 23.Table 992.331 0.3 Lot 2 2.265 0.433 52.328 0.946 0.5 Lot 2 1.227 0.901 0. coli Dry egg Low 1.898 0.355 0.592 21.7 Lot 2 2.801 0.  2000 AOAC INTERNATIONAL .2 Nut meats Low 1.418 29.341 0.1 a Frozen broccoli.315 0.319 0.30 Interlaboratory study results for confirmed total coliform and E.931 0.2 Lot 3 3.364 0.5 Lot 3 3. % RSDR.571 0.342 0.8 High 1.2 46.361 0.2 High 2.3 43.8 Lot 3 2.7 24.308 0.6 43.1 20.1 Raw liquid milk Lot 1 2.194 0.423 346 504 Medium 0.362 0.327 0.391 0.817 0.173 0.9 High 2.287 0.725 0.7 47.1 22.767 0.288 0.631 16.671 0.0 High 0.696 42.1 Nut meats Low 0.570 0.562 36.8 43.6 32.1 57.596 8.8 21.3 Lot 3 3.227 0.347 20.126 0.491 36.084 0.941 12.3 Lot 2 2.8 36.1 Frozen vegetablesa Low 0.242 0.3 Lot 3 1.363 0.9 Lot 2 2.280 13.2 Lot 2 3.976 0.1 48.619 0.551 15.328 0.356 0.693 0.522 13.663 0.601 0.834 0.650 19.398 0.506 35.354 27.8 45.84 20.250 0.88 Pork sausage (run 2) Lot 1 0.051 0.325 0.0 27.074 0.398 6.587 0.0 29.325 0.2 21.46 17.4 Ground beef Lot 1 3.8 19.8 36.6 E.9 17.6 35.8 Medium 1.332 11.5 Raw liquid milk Lot 1 1.78 18.1 29.558 13.500 14.567 14.939 0.3 20.680 0.367 21.8 30.829 0.8 23.3 Lot 2 1.626 18.671 0.253 0.270 0.6 Lot 3 1.114 0.666 29.8 28.577 27.7 28.334 0.605 0.3 Medium 1.9 Lot 3 1.436 0.751 0.621 25.988 0.3 37.5 Pork sausage (run 1) Lot 1 2.007 0.5 23.403 0.325 0.1 Pork sausage (run 1) Lot 1 2.458 0.720 0.559 0.7 Medium 1.3 21.4 37.189 0.8 37 3 Lot 2 2. % Total coliforms Dry egg Low 1.147 0.833 14.178 0.010 0.9 Pork sausage (run 2) Lot 1 0.221 0.5 Medium 1.814 0.498 10.7 17.351 0.443 0.302 0.596 7.302 18.757 12.248 34.1 Lot 3 2.681 0. ColicompleteÒ substrate supporting disc method Product Level Mean log/g sr sR RSDr.79 10.4 67.3 21.399 0.309 24.4 High 2.1 Medium 2.7 33.147 4.343 0.725 71.679 42. coli in all foods.226 0.436 20.88 4.396 0.661 0.7 Ground beef Lot 1 1.291 0.952 10.8 High 2.667 9.457 13.