AK #2 Agar

Cat. no. U189 AK #2 Agar, 8oz. Glass Bottle, 200ml 12 bottles/box

INTENDED USE

Hardy Diagnostics AK #2 Agar is recommended for use as a culture medium for the preparation of spore suspensions used in the detection of antibiotic residues (such as penicillin and sulfa drugs) in milk and dairy products.

SUMMARY

Arret and Kirshbaum developed AK #2 Agar for use in the production of spores of Bacillus subtilis , ATCC ® 6633 for use in the Penicillin Milk Test. (3) The medium is also detailed in the spore preparation phase of the American Public Health Association (APHA) disk assay procedure for the detection of sulfa drugs and antibiotics in milk. (1)

Hardy Diagnostics' AK #2 Agar contains peptones and beef extract for nitrogen, sulfur, amino acids, essential vitamins and minerals. Yeast extract is a source of B vitamins. Dextrose provides the energy source for replication and manganous sulfate is important in promoting sporulation.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Gelatin 6.0gm
Pancreatic Digest of Casein 4.0gm
Yeast Extract 3.0gm
Beef Extract 1.5gm
Dextrose 1.0gm
Manganous Sulfate 0.3gm
Agar 15.0gm

Final pH 6.6 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

In the preparation of spore suspensions when performing the FDA procedure for the Penicillin Milk Test: (3,4)

1. Transfer a culture of Bacillus subtilis , ATCC ® 6633 monthly to a fresh plate or slant of AK #2 Agar and incubate cultures at 35 +/- 2ºC. for 18 hours.

2. Wash the growth with 2 to 5ml of Saline, 0.85% (Cat. no. K59) onto the agar surface and spread the sample across the entire surface of the medium. Note: The use of sterile glass beads may facilitate the removal of growth from the agar surface.

3. Incubate the sample for 18-24 hours at 35 +/- 2ºC. and then at room temperature for the remainder of 1 week (6 days).

4. Harvest the spores and cells by washing the growth from the agar surface with 5ml Saline, 0.85% (Cat. no. K59) into a sterile centrifuge tube. Gently swirl the container to loosen the growth, being careful not to break the agar. Sterile glass beads may be used to facilitate the removal of growth from the agar surface.

5. Centrifuge the suspension, and decant and discard the supernatant. Resuspend the sediment in Saline, 0.85% and heat shock the cells at 70ºC. for 30 minutes. The resulting spore suspension can be stored refrigerated for several months. Consult listed reference for testing procedures utilizing the spore suspension. (3)

In the preparation of spore suspension in performing the APHA procedure for detection of sulfa drugs and antibiotics in milk: (1,4)

1. Transfer a culture of Bacillus megaterium , ATCC ® 9855 to the surface of AK #2 Agar and streak the inoculum evenly to distribute the cells.

2. Incubate the sample at 35 +/- 2ºC. for 18-24 hours and then at room temperature for the remainder of 1 week (6 days).

3. Harvest the spores and cells by washing the growth from the agar surface with 2 to 5ml of Butterfield's Phosphate Buffer (Cat. no. K109). Gently swirl the container to loosen the growth, being careful not to break the agar. Note: The use of sterile glass beads may facilitate the removal of growth from the agar surface.

4. Aseptically transfer the suspension into a sterile centrifuge tube and heat the tube in a boiling waterbath (100ºC.) for 10 minutes.

5. Centrifuge the suspension at 5ºC. for 20 minutes at 20,000 x G and decant the supernatant. Wash the sediment and resuspend it using fresh Butterfield's Phosphate Buffer and centrifuge again under the same parameters. Repeat the wash step two more times.

6. Store the suspension in Butterfield's Phosphate Buffer and refrigerate until use. Consult listed reference for the proper procedure before use. (1)

INTERPRETATION OF RESULTS

Consult listed references for more information. (1,3) It is expected that AK #2 Agar will yield large numbers of bacterial spores.

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, saline 0.85% (Cat. no. K59), Butterfield's Phosphate Buffer (Cat. no. K109), incinerators, waterbath, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bacillus subtilis
ATCC ® 6633
A 18-48hr 35°C Aerobic Growth

USER QUALITY CONTROL

PHYSICAL APPEARANCE

AK #2 Agar should appear clear with a slight opalescence, and light to medium amber color.

REFERENCES

1. American Public Health Association. 1993. Standard Methods for the Examination of Dairy Products, 16th ed. APHA, Washington, D.C.

2. Anderson, N.L., et al. 2005. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

3. Arret, B. and A. Kirshbaum. 1959. A Rapid Disc Assay Method for Detecting Penicillin in Milk. J. Milk Food Technol. ; 22:329-331.

4. Dey, B.P., C.A. White, R.H. Reamer and N.H. Thaker. 1998. USDA/FSIS Microbiology Laboratory Guidebook, 3rd ed. www.fsis.usda.gov/science/microbiological_Lab_Guidebook/ .

5. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.


ATCC is a registered trademark of the American Type Culture Collection.

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