STANDARD METHODS AGAR WITH BROMCRESOL PURPLE

Cat. no. G143 Standard Methods Agar with Bromcresol Purple,
15x100mm Plate, 18ml
10 plates/bag

INTENDED USE

Hardy Diagnostics Standard Methods Agar with Bromcresol Purple is a non-selective medium recommended for the growth, enumeration and differentiation of acid producing bacteria. This media is recommended by the American Public Health Association (APHA) to recover sub-lethally injured Lactobacilli from pasteurized dairy products.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Standard Methods Agar (SMA) is a modified formulation of Tryptone Glucose Skim Milk Agar developed by Bowers and Hucker. (5) Yale showed that SMA is more effective in plate count procedures on milk and dairy products than Tryptone Glucose Skim Milk Agar. In addition, SMA is equivalent to Plate Count Agar (Tryptone Glucose Yeast Agar) as listed by the APHA and USP. (1-4) When combined with a dye indicator such as Bromcresol Purple, Standard Methods Agar is useful in detecting microorganisms in cultured dairy products.

Lactic acid bacteria found in dairy products are a broad and diverse group, consisting primarily of Streptococcus, Lactococcus, Leuconostoc, and fermentative species of Lactobacillus . Lactobacilli may become sub-lethally injured after pasteurization, making them difficult to culture and isolate. Consequently, the APHA in the Standard Methods for the Examination of Dairy Products recommends the use of SMA with Bromcresol Purple for differentiating lactic acid bacteria using the "standard plate count" method on dairy products. (6) Furthermore, lactobacilli in young cheeses and dairy products, such as liquid milks, cottage cheeses, hard and soft cheeses, cultured milks, uncultured products containing added cultures and lactic starter cultures, should be enumerated and differentiated using a relatively non-selective medium, such as Standard Methods Agar with Bromcresol Purple, to retrieve potentially heat-injured cells.

Hardy Diagnostics Standard Methods Agar with Bromcresol Purple contains peptone, yeast extract and glucose which promote the growth of bacterial colonies. B-complex vitamins are primarily supplied by yeast extracts. Bromcresol purple is a colorimetric indicator used to facilitate the differentiation of acid producing bacteria.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 5.0gm
Yeast Extract 2.5gm
Glucose 1.0gm
Bromcresol Purple 0.02gm
Agar 15.0gm

Final pH 7.0 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. 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

Specimen Collection: Consult listed references for information on specimen collection and processing of food, dairy, water samples, and other materials of sanitary significance. (1-8)

Samples suspected of containing lactic acid bacteria must be handled with care as these microorganisms are easily injured by adverse environments. Prepare dilutions of dairy products using peptone water.

Prior to inoculation, allow prepared media to warm to room temperature.

Spread Plate Method:

1. Prepare decimal dilutions using sterile diluent to obtain a final plate containing 30-300 CFUs.

2. Aseptically inoculate the agar surface with 0.1ml of well mixed desired dilution sample.

3. Using a sterile spreading device, distribute the inoculum evenly over the entire surface of the medium.

4. Incubate plates in a 5-10% CO 2 atmosphere for 48 +/- 2 hours at 35ºC.

INTERPRETATION OF RESULTS

Following incubation, examine plates for growth. Count the number of colonies and express in number of colony forming units (CFU) per gram or milliliter of sample, taking into account the dilution factor. If duplicate plates were used, express an average for the two plates in terms of the number of microbes per gram or milliliter of sample. Consult listed references for additional information on interpretation and enumeration of microbial growth. (1-8)

Acid produced during fermentation will lower the pH of the medium and yield a color change. The transition range for bromcresol purple is pH 5.2 to 6.8. At low pH, bromcresol purple will show colonies growing on a yellow medium. At a high pH, the medium will remain purple.

LIMITATIONS

Standard Methods Agar with Bromcresol Purple is a non-selective medium. Therefore, it is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, buffered peptone, sterile diluent, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Lactobacillus acidophilus
ATCC ® 4356
A 48hr 35°C CO 2 ** Growth; yellow
Lactobacillus casei
ATCC ® 393
A 48hr 35°C CO 2 ** Growth; yellow
Lactobacillus fermentum
ATCC ® 9338
A 48hr 35°C CO 2 ** Growth; yellow

** Atmosphere of incubation is enriched with 5-10% CO 2 .

USER QUALITY CONTROL

Physical Appearance

Hardy Diagnostics Standard Methods Agar with Bromcresol Purple should appear clear, slightly opalescent, and purple in color.

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Water and Wastewater , APHA, Washington, D.C.

2. Association of Official Agricultural Chemists, 10th ed. p. 737; 1965.

3. Association of Official Analytical Chemists. Official Methods of Analysis , AOAC, Washington, D.C.

4. The United States Pharmacopeia , 26th rev. 2002. United States Pharmacopeial Convention, Rockville, MD.

5. Bowers and Hucker. 1944. Tech. Bull ., p. 228. N.Y. State Exp. Station.

6. Richardson, Standard Methods for the Examination of Dairy Products , 16th ed. 1992. APHA, Washington, D.C.

7. Speck. Compendium of Methods for the Microbiological Examination of Foods , 3rd ed. 1992. APHA, Washington, D.C.

8. U.S. Food and Drug Administration. 1995. Bacteriological Analytical Manual , 8th ed. AOAC, Arlington, VA.


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