LACTOBACILLI MRS AGAR

Cat. no. G117 Lactobacilli MRS Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. G197 Lactobacilli MRS Agar, 15x60mm Plate, 12ml 10 plates/bag
Cat. no. G179 Lactobacilli MRS Agar with Cycloheximide, 15x60mm Plate, 11ml 10 plates/bag
Cat. no. U204 Lactobacilli MRS Agar, 500ml Polycarbonate Bottle, 400ml 10 bottles/box
Cat. no. U276 Lactobacilli MRS Agar, 1L Polycarbonate Bottle, 500ml 10 bottles/box

INTENDED USE

Hardy Diagnostics Lactobacilli MRS Agar is recommended for the isolation, enumeration and cultivation of Lactobacillus spp.

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

SUMMARY

Lactobacilli MRS Agar was developed by researchers deMan, Rogosa and Sharpe as an alternative non-selective media for the cultivation of fastidious lactobacilli. Previous media for the cultivation of lactobacilli employed the use of tomato juice, however, tomato juice agar was undesirable because of its variability and difficulty in preparation. A media described by Rogosa, Mitchell and Wiseman, although adequate for most lactobacilli, was found to be unsatisfactory for use with some dairy lactobacilli organisms. With this in mind, deMan, Rogosa and Sharpe hoped to develop a new and general non-selective growth medium for lactobacilli. They found that the inclusion of Tween ® 80, citrate and acetate resulted in improved growth for lactobacilli. Manganese and magnesium are inorganic ions necessary for growth in the presence of citrate. (1) This media shows a low degree of selectivity; therefore, secondary accompanying microflora may grow well and compete for nutrients. However, most of these accompanying microorganisms can be inhibited by the addition of various concentrations of selective agents, such as cycloheximide, polymyxin, thallium acetate, sorbic acid, acetic acid, or sodium nitrite to the medium.

Lactobacilli are long, slender, non-sporeforming, gram-positive rods that are generally facultatively anaerobic, most of which grow well with reduced oxygen tension and increased CO 2 . (2) Lactobacilli are important microorganisms for the dairy, food, and beverage industry. Microbial spoilage of fruit juice is most commonly due to aciduric organisms such as lactic acid bacteria and yeast. (3) Lactic acid organisms are important to the dairy industry for determining the cause of acid defects in dairy products as well as in evaluating the lactic starter cultures in cured cheese and cultured milks. (4) Lactobacillus brevis is a contaminating organism in the production of beer that, if present, can be responsible for its spoilage. (6) These lactobacilli damage beer by causing turbidity and a poor flavor due to diacetyl, a strongly flavored by-product of their metabolism. (5) Finally, lactobacilli are used by the vegetable food industry for the fermentation of cabbage to sauerkraut. (5)

FORMULA

Ingredients per liter of deionized water:*

Dextrose 20.0gm
Peptic Digest of Animal Tissue 10.0gm
Beef Extract 10.0gm
Yeast Extract 5.0gm
Sodium Acetate 5.0gm
Dipotassium Phosphate 2.0gm
Ammonium Citrate 2.0gm
Tween ® 80 1.0gm
Magnesium Sulfate 0.1gm
Manganese Sulfate 0.05gm
Agar 15.0gm

In addition, Lactobacilli MRS Agar with Cycloheximide contains the following ingredient per liter:*

Cycloheximide 0.1gm

Final pH 6.5 +/- 0.3 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

Hardy Diagnostics Lactobacilli MRS Agar can be used in several ways. These plates can be streaked directly or used with a membrane filter for plate counts. Lactobacilli MRS Agar plates are incubated at 35ºC. for 24-72 hours in a CO 2 incubator or under microaerophilic conditions (5% carbon dioxide, 5-10% oxygen). Lower temperatures of 22-25ºC. may be used for psychrotrophic counts or 42ºC. for thermophilic counts.

It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification.

INTERPRETATION OF RESULTS

Lactobacilli appear as large clear colonies after 24-72 hours incubation at 35ºC. in an enhanced CO 2 environment.

LIMITATIONS

Lactobacilli MRS Agar has a low degree of selectivity allowing for the growth of other lactic acid organisms such as Pediococcus and Leuconostoc species. Presumptive Lactobacillus colonies can be subcultured to Hardy Diagnostics Lactobacilli MRS Broth for further identification.

Lactobacilli MRS Agar with cycloheximide can be used to inhibit the overgrowth of accompanying microbial flora such as yeasts and molds.

It is important to maintain the appropriate moisture content of the plates during incubation. Do not allow the surface of the plates to dry out as this will inhibit the growth of lactobacilli due to an increasing concentration of acetate on the agar surface.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, 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 delbrueckii
ATCC ® 7830
A 24-72hr 35°C CO 2 ** Growth; clear colonies
Lactobacillus fermentum
ATCC ® 9338
A 24-72hr 35°C CO 2 ** Growth; clear colonies
Lactobacillus acidophilus
ATCC ® 4356
A 24-72hr 35°C CO 2 ** Growth; clear colonies
Lactobacilli MRS Agar with Cycloheximide is also tested with the following organisms:
Candida albicans
ATCC ® 10231
A 24-72hr 35°C CO 2 ** Growth
Staphylococcus aureus
ATCC ® 25923
A 24-72hr 35°C CO 2 ** Partial to complete inhibition

User Quality Control

PHYSICAL APPEARANCE

Lactobacilli MRS Agar should appear slightly opalescent, and medium amber in color.
Lactobacilli MRS Agar with Cycloheximide should appear slightly opalescent, and light to medium amber in color.

L. acidophilus growing on Lactobacilli MRS Agar

Lactobacillus acidophilus (ATCC ® 4356) colonies growing on Lactobacilli MRS Agar (Cat. no. G117). Incubated in CO 2 for 72 hours at 35ºC.

Lactobacilli MRS Agar

Uninoculated plate of Lactobacilli MRS Agar (Cat. no. G117).

REFERENCES

1. De Man, J.C., Rogosa, M., Sharpe, M. Elisabeth. 1960. A Medium for the Cultivation of Lactobacilli. J. Appl. Bact.; 23:130-135.

2. MacFaddin, Jean F. Biochemical Tests for Identification of Medical Bacteria, Lippincott Williams & Wilkins, Philadelphia, PA.

3. Vanderzant, Carl., and Splittstoesser, Don F. 1992. Compendium of Methods for the Microbiological Examination of Foods, 3rd ed. American Public Health Association, Washington, D.C.

4. Marshall, Robert T. 1992. Standard Methods for the Examination of Dairy Products, 16th ed. American Public Health Association, Washington, D.C.

5. Doyle, Michael P., Beuchat, Larry R., and Montville, Thomas J. 1997. Food Microbiology Fundamentals and Frontiers, 1st ed. American Society for Microbiology, Washington, D.C.

6. Yasui, T., and Yoda, Y. 1997. Imaging of Lactobacillus brevis Single Cells and Microcolonies with a Microscope by an Ultrasensitive Chemiluminescent Enzyme Immunoassay with a Photon-counting Television Camera. Appl. Environ. Microbiol.; 63:4528-4533.

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

8. Committee of Revision for The United States Pharmacopeia. 2000. The United States Pharmacopeia, 24th rev. United States Pharmacopeial Convention, Rockville, MD.

9. American Public Health Association. 1992.

10. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.

ATCC is a registered trademark of the American Type Culture Collection.
Tween is a registered trademark of ICI Americas, Inc.

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