OXFORD MEDIUM, MODIFIED

Cat. no. G46 Oxford Medium, Modified, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. GA46 Oxford Medium, Modified, 15x100mm Plate, no logo, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Oxford Medium, Modified is recommended for the selective isolation of Listeria monocytogenes in food.

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

SUMMARY

Listeria spp. are microaerophilic, gram-positive regular, short motile rods or coccobacilli that are asporogenous, non-encapsulated, and non-branching. Motility is best observed at 20-25ºC. Listeria monocytogenes is a pathogenic organism for humans and a large number of animal species. The members of the population most at risk are neonates, the elderly and those compromised by pregnancy or an underlying illness such as malignancy, alcoholism or some condition which requires immunosuppressive procedures. Intrauterine infection of the fetus results in death, or an acutely ill infant with a septic disseminated form of listeriosis. Papular lesions of the skin may be found in listeriosis of the newborn. A similar cutaneous form has been reported in veterinarians working with infected animals. (1)

A common vehicle for Listeria monocytogenes is pasteurized milk; since the induction of the Pasteurized Milk Ordinance in 1924, there have been fewer reported cases of milk contaminants other than Listeria spp. In Massachusetts in 1983, pasteurized milk spread Listeria monocytogenes to forty-nine people, 14 of which died of septicemia. Another incidence in California in 1985, was due to contamination of a soft Mexican cheese which caused 85 deaths of 300 infected patients. This led to a re-evaluation of pasteurization and aging techniques; however, the ability of L. monocytogenes to grow between 4 and 10ºC. and over a wide pH range (4.4-9.6) complicates the issue. The most effective containment still involves post-pasteurization pathogen detection. (2)

Other types of food that have been found to contain Listeria species as a contaminant are raw milk, raw vegetables, fish, poultry, and both fresh and processed meats. Ice cream has also served as a vehicle of transmission and in 1994 shrimp from a party in New York City infected ten people including two pregnant women. The CDC recommends, for immunocompromised, pregnant or elderly individuals, that foods to avoid are: soft cheeses, cold cuts and salami. There are also some reports of nosocomial infections of Listeria monocytogenes usually among infants or immunosuppressed adults. (1)

Listeria monocytogenes is ubiquitous in nature and has been isolated from soil, mud, sewage, decaying vegetation, silage, feces, and river water. Many animal species are vulnerable to infection by Listeria species and some lactating mammals can function as carriers (with no visible symptoms) while still excreting the organisms in their milk. Sheep, cattle and goats have also been found to shed Listeria monocytogenes in their feces. Listeriosis was caused by a meat product (hot dogs) in 1999 in the United States when 101 infections caused 21 deaths. Other contaminated foods include: coleslaw, pate, jellied pork tongue, cooked chicken and smoked mussels. (3)

This Modified Oxford Medium formulation contains moxalactam (to inhibit staphylococci, bacilli and Proteus species) and colistin sulfate (to inhibit gram-negative bacilli); this is especially useful when attempting to isolate Listeria monocytogenes from samples containing a mixed bacterial flora. The selectivity is also enhanced by lithium chloride which negatively affects the growth of enterococci. The Columbia Agar Base provides amino acids, carbon, vitamins and nitrogen. The esculin in the media is hydrolyzed by Listeria spp. and the resulting compound reacts with ferric ions (from the ferric ammonium citrate) to produce 6,7-dihydroxycoumarin and blackening of the media surrounding the colonies. Agar is used in solidification of the media.

FORMULA

Ingredients per liter of deionized water:*

Columbia Agar Base 39.0gm
Lithium Chloride 15.0gm
Esculin 1.0gm
Ferric Ammonium Citrate 0.5gm
Moxalactam 20.0mg
Colistin Sulfate 10.0mg
Agar 2.0gm

Final pH 6.8 +/- 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

Consult listed references for information regarding sample preparation and processing. (3,4)

USDA METHOD

For raw meat and poultry samples:

1. Add 25gm of meat sample to 225ml of primary enrichment broth in a stomacher bag.

2. Mix in a stomacher for 2 minutes with some air trapped in the bag.

3. Incubate for 20 to 24 hours at 28 to 32ºC.

4. Streak onto Hardy Diagnostics Modified Oxford Medium and incubate at 35ºC.

5. Examine plates at 26-28 and 48 hours.

As a concurrent secondary test, 0.08 to 0.12ml of the enrichment broth may be pipetted into 10ml of Fraser Broth and examined for blackening after 24 and 48 hours of incubation at 35ºC. Positive cultures are then subcultured onto Oxford Medium, Modified and incubated for another 24 hours.

INTERPRETATION OF RESULTS

Observe plates for round 1mm colonies with a blackening of the media around them. Suspect colonies may be confirmed by CAMP test on 5% Blood Agar, by further biochemical testing, use of a macroscopic tube rapid slide test, or other means of definitive serological identification.

LIMITATIONS (3)

Since the nutritional requirements of organisms vary, some strains of Listeria may fail to grow or grow poorly on Oxford Medium, Modified.

Oxford Medium, Modified is a partially selective medium. Growth of some contaminating strains will be markedly but not totally inhibited. Poor growth and a weak esculin reaction may be seen after 40 hours incubation for some enterococci.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as stomacher, stomacher bags, loops, swabs, applicator sticks, other culture media, 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
Listeria monocytogenes
ATCC ® 7644
A 24-48hrs 35°C Aerobic Growth; blackening of media around colonies
Escherichia coli
ATCC ® 25922
B 24-48hrs 35°C Aerobic Partial to complete inhibition
Staphylococcus aureus
ATCC ® 25923
B 24-48hrs 35°C Aerobic Partial to complete inhibition

USER QUALITY CONTROL

Physical Appearance

Hardy Diagnostics Oxford Medium, Modified should appear clear, slightly opalescent, and amber in color.

REFERENCES

1. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

2. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

3. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

4. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

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

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