Buffered Listeria Enrichment Broth

Cat. no. U233 Buffered Listeria Enrichment Broth Base, 12oz Wide Mouth Jar, 225ml 12 jars/box
Cat. no. K183 BLEB Selective Supplement, 15x103mm Tube, 3.4ml** 3.4ml
** Sold separately.

INTENDED USE

Hardy Diagnostics Buffered Listeria Enrichment Broth is recommended for the selective enrichment of Listeria monocytogenes from food samples.

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.(1,2)

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 to 9.6) further complicates the issue. The most effective containment still involves post-pasteurization pathogen detection.(3)

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.(4)

The Buffered Listeria Enrichment Broth follows the Food and Drug Adminstration Bacteriological Analytical Manual 8th edition formulation.(5) This medium contains digests of soybean meal and casein, which provide amino acids and other nitrogenous substances. Sodium chloride is added to maintain the osmotic equilibrium. Dextrose is incorporated as an energy source. The dipotassium phosphate, monopotassium phosphate and disodium phosphate are included in the formulation as buffers to maintain the pH. Protein, B vitamins and other growth nutrients are supplied by yeast extract. Pyruvic acid aids in resuscitating organisms that are stressed or injured. Cycloheximide inhibits saprophytic fungi while the selective agents, nalidixic acid, and acriflavine act to inhibit gram-negative and gram-positive organisms, respectively.

FORMULA

Ingredients per liter of deionized water:*

Buffered Listeria Enrichment Broth (Cat. no. U233):
Pancreatic Digest of Casein 17.0gm
Sodium Chloride 5.0gm
Papaic Digest of Soybean Meal 3.0gm
Dextrose 2.5gm
Dipotassium Phosphate 2.5gm
Yeast Extract 6.0gm
Monopotassium Phosphate 1.35gm
Disodium Phosphate 9.6gm
Pyruvic Acid 1.1gm

Final pH 7.3 +/- 0.2 at 25ºC.

Ingredients per liter (after addition to the Buffered Listeria Enrichment Broth):*

BLEB Selective Supplement (Cat. no. K183) not included:**
Nalidixic Acid 40.0mg
Cycloheximide 50.0mg
Acriflavine HCl 10.0mg

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

** Sold separately.

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, 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 (5)

1. Sample possibly contaminated food lots.

2. If required by FDA field laboratory instructions, sub-samples may be composited.

3. Pre-enrich by placing the 25gm food portion in Hardy Diagnostics Buffered Listeria Enrichment Broth Base for four hours at 30ºC.

4. At the fourth hour, add 3.4ml of the BLEB Selective Supplement (Cat. no. K183 sold separately).

5. Continue incubation for selective enrichment at 30ºC. for a total of 48 hours.

6. At 24 and 48 hours, streak BLEB culture onto one of the following esculin-containing selective isolation agars: either Modified Oxford Medium (Cat. no. G46) or PALCAM (Cat. no. G149) or LPM (Cat. no. G92).

INTERPRETATION OF RESULTS(5)

Listeria colonies will appear black with a black halo on esculin-containing media.

For further identification of purified isolates, consult listed references for details on the following optional tests:

1. Perform a motility test either by wet mount or using Motility Test Medium (Cat. no. Q10) or SIM medium (Cat. no. Q30). The motility pattern should give a typical umbrella-shaped growth pattern. Pick colonies from cultures incubated at 30 degrees C. or less. Listeria spp. are slim, short rods with slight rotating or tumbling motility. Always compare with known culture. Cocci, large rods, or rods with rapid, swimming motility are not Listeria spp.

2. Test for catalase using catalase reagent (Cat. no. Z62 and Z76). Listeria species are catalase-positive.

3. Gram stain 16 to 24 hour cultures. All Listeria spp. are short, gram-positive rods; however, with cultures older than 24 hours, the Gram stain reaction can be variable.

4. Perform xylose-rhamnose reaction tests. Inoculate the following carbohydrates as 0.5% solutions in purple carbohydrate broth (with Durham tubes): dextrose (Cat. no. Y104), maltose (Cat. no. Y109), rhamnose (Cat. no. Y112), mannitol (Cat. no. Y110), and xylose (Cat. no. Y117). Incubate 7 days at 35ºC. Listeria spp. produce acid with no gas and a positive reaction. All species should be positive for dextrose and maltose. All Listeria spp. except L. grayi should be mannitol-negative. L. monocytogenes does not utilize xylose and is positive for rhamnose utilization.

5. Perform CAMP (Christie-Atkins-Munch-Peterson) test. Other organisms needed are a beta-hemolytic Staphylococcus aureus and/or Rhodococcus equi . Examine plates for hemolysis after incubation. L. monocytogenes and L. seeligeri should show increased hemolysis near the S. aureus streak. L. ivanovii has enhanced hemolysis near the R. equi streak.The other species are non-hemolytic. Quality control with known a isolate of Listeria spp. on a separate sheep blood agar plate is encouraged to verify the procedure was done correctly.

6. Consult list of references for approved rapid methods of identification.

7. Listeria monocytogenes isolates may require subtyping by the FDA (serological or genetic).

LIMITATIONS

Other bacterial species may exhibit similar black to brown coloring on the esculin-containing media but will take longer than 48 hours to do so.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, BLEB Selective Supplement (Cat. no. K183), 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 24-48hr 35°C Aerobic Growth
Enterococcus faecalis
ATCC ® 29212
B 18-24hr 35°C Aerobic Partial inhibition
Escherichia coli
ATCC ® 25922
B 24-48hr 35°C Aerobic Inhibited
Saccharomyces cerevisiae
ATCC ® 9763
B 24-48hr 35°C Aerobic Inhibited

USER QUALITY CONTROL

Physical Appearance

Buffered Listeria Enrichment Broth should appear clear, and light to medium amber in color, with a slight precipitate.

BLEB Selective Supplement should appear clear and yellow in color, with a possible precipitate. (sold separately)

REFERENCES

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

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

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

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

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

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

012617gr