PALCAM AGAR

Cat. no. G149 PALCAM Agar, 15x100mm Plate, 19ml. 10 plates/bag

INTENDED USE

Hardy Diagnostics PALCAM Agar is recommended for use as a selective and differential growth medium for the cultivation, isolation and differentiation of Listeria species from food and dairy products.

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

SUMMARY

Listeria spp. are microaerophilic, gram-positive, 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 that affects humans and a large number of animal species. 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 requiring immunosuppressive procedures. There are some reports of nosocomial infections of Listeria monocytogenes , usually among infants or immunosuppressed adults. Intrauterine infection of the fetus results in death, or an acutely ill infant with a septic disseminated form of listeriosis. (1) 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)

Pasteurized milk is common vehicle for Listeria monocytogenes . Since the induction of the Pasteurized Milk Ordinance in 1924, there have been fewer reported cases of milk contaminants other than Listeria spp. 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 prevention method still involves post-pasteurization pathogen detection. (2)

Other types of food that have been found to contain Listeria species are raw milk, raw vegetables, fish, poultry, and both fresh and processed meats.The CDC recommends that immunocompromised, pregnant or elderly individuals avoid soft cheeses, cold cuts and salami.

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. Some lactating mammals can function as asymptomatic carriers while still excreting the organisms in their milk. Sheep, cattle and goats have been found to shed Listeria monocytogenes in their feces.

Van Netten et al. described a modification of Columbia Agar that provides a selective and differential medium for culturing Listeria species. (4) Columbia Agar base provides the essential nutrients required for growth. The addition of lithium chloride, acriflavine, polymyxin b and ceftazidime makes the medium selective for Listeria species. The selective agents suppress the growth of most common bacteria found in food. The addition of esculin, ferric ammonium citrate, phenol red, and mannitol make the medium differential. Listeria species hydrolyze esculin to dextrose and esculetin, which forms a dark brown to black precipitate with ferric citrate. Mannitol fermenting organisms, such as Staphylococcus , will produce acid products from mannitol. The acidic end products will turn the colonies yellow and/or produce a yellow halo around the colonies.

FORMULA

Ingredients per liter of deionized water:*

Columbia Agar Base 39.0gm
Mannitol 10.0gm
Dextrose 0.5gm
Esculin 1.0gm
Ferric Ammonium Citrate 0.5gm
Lithium Chloride 15.0gm
Phenol Red 0.08gm
Acriflavine HCL 5.0mg
Polymyxin B Sulfate 10.0mg
Ceftazidime 40.0mg
Agar 2.0gm

Final pH 7.2 +/- 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

Sample collection and preparation should be performed following appropriate standards and guidelines. (2-6)

Enrich and culture sample according to US Food and Drug Administration (FDA), US Department of Agriculture (USDA), or ISO Standards appropriate for the sample type . (2,3,5)

Method of Use: Allow plates to warm to room temperature. The agar surface should be dry before inoculating. Inoculate and streak the specimen for isolation. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface. Streak for isolation with a sterile loop. Incubate plates aerobically at 35-37 ºC. for 24-48 hours. Examine plates for colonial morphology.

INTERPRETATION OF RESULTS

Listeria colonies are gray-green with a black precipitate. Mannitol fermenting organisms have yellow colonies and/or a yellow halo around their colonies. Other organisms that develop on the media may appear as gray colonies with a brown-green halo.

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as 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 24-48hr 35°C Aerobic Growth. Gray-green with black precipitate
Escherichia coli
ATCC ® 25922
B 24-48hr 35°C Aerobic Partial to complete inhibition
Staphylococcus aureus
ATCC ® 25923
B 24-48hr 35°C Aerobic Partial to complete inhibition
Enterococcus faecalis
ATCC ® 29212
B 24-48hr 35°C Aerobic Partial to complete inhibition

USER QUALITY CONTROL

Physical Appearance

PALCAM Agar should appear slightly opalescent, and dark red in color with a slight precipitate.

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. Van Netten, Perales, Van de Moosalijk, Curtis, and Mossel. 1989. Int. J. Food Microbiol. 8:299.

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

6. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

7. 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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