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LISTERIA SELECTIVE AGAR (OXFORD FORMULATION)

Code: CM0856

A selective and diagnostic medium for the detection of Listeria monocytogenes, when prepared from Listeria Selective Agar Base and Listeria Selective Supplement SR0140 or Modified Listeria Selective Supplement (Oxford) SR0206

Typical Formula*

gm/litre

Columbia Blood Agar Base

39.0

 Aesculin

1.0

 Ferric ammonium citrate

0.5

 Lithium chloride

15.0

 pH 7.0 ± 0.2 @ 25°C

 

* Adjusted as required to meet performance standards 

LISTERIA SELECTIVE SUPPLEMENT (OXFORD FORMULATION)

Code: SR0140

Vial contents (each vial is sufficient for 500ml of medium)

per vial

per litre
Cycloheximide  
200mg
400mg

Colistin sulphate

10.0mg

20.0mg

Acriflavine

2.5mg

5.0mg

Cefotetan

1.0mg

2.0mg

Fosfomycin

5.0mg

10.0mg

MODIFIED LISTERIA SELECTIVE SUPPLEMENT (OXFORD)

Code: SR0206

Vial contents (each vial is sufficient for 500ml of medium)

per vial

per litre
Amphotericin B  
5.0mg
10.0mg

Colistin sulphate

10.0mg

20.0mg

Acriflavine

2.5mg

5.0mg

Cefotetan

1.0mg

2.0mg

Fosfomycin

5.0mg

10.0mg

Directions
Suspend 27.75 of the Listeria Selective Agar Base (Oxford Formulation) in 500ml of distilled water. Bring gently to the boil to dissolve. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and aseptically add the contents of one vial of Listeria Selective Supplement (Oxford Formulation) or Modified Listeria Selective Supplement (Oxford) SR0206, SR0140 reconstituted with 5ml of 70% ethanol. Mix well and pour into sterile Petri dishes.

Description
Foodborne infection by Listeria monocytogenes has prompted increased concern for detecting this organism in foods, in the environment and in pathological specimens from both human and animal subjects.

Most infections in adult humans are symptomless and result in intestinal, vaginal and cervical carriage. Infection during pregnancy may cause abortion, premature delivery and neonatal infection. The possibility of listeriosis should be considered in any woman with unexplained recurrent miscarriage, premature labour or foetal death. The organism should be sought in blood cultures and genital-tract swabs1.

The most common clinical manifestation in both adults and neonates is meningitis. Widely disseminated infection, abscesses, sub-acute bacterial endocarditis and opportunistic infections in immunosuppressed patients occur less frequently.

Birds, fish and other animals are all susceptible to infection with Listeria. It is of particular importance in domestic farm animals. In the Federal Republic of Germany reporting of listeriosis in animals is compulsory and meat inspection law in the same country requires examination for Listeria because of its significance in meat hygiene.

Listeria monocytogenes is very widespread in the environment. Isolation has been reported from milk2,3, cheese4, sewage and riverwater5, and silage6. Because Listeria is so widespread sources of infections are numerous. Uncooked vegetable foods have been implicated; an episode associated with consumption of coleslaw 7 was linked with cabbage from a farm using sewage fertiliser. In outbreaks caused by dairy products, cattle with mastitis may be the source of the organism. Of great importance to veterinarians is the considerable increase amongst sheep of infection manifesting as abortion or encephalitis due largely to changing practices in silage manufacture8.

The ability to isolate the organism has been impeded in the past by lack of an effective selective medium, as Listeria monocytogenes can be easily and completely overgrown by competing flora.

Listeria Selective Medium (Oxford Formulation) is based on the formulation described by Curtis et al.9 and is recommended for the detection of Listeria monocytogenes from clinical and food specimens.

The medium utilises
(i) the selective inhibitory components lithium chloride, acriflavine, colistin sulphate, cefotetan, cycloheximide or amphotericin B and fosfomycin,
(ii) the indicator system aesculin and ferrous iron for the isolation or differentiation of Listeria monocytogenes.

Listeria monocytogenes hydrolyses aesculin, producing black zones around the colonies due to the formation of black iron phenolic compounds derived from the aglucon. Gram-negative bacteria are completely inhibited. Most unwanted Gram-positive species are suppressed, but some strains of enterococci grow poorly and exhibit a weak aesculin reaction, usually after 40 hours incubation. Some staphylococci may grow as aesculin-negative colonies.

Typical Listeria monocytogenes colonies are almost always visible after 24 hours, but incubation should be continued for a further 24 hours to detect slow-growing strains.

Techniques for isolation vary with the author and the material under examination10,11. For all specimens selective enrichment and cold enrichment have been shown to increase isolation rates significantly 12,13,14. The efficacy of Listeria Selective Medium (Oxford Formulation) has been confirmed for various foods15,16 following the methodology and using selective enrichment media described in the literature16,17,18,19.
Oxford agar is a specified plating medium in the FDA/BAM isolation procedure20 and in the standardised testing methods of other national and international bodies 21.

Oxford agar base was used by Al-Zoreki and Sandine as the basal medium for their ASLM agar which incorporates ceftazidime, moxalactam and cycloheximide as selective agents 22.

Technique
Faecal and biological specimens
The sample is homogenised in 0.1% Peptone Water CM0009 (1 part to 9 parts peptone water).

Direct surface plate method
1. Inoculate 0.1ml of the homogenised specimen onto the Listeria Selective Medium plates.
2. Incubate at 35°C for up to 48 hours.
3. Examine for typical colonies of Listeria after 24 and 48 hours incubation.

Selective Enrichment Method
1. Add the homogenised specimen to the selective enrichment broth and incubate at 30°C for up to 7 days.
2. Inoculate 0.1ml of the selective enrichment broth, after 24 hours, 48 hours and 7 days, onto the Listeria Selective Medium plates.
3. Incubate the plates at 35°C for up to 48 hours.
4. Examine for typical colonies of Listeria after 24 and 48 hours incubation.

Food and Environmental Samples
Techniques for isolation vary with the author, material and authorities. For detection of Listeria monocytogenes when present in small numbers, the test samples must be inoculated into an enrichment broth to allow multiplication before isolation and identification. Depending on the type of sample under test, an appropriate method and selective enrichment broth should be chosen prior to inoculation onto the Listeria Selective Medium plates.
1. Inoculate 0.1ml of the selective enrichment broth onto the Listeria Selective Medium plates.
2. Incubate at 35°C for up to 48 hours.
3. Examine for typical colonies after 24 and 48 hours incubation.

Colonies presumptively identified as Listeria monocytogenes must be confirmed by biochemical and serological testing 23.

Note
Differences in susceptibility of Listeria monocytogenes, Listeria seeligeri and Listeria ivanovii to b-lactam antibiotics and fosfomycin have been observed dependent on whether incubation is at 30°C or 35-37°C 24.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates at 2-8°C in the dark.

Appearance
Dehydrated medium: Straw-coloured, free-flowing powder
Prepared medium: Pale green-coloured gel

Quality control

Positive control:

Expected results

Listeria monocytogenes ATCC® 7644 *

Good growth; brown coloured colonies with aesculin hydrolysis

Negative control:

 

Entercoccus feacalis ATCC® 29212 *

No growth

* This organism is available as a Culti-Loop®

Precautions
Listeria monocytogenes is in ACDP Group 2 i.e. `might be a hazard to laboratory workers’ and should be handled in a suitable environment only. It is also recommended that pregnant staff should be excluded from working with known cultures of Listeria.
Listeria media containing acriflavine should be protected from light because photo-oxidation makes it inhibitory to Listeria.
Supplement SR0140 used in this medium contains a toxic concentration of cycloheximide. Note the precautions to be taken under HAZARDS.

References
1. Lancet (1985 [2]) August 17. 364-365.
2. Hayes et al (1986) Appl. Env. Microbiol. 50. 438-440.
3. Fernandez Garayzabal J.F. et al (1986) Can. J. Microbiol. 32. 149-150.
4. James S.M., Ferrin S.L. and Agee B.A. (1985) MMWR 34. 357-359.
5. Watkins J. and Sleath K.P. (1981).
6. Gitter M. (1983) Vet. Rec. 112, 314.
7. Schlech W.F., Lavigne P.M. and Bortolussi R.A. (1983) N. Eng. J. Med. 308. 203-206.
8. Appleyard W. (1986) Communicable Diseases, Scotland. April 1986. CDS 86/13.
9. Curtis G.D.W., Mitchell R.G., King A. F. and Griffin E.J. (1989) Letters in Appl. Microbiol. 8. 95-98.
10. van Netten P., van de Ven A., Perales I. and Mossel D.A.A. (1988) Int. J. Food Microbiol. 6. 187-198.
11. Prentice G.A. and Neaves P. (1988) Bulletin of the International Dairy Federation No. 223.
12. Hayes P.S., Feeley J.C. Graves L.M., Ajello G.W. and Fleming D.W (1986) Appl. & Environ. Microbiol. 51. 438-440.
13. Garayzabal J.F.F. Rodriquez L.D., Boland J.A.V. Cancelo J.L.B. and Fernendez G.S. (1986) Can. J. Microbiol. 32. 149-150.
14. Doyle M.P., Meske L.M. and Marth E.H. (1985) J. of Food Protection, 48. 740-742.
15. Crowther J.S. (1988) Personal Communication, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, U.K.
16. Neaves P. and Prentice G.A. (1988) Personal Communication, Technical Division, Milk Marketing Board, Thames Ditton, Surrey.
17. Lovett J., Francis D.W. and Hunt J.M. (1987) J. Food Prot. 50. 188-192.
18. Donelly C.W. and Baigent G.J. (1986) Appl. and Environ. Microbiol. 52. 689-695.
19. Hammer P., Hahn G. and Heeschen W. (1988) Deutsch Mock-Zeit. 50. 1700-1706.
20. Food and Drug Administration (FDA) Bacteriological Analytical Manual 7th Edition 1992, AOAC Int. Publishers Arlington V.A.
21. Foodborne Pathogens. Monograph Number 2 -- Listeria, page 7. Oxoid Ltd, Wade Road, Basingstoke, Hampshire, U.K.
22. Al-Zoreki N. and Sandine W.E. (1990) Appl. Env. Microbiol. 56. 3154-3157.
23. Bille J. and Doyle M.P. (1991) ``Listeria and Erysipelothrix’’, 287-295 in Balows A., Hauster W.J. Jnr., Herrman K.L., Isenberg H.D. and Shadomy H. J. (Editors), Manual of Clinical Microbiology, 5th Edition, American Society for Microbiology, Washington, D.C.
24. Curtis G.D.W., Nichols W.W. and Falla T.J. (1989) Letters in Appl. Microbiol. 8. 169-172.

 
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