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Culture Media Supplements

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PALCAM AGAR BASE

Code: CM0877

a selective and differential diagnostic medium for the detection of Listeria monocytogenes

Typical Formula*

gm/litre

Columbia Blood Agar Base

39.0

Yeast extract

3.0

Glucose

0.5

Aesculin

0.8

Ferric ammonium citrate

0.5

Mannitol

10.0

Phenol red

0.08

Lithium chloride

15.0

pH 7.2 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

PALCAM SELECTIVE SUPPLEMENT

Code: SR0150

Typical Formula*

per litre

Polymyxin B

10.0mg

Acriflavine hydrochloride

5.0mg

Ceftazidime

20.0mg

* Adjusted as required to meet performance standards
Product order code

 Volume of medium

SR0150E

10 x 500ml medium

SR0150B

10 x 2.5 litres medium

Directions
Suspend 34.5g per 500ml of distilled water. Bring gently to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C, and aseptically add PALCAM Selective Supplement (SR0150), reconstitutes as directed. Mix well and pour into sterile Petri dishes.

The addition of 2.5% (v/v) Egg Yolk Emulsion (SR0047) to the medium may aid the recovery of damaged Listeria.

Description
PALCAM Medium is based on the formulation described by Van Netten et al.1 and is recommended for the isolation of Listeria monocytogenes from foods.

The heightened awareness and concern surrounding the presence of Listeria monocytogenes in food has resulted in the development of many media for its isolation2,3,4,5. However, Cassiday and Brackett6 conclude that no single method currently available at the time was suitable for use with all types of food.

PALCAM Medium is highly selective due to the presence of lithium chloride, ceftazidime, polymyxin B and acriflavine hydrochloride. It allows the easy differential diagnosis of Listeria monocytogenes by utilising the double indicator system.

  1.  aesculin and ferrous iron
  2.  mannitol and phenol red

Listeria monocytogenes hydrolyses aesculin resulting in the formation of a black halo around colonies. Listeria monocytogenes does not ferment mannitol so easy differentiation from contaminants, such as enterococci and staphylococci, can be made as these will ferment mannitol. This formulation produces a change from red to yellow in the pH indicator phenol red. The addition of egg yolk to PALCAM medium has been reported to aid repair of damaged cells3.

Incubation under microaerophilic conditions serves to inhibit strict aerobes such as Bacillus spp. and Pseudomonas spp. that might otherwise appear on the medium. A modification to PALCAM medium in which incubated plates are overlaid with medium containing blood enables haemolytic Listeria spp. to be differentiated and enumerated7. Incubation under microaerobic conditions serves to inhibit strict aerobes such as Bacillus spp. and Pseudomonas spp. that might otherwise appear on the medium.

Technique
Techniques for the isolation of Listeria monocytogenes will depend on the material under test. It is usual for the test sample to be first inoculated into an enrichment broth to allow multiplication before isolation and identification. Depending on the type of sample used, the appropriate method and selective enrichment broth should be used prior to inoculation onto PALCAM Medium plates. As a general rule, use Listeria Selective Enrichment Medium (CM0862 and SR0149) for dairy products, and Listeria Selective Enrichment Media UVM ( CM0863, SR0142 and SR0143) and Fraser Broth (CM0895 and SR0156) for meats and poultry.

  1. Inoculate one loopful of the selective enrichment broth onto the PALCAM Medium plates.
  2. Incubate at 37°C for 48 hours under micro-aerophilic conditions. The micro-aerophilic condition can be best achieved by using Oxoid Campylobacter Gas Generating Kit (BR0056) in conjunction with the Oxoid Anaerobic Jar and an active catalyst (BR0042). For jars of smaller capacity (2.5 litres) use the Oxoid Campylobacter Gas Generating Kit (BR0060). Alternatively use CampyGen (CN0025 or CN0035). CampyGen does not require the addition of water or a catalyst.
  3. Examine for typical colonies of Listeria after 48 hours incubation.
  4. Colonies identified as presumptive Listeria spp. must be confirmed by biochemical and serological testing8.

After 48 hours incubation, typical Listeria spp. form colonies that are approximately 2mm in diameter, grey-green in colour with a black sunken centre and a black halo against a cherry-red medium background. Occasional enterococci or staphylococci develop on PALCAM Medium to forming grey colonies with a brown-green halo or yellow colonies with a yellow halo.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the selective supplement in the dark at 2-8°C and use before the expiry date on the label.
The prepared medium may be stored for up to 4 weeks at 2-8°C in the dark.

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

Quality control

Positive control:

Expected results

Listeria monocytogenes ATCC® 7644 *

Good growth; dimpled brown/black coloured colonies with black halo

Negative control:

 
Enterococcus faecalis ATCC® 29212 *Inhibited
* This organism is available as a Culti-Loop®

Precautions
Acriflavine hydrochloride is activated by light which may cause it to become inhibitory to Listeria growth.

References
1. van Netten P. et al. (1989) Int. J. Food Microbiol. 8. (4) 299-316.
2. Farber J.M. and Peterkin P. (1991) Microbiol. Rev. 55. 476-511.
3. in’t Veld P.H. and de Boer E. (1991) Int. J. Food Microbiol. 13. 295-300.
4. Gunasinghe C.P.G.L. Henderson C and Rutter M.A. (1994) Lett. Appl. Microbiol. 18. 156-158.
5. Lund A.M., Zottola E.A. and Pusch D.J. (1991) J. Food Prot. 54. 602-606.
6. Cassiday P.K. and Brackett R.E. (1989) J. Food Prot. 52. 207-214.
7. van Netten P., van Gaal B. and Mossel D.A.A. (1991) Lett. Appl. Microbiol. 12. 20-22.
8. Bille J. and Doyle M.P. (1991) ``Listeria and Erysipelothrix’’ 287-295 in Balows A., Hausler W.J. Jnr., Herrman K.L. Isenberg H.D. and Shadomy H.J. (Eds) Manual of Clinical Microbiology, 5th Edition, American Society for Microbiology, Washington D.C.

 
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