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Dehydrated Culture Media

LISTERIA ENRICHMENT BROTH BASE (UVM FORMULATION)

Code: CM0863

A two step selective enrichment medium for USDA-FSIS method.

Typical Formula*

gm/litre

Proteose peptone

5.0

Tryptone

5.0

`Lab-Lemco’ powder

5.0

Yeast extract

5.0

Sodium chloride

20.0

Disodium hydrogen phosphate

12.0

Potassium dihydrogen phosphate

1.35

Aesculin

1.0

pH 7.4 ± 0.2 @ 25°C

 

* Adjusted as required to meet performance standards 

LISTERIA PRIMARY SELECTIVE ENRICHMENT SUPPLEMENT (UVM I)

Code: SR0142

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

per vial
per litre

Nalidixic acid

10.0mg

20.0mg

Acriflavine hydrochloride

6.0mg

12.0mg


LISTERIA SECONDARY SELECTIVE ENRICHMENT SUPPLEMENT (UVM II)

Code: SR0143

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

Nalidixic acid

10.0mg

20.0mg

Acriflavine hydrochloride

12.5mg

25.0mg

Directions
Suspend 27.2g in 500ml of distilled water. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C.

To Prepare Listeria Primary Selective Enrichment Medium (UVM I)
Aseptically add the contents of 1 vial of Listeria Primary Selective Enrichment Supplement (UVM I) SR0142 reconsituted as directed. Mix well and distribute into sterile containers.

To Prepare Listeria Secondary Selective Enrichment Medium (UVM II)
Aseptically add the contents of 1 vial of Listeria Secondary Selective Enrichment Supplement (UVM II) SR0143 reconsituted as directed. Mix well and distribute into sterile containers.

Description
The Listeria Selective Enrichment Media (UVM formulations) are based on the original formulation described by Donnelly and Baigent1, and its subsequent modification2 which reduced the nalidixic acid concentration in both the primary and secondary selective enrichment media and increased the concentration of acriflavine hydrochloride in the secondary selective enrichment medium.

This modification, and the two step selective enrichment method developed (USDA-FSIS method)2, results in a higher detection rate of Listeria monocytogenes from meat products and has the added advantage of only taking 3-4 days.

UVM Broth has been recommended as a primary enrichment broth for recovery of heat-injured Listeria monocytogenes3.

Care must be taken when using UVM broth with DNA probe methodology because the high salt content of the medium may have an inhibitory effect on detection4.

Technique

Primary Enrichment
1. Add 25g or 25ml samples to 225ml of Listeria Primary Selective Enrichment Medium (UVM I). Homogenise in a Stomacher for 2 minutes.
2. Incubate the prepared sample in the Stomacher bag at 30°C.
3. From this bag, carry out the following procedures: After 4 hours incubation, spread 0.2ml on Listeria Selective Agar plates (see Note).
After 24 hours incubation,
(i) transfer 0.1ml to 10ml of Listeria Secondary Enrichment Medium (UVM II), and
(ii) transfer 1ml to 4.5ml KOH solution. Vortex mix and within one minute subculture onto Listeria Selective Agar plates. For details of KOH preparation see below.

Secondary Enrichment
4. Incubate the inoculated Listeria Secondary Selective Enrichment Medium (UVM II) at 30°C. See 3(i).
5. After 24 hours incubation,
(i) spread 0.2ml onto Listeria Selective Agar plates.
(ii) transfer 1ml to 4.5ml KOH solution. Vortex mix and within one minute subculture this mixture onto Listeria Selective Agar plates.

Preparation Of KOH Solution
Dissolve 2.5g of KOH and 20g of NaCl in 1000ml of distilled water. Sterilise by autoclaving at 121°C and ensure that the pH is above 12.0 before use.

Note
The Listeria Selective Agar recommended for use in the USDA method2 is LPM plating medium3. However, Oxoid laboratory studies5 have shown that comparable results can be achieved with Listeria Selective Medium (Oxford formulation) CM0856 and SR0140.

Updated USDA methodology6 has replaced LPM medium with Modified Oxford Medium (MOX). There is no longer a requirement to treat enrichment culture with potassium hydroxide before plating.

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 medium at 2-8°C.

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

Quality control

Positive control:

Expected results

Listeria monocytogenes ATCC® 7644 *

Turbid growth

Negative control:

 

Enterococcus faecalis ATCC® 29212 *

No change

* This organism is available as a Culti-Loop®

Precautions
Note the precautions stated under Listeria Selective Medium (Oxford) CM0856 and SR0140.
Broth cultures are more dangerous than colonies on agar plates.
Store prepared medium away from light. Acriflavine can photo-oxidise to form inhibitory compounds against listeriae.

References
1. Donnelly C.W. and Baigent G.J. (1986) Appl. Environ. Microbiol. 52. 689-695.
2. McClain D. and Lee W.H. (1988) Assoc. Off. Anal. Chem. 71. 660-664.
3. Bailey J.S., Fletcher D.L. and Cox N.A. (1990) J. Food Prot. 53. 473-477.
4. Partis L., Newton L., Marby J. and Wells R.J. (1994) Appl. Environ. Microbiol. 60. 1693-1694.
5. Sawhney D.R. and Dodds L. (1988) Internal project report. Oxoid R&D Laboratory.
6. McLain D. and Lee W.H. (1989) FSIS Method for the isolation and identification of Listeria monocytogenes from processed meat and poultry products. Laboratory Communications number 57.

 
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