LISTERIA ENRICHMENT BROTH BASE (UVM FORMULATION)

Code: CM0863

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

用于 USDA-FSIS 方法的两步选择性富集培养基。

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

方向
将 27.2g 悬浮在 500ml 蒸馏水中。通过在 121°C 高压灭菌 15 分钟进行灭菌。冷却至 50°C。

制备李斯特菌初级选择性富集培养基 (UVM I)
无菌添加 1 瓶按照指示重新配制的李斯特菌初级选择性富集补充剂 (UVM I) SR0142 的内容物。充分混合并分配到无菌容器中。

制备李斯特菌二级选择性富集培养基 (UVM II)
无菌添加 1 瓶按照指示重新配制的李斯特菌二级选择性富集补充剂 (UVM II) SR0143 的内容物。充分混合并分配到无菌容器中。

描述
李斯特菌选择性富集培养基（UVM 配方）基于 Donnelly 和 Baigent1 描述的原始配方，以及随后的修改 2，降低了初级和次级选择性富集培养基中的萘啶酸浓度并增加了次级中盐酸吖啶黄的浓度选择性富集培养基。

这种修改以及开发的两步选择性富集方法（USDA-FSIS 方法）2 提高了肉制品中单核细胞增生李斯特菌的检出率，并具有仅需 3-4 天的额外优势。

UVM 肉汤已被推荐作为主要的浓缩肉汤，用于恢复热损伤的单核细胞增生李斯特菌3。

使用带有 DNA 探针方法的 UVM 肉汤时必须小心，因为培养基的高盐含量可能会对检测产生抑制作用。

技术

初级浓缩
1. 将 25g 或 25ml 样品添加到 225ml 李斯特菌初级选择性富集培养基 (UVM I) 中。在 Stomacher 中均质化 2 分钟。
2. 将准备好的样品放入 Stomacher 袋中，在 30°C 下孵育。
3. 从这个袋子中，执行以下程序：孵育 4 小时后，将 0.2 毫升涂抹在李斯特菌选择性琼脂板上（见注）。
孵育 24 小时后，
(i) 将 0.1 毫升转移到 10 毫升李斯特菌二级富集培养基 (UVM II)，和
(ii) 将 1ml 转移至 4.5ml KOH 溶液。涡旋混合并在一分钟内传代到李斯特菌选择性琼脂板上。有关 KOH 制备的详细信息，请参见下文。

二次浓缩
4. 在 30°C 下孵育接种的李斯特菌二级选择性富集培养基 (UVM II)。见 3(i)。
5. 孵育 24 小时后，
(i) 将 0.2 毫升涂抹在李斯特菌选择性琼脂板上。
(ii) 将 1ml 转移至 4.5ml KOH 溶液。涡旋混合并在一分钟内将该混合物继代培养到李斯特菌选择性琼脂板上。

KOH溶液的制备
将 2.5g KOH 和 20g NaCl 溶解在 1000ml 蒸馏水中。使用前在 121°C 高压灭菌并确保 pH 值高于 12.0 进行灭菌。

笔记
推荐用于 USDA 方法 2 的李斯特菌选择性琼脂是 LPM 电镀培养基 3。然而，Oxoid 实验室研究 5 表明，使用李斯特菌选择性培养基（牛津配方）CM0856 和 SR0140 可以获得类似的结果。

更新后的 USDA 方法 6 已将 LPM 培养基替换为改良牛津培养基 (MOX)。不再需要在电镀前用氢氧化钾处理富集培养物。

储存条件和保质期
将脱水培养基储存在 10-30°C 并在标签上的有效期之前使用。
将准备好的培养基储存在 2-8°C。

外貌
脱水介质：稻草色、自由流动的粉末
制备培养基：稻草色溶液

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.