MRS AGAR (DE MAN, ROGOSA, SHARPE)

Code: CM0361

A solidified version of MRS Broth for the culture of `lactic acid bacteria’.

用于培养“乳酸菌”的 MRS 肉汤的固化版本。

* Adjusted as required to meet performance standards

Directions
Suspend 62g in 1 litre of distilled water. Boil to dissolve the medium completely. Dispense into tubes, bottles or flasks and sterilise by autoclaving at 121°C for 15 minutes.

Description
The MRS formulation was developed by de Man, Rogosa and Sharpe¹ to replace a variable product (tomato juice) and, at the same time, provide a medium which would support good growth of lactobacilli in general, even those strains which showed poor growth in existing media. MRS medium is superior to the tomato juice medium of Briggs² and the meat extract tomato juice medium of de Man. It gives more profuse growth of all strains of lactobacilli, especially the difficult and slow growing strains of Lactobacillus brevis and Lactobacillus fermenti.

MRS Agar and Broth were designed to encourage the growth of the `lactic acid bacteria’ which includes species of the following genera: Lactobacillus, Streptococcus, Pediococcus and Leuconostoc. All these species can produce lactic acid in considerable amounts. They are Gram-positive, catalase and oxidase negative and are fastidious in their nutritional requirements. Growth is enhanced considerably by microaerobic conditions. Generally the `lactic acid bacteria’ show delayed growth and smaller colony size than other micro-organisms. They may be overgrown in non-selective media, especially if incubation is required for 2-4 days.

MRS medium is selective for lactobacilli but some growth of leuconostocs and pediococci may occur. Selectivity can be altered by pH adjustment. Lactobacilli will tolerate lower pH levels than streptococci (pH 5.0-6.5) with pediococci and leuconostocs growing best within this range. Inhibitors of the main groups of competitor microflora include thallous acetate, sodium acetate, sorbic acid, acetic acid, sodium nitrite, cycloheximide and polymyxin. These substances can be used at varying concentrations and combinations but, inevitably, a compromise has to be reached between selectivity and productivity of the organism(s) sought³. MRS Agar with sorbic acid has been described^3,4. This is MRS medium with its pH reduced to 5.7 and 0.14% sorbic acid added (equating to 0.2% potassium sorbate).

An evaluation of media for selective enumeration of Lactobacillus acidophilus and Bifidobacterium species showed that minor adjustments to the basic formula of MRS Agar can readily be made to optimise its performance for determining the content of Lactobacillus acidophilus and Bifidobacterium spp. in the presence of other lactic acid bacteria which are present in yoghurt⁵.

Lactobacilli are microaerophilic and generally require layer plates for aerobic cultivation on solid media. Submerged or surface colonies may be compact or feathery, and are small, opaque and white.

Technique
1. Products to be examined for lactobacilli content are macerated or diluted in a diluent such as quarter-strength Ringer solution, and further dilutions are made in MRS Broth.
2.  Diluted sample in 1ml volumes are added to sterile dishes, and molten MRS Agar (45°C) is poured into the dish and mixed thoroughly.
3. When the medium has set, another layer of uninoculated MRS Agar is poured over the surface to produce a layer-plate.
4. Plates are incubated as described below. It is important that adequate moisture vapour is present in the atmosphere above the agar because drying of the plates during incubation will concentrate the selective factors on the surface and make the medium inhibitory. The presence of carbon dioxide stimulates growth and plates should be incubated in an atmosphere of 5% CO₂.

方向
将 62g 悬浮在 1 升蒸馏水中。煮沸以完全溶解培养基。分装到试管、瓶子或烧瓶中，然后在 121°C 高压灭菌 15 分钟进行灭菌。

描述
MRS 配方是由 de Man、Rogosa 和 Sharpe1 开发的，用于替代可变产品（番茄汁），同时提供一种支持乳酸杆菌良好生长的培养基，即使是那些在现有培养基中生长不良的菌株媒体。 MRS 培养基优于 Briggs2 的番茄汁培养基和 de Man 的肉汁番茄汁培养基。它使所有乳酸杆菌菌株的生长更加旺盛，尤其是难以生长且生长缓慢的短乳杆菌和发酵乳杆菌菌株。

MRS 琼脂和肉汤旨在促进“乳酸菌”的生长，其中包括以下属的物种：乳酸杆菌、链球菌、片球菌和明串珠菌。所有这些物种都可以产生大量的乳酸。它们是革兰氏阳性、过氧化氢酶和氧化酶阴性的，并且对营养需求很挑剔。微需氧条件显着促进生长。一般来说，与其他微生物相比，“乳酸菌”的生长延迟和菌落大小更小。它们可能在非选择性培养基中过度生长，特别是如果需要孵育 2-4 天。

MRS 培养基对乳酸杆菌具有选择性，但可能会出现一些明串珠菌和片球菌生长。选择性可以通过调节 pH 值来改变。与链球菌 (pH 5.0-6.5) 相比，乳杆菌能耐受更低的 pH 值，而片球菌和明串珠菌在此范围内生长最好。主要竞争菌群的抑制剂包括醋酸亚铁、醋酸钠、山梨酸、醋酸、亚硝酸钠、放线菌素和多粘菌素。这些物质可以以不同的浓度和组合使用，但不可避免地，必须在所寻求的生物体的选择性和生产力之间达成妥协3。已经描述了含有山梨酸的 MRS 琼脂 3,4。这是 MRS 培养基，其 pH 值降至 5.7，并添加了 0.14% 山梨酸（相当于 0.2% 山梨酸钾）。

对选择性计数嗜酸乳杆菌和双歧杆菌的培养基的评估表明，可以轻松地对 MRS 琼脂的基本配方进行微调，以优化其确定嗜酸乳杆菌和双歧杆菌含量的性能。在酸奶中存在其他乳酸菌的情况下。

乳酸杆菌是微需氧的，通常需要层板在固体培养基上进行需氧培养。淹没或表面的菌落可能是紧凑的或羽毛状的，并且是小的、不透明的和白色的。

技术
1. 待检测乳酸杆菌含量的产品在稀释剂（如四分之一浓度的林格溶液）中浸渍或稀释，并在 MRS 肉汤中进一步稀释。
2. 将 1 ml 体积的稀释样品加入无菌培养皿中，将熔化的 MRS 琼脂 (45°C) 倒入培养皿中并充分混合。
3. 培养基凝固后，将另一层未接种的 MRS 琼脂倒在表面上，形成层板。
4. 如下所述培养板。重要的是在琼脂上方的大气中存在足够的水蒸气，因为在培养过程中干燥板会使选择因子集中在表面上并使培养基具有抑制性。二氧化碳的存在会刺激生长，培养板应在 5% CO2 的气氛中培养。

Incubation method

Incubation carried out under anaerobic or microaerophilic conditions
To identify the presumptive Lactobacillus colonies, select isolated colonies on the agar medium. Stain a smear from each  and pick off into MRS Broth (CM0359), indivdually. An advantage of this broth is that any other micro-organisms, originally lying dormant in the selective agar, are not given the opportunity to multiply, as may occur in a non-selective broth. Incubate the broths at temperatures and times similar to those used for the MRS Agar; they can then be examined microscopically and further sub-cultured to MRS Agar for subsequent confirmation and identification of species.

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.

Appearance
Dehydrated medium: Dark straw coloured powder
Prepared medium: Amber coloured gel

在厌氧或微需氧条件下进行孵化
要识别假定的乳酸菌菌落，请在琼脂培养基上选择分离的菌落。 将每个涂片染色，然后逐个提取到 MRS Broth (CM0359) 中。 这种肉汤的一个优点是，原本在选择性琼脂中处于休眠状态的任何其他微生物都没有机会繁殖，就像在非选择性肉汤中可能发生的那样。 在与 MRS 琼脂相似的温度和时间下培养肉汤； 然后可以在显微镜下检查它们并进一步传代培养到 MRS 琼脂中，以进行后续的物种确认和鉴定。

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。
将准备好的板储存在 2-8°C。

外貌
脱水介质：深稻草色粉末
制备培养基：琥珀色凝胶

Quality control

* This organism is available as a Culti-Loop®

References
1. de Man J. C., Rogosa M. and Sharpe M. Elisabeth (1960) Appl. Bact. 23. 130-135.
2. Briggs M. (1953) J. Dairy Res. 20. 36-40.
3. Reuter G. (1985) Intern. J. Food Microbiol. 2. 55-68.
4. ISO/TC 34/SC 6/WG 15, No.3 and 5 (1984) Enumeration of Lactobacteriaceae in meat and meat products.
5. Lankaputhra W.E.V., Shah N.P. and Britz M.L. (1996) Food Australia 48. 113-118.