Oxoid CM0777B Raka-Ray培养基RAKA-RAY MEDIUM成分表

RAKA-RAY AGAR

Code: CM0777

The addition of phenylethanol and cycloheximide forms a selective medium for the isolation of lactic acid bacteria in beer and brewing processes.

苯乙醇和放线菌酮的加入形成了一种选择性培养基，用于啤酒和酿造过程中乳酸菌的分离。

Typical Formula*	gm/litre
Yeast extract	5.0
Tryptone	20.0
Liver concentrate	1.0
Maltose	10.0
Fructose	5.0
Glucose	5.0
Betaine HCL	2.0
Diammonium hydrogen-citrate	2.0
L-Aspartic acid†	2.5
L-Glutamic acid††	2.5
Magnesium sulphate. 7H2O	2.0
Manganese (II) sulphate. 4H2O	0.66
Potassium phosphate	2.0
N-Acetyl glucosamine	0.5
Agar	17.0
pH 5.4 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards
† L-Aspartic acid = potassium aspartate
†† L-Glutamic acid = potassium glutamate

Supplement	per litre
0.1% cycloheximide solution †	7.0ml
Sorbitan mono-oleate	10ml
2-Phenylethanol	3.0g

† 0.1% Cycloheximide Solution SR0222

Suspend 77.1g in 1 litre of distilled water. Add 10ml of sorbitan mono-oleate and 7ml of 0.1% Cycloheximide Solution (SR0222). Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50-55°C and aseptically add 3g of phenylethanol. Pour into sterile Petri dishes or distribute into 4ml volumes held at 55°C if the overlay technique is to be used.

将 77.1g 悬浮在 1 升蒸馏水中。 加入 10ml 脱水山梨糖醇单油酸酯和 7ml 0.1% 放线菌酮溶液 (SR0222)。 通过在 121°C 高压灭菌 15 分钟进行灭菌。 冷却至 50-55°C 并在无菌条件下加入 3g 苯乙醇。 如果要使用覆盖技术，则倒入无菌培养皿中或分装成 4ml 保持在 55°C 的体积。

Description
Raka-Ray Agar, is based on the formula of Saha, Sondag and Middlekauff for the detection of lactic acid bacteria in beer and brewing processes¹. Its use is recommended by the American Society of Brewing Chemists (ASBC) ², and the European Brewing Convention (EBC)³.

Members of the family Lactobacillaceae occurring in the brewing process are important spoilage organisms because products arising from their growth and metabolism are often seriously detrimental to flavour. Detection is complicated by the diverse nutritional and environmental requirements of the family and a considerable number of formulations have been described arising from attempts to optimise conditions.

Raka-Ray 3 Medium¹ was developed to enable brewers to monitor in-process beer quickly and accurately for a wide range of organisms including pediococci.

Investigations in which various combinations of growth stimulating agents were added to Universal Beer Agar led to the recognition of a number of agents including sorbitan mono-oleate, liver extract, yeast extract and N-acetyl glucosamine which gave superior results in respect of colony size, colony numbers and incubation time when compared with unmodified Universal Beer Agar.

These investigations provided the basis for the formula of Raka-Ray 3 Medium in which sorbitan mono-oleate is included as a stimulant for lactic acid bacteria in general⁴. Fructose is present as the essential carbohydrate source for Lactobacillus fructivorans ⁵ while maltose is present to detect lactobacilli which cannot utilise glucose⁶.

Detailed changes to the published Raka-Ray 3 formula are common, arising from attempts to further improve the performance for particular organisms and strains. Pediococci appear to have a universal ability to utilise glucose⁷. The value of partial substitution by glucose of the fructose content has been noted.

Selectivity is achieved by the addition of 3 gm/litre of 2-phenylethanol to inhibit Gram-negative organisms and 7mg of cycloheximide to inhibit yeasts⁸.

In a review of the performance of various media, Van Keer et al.⁵ found that Raka-Ray 3 yielded the highest colony count and allowed the enumeration of the greatest number of strains within 48 hours from a total of 30 strains of Lactobacillus taken from different origins and incubated under semi-anaerobic conditions.

Hsu and Taparowsky ⁹, when comparing Raka-Ray 3 and MRS Agar found the Raka-Ray formulation to be superior for Pediococcus cerevisiae although it was not as efficient for Lactobacillus gayonii. In another study Hug, Schlienger and Pfenniger¹⁰ compared Raka-Ray 3 with a number of other Lactobacillus media including MRS and sucrose agars and concluded that Raka-Ray 3 and MRS were the best.

描述
Raka-Ray 琼脂基于 Saha、Sondag 和 Middlekauff 的配方，用于检测啤酒和酿造过程中的乳酸菌1。美国酿造化学家协会 (ASBC) 2 和欧洲酿造公约 (EBC)3 推荐使用它。

酿造过程中出现的乳杆菌科成员是重要的腐败生物，因为它们的生长和新陈代谢产生的产品通常会严重损害风味。由于该家族的不同营养和环境要求，检测变得复杂，并且已经描述了由于尝试优化条件而产生的大量配方。

Raka-Ray 3 Medium1 的开发使啤酒制造商能够快速准确地监测过程中啤酒中的各种微生物，包括片球菌。

在 Universal Beer Agar 中添加各种生长刺激剂组合的研究导致了对包括山梨糖醇单油酸酯、肝提取物、酵母提取物和 N-乙酰氨基葡萄糖在内的许多试剂的认可，这些试剂在菌落大小方面产生了优异的结果，与未修饰的通用啤酒琼脂相比，菌落数和孵育时间。

这些研究为 Raka-Ray 3 培养基的配方提供了基础，其中脱水山梨糖醇单油酸酯通常作为乳酸菌的兴奋剂 4。果糖是果糖乳杆菌 5 的必需碳水化合物来源，而麦芽糖则用于检测不能利用葡萄糖的乳杆菌 6。

对已发布的 Raka-Ray 3 公式的详细更改很常见，这是为了进一步提高特定生物体和菌株的性能。片球菌似乎具有利用葡萄糖的普遍能力7。已经注意到果糖含量被葡萄糖部分取代的价值。

通过添加 3 克/升 2-苯乙醇来抑制革兰氏阴性菌和 7 毫克放线菌酮来抑制酵母菌，可以实现选择性。

在审查各种培养基的性能时，Van Keer 等人 5 发现 Raka-Ray 3 产生了最高的菌落数，并允许在 48 小时内从总共 30 株乳酸杆菌中计数最多的菌株。不同来源并在半厌氧条件下培养。

Hsu 和 Taparowsky 9 在比较 Raka-Ray 3 和 MRS 琼脂时发现，Raka-Ray 配方对酿酒小球菌的效果更好，尽管它对 Gayonii 乳杆菌的效率不高。在另一项研究中，Hug、Schlienger 和 Pfenniger10 将 Raka-Ray 3 与包括 MRS 和蔗糖琼脂在内的许多其他乳酸菌培养基进行了比较，并得出结论认为 Raka-Ray 3 和 MRS 是最好的。

Technique
Surface inoculation
0.1ml of the sample is spread on agar plates. Incubate at 25-30°C under anaerobic conditions using the Oxoid Gas Generating Kit BR0038 with the Oxoid Anaerobic Jar, alternatively use Anaerogen AN0025 or AN0035. Anaerogen does not require the addition of water or the use of an active catalyst. Alternatively, the specimen can be filtered and the membrane placed on the agar surface for incubation.

Overlay technique
Aseptically dispense 4ml volumes of Raka-Ray Agar into small test tubes and keep molten in a water bath at 55°C.
Mix 1ml of the test sample with 4ml of molten agar and immediately pour the contents into a petri dish containing 15-20ml of solid Raka-Ray Agar to give well distributed colonies. Incubate under anaerobic conditions at 25-30°C.
Because the agar layer is very thin, individual colonies can be picked easily for further examination.

Incubation conditions
An incubation period of 4 days is generally sufficient but slower growing organisms may require up to 7 days.
Because of the diversity of environmental conditions required for growth of lactic acid bacteria a semi-anaerobic atmosphere may be needed. This is achieved using Oxoid Gas Generating Kit BR0056 in the Oxoid Anaerobic Jar. Alternatively use CampyGen CN0025 or CN0035. CampyGen does not require the addition of water or the use of an active catalyst.

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 gel

Quality control

Positive control:	Expected results
Lactobacillus brevis ATCC® 14869	Good growth; cream/white coloured colonies
Negative control:
Escherichia coli ATCC® 25922 *	Inhibited

* This organism is available as a Culti-Loop®

Precautions
Although the concentration of cycloheximide in the medium is below toxic levels, precautions should be observed as detailed under HAZARDS section.

References
1. Saha R. B., Sondag R. J. and Middlekauff J. E. (1974) Proceedings of the American Society of Brewing Chemists, 9th Congress, 1974.
2. Methods of Analysis of the ASBC (1976) 7th Edition. The Society, St. Paul. Mn. USA.
3. European Brewing Convention, EBC Analytica Microbiologica: Part II J. Inst. Brewing (1981) 87. 303-321.
4. Mauld B. and Seidel H. (1971) Brauwissenschaft 24. 105.
5. Van Keer C., Van Melkebeke L., Vertriest W., Hoozee G. and Van Schoonenberghe E. (1983) J. Inst. Brewing 89. 361-363.
6. Lawrence D. R. and Leedham P. A. (1979) J. Inst. Brewing 85. 119.
7. Coster E. and White H.R. (1951) J. Gen. Microbiol. 37. 15.
8. S. Shaw – Personal communication.
9. Hsu W. P. and Taparowsky J. A. (1977) Brewers Digest 52. 48.
10. Hug H., Schlienger E. and Pfenniger H. (1978) Braueri-Rundschau 89. 145.