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.

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.

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.

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