LYSINE MEDIUM

Code: CM0191

A synthetic medium for the isolation and enumeration of wild yeasts encountered in brewing. On this medium, pitching yeasts are suppressed.

Typical Formula*	gm/litre
Glucose	44.5
Potassium dihydrogen phosphate	1.78
Magnesium sulphate	0.89
Calcium chloride fused	0.178
Sodium chloride	0.089
Adenine	0.00178
DL-methionine	0.000891
L-histidine	0.000891
DL-tryptophane	0.000891
Boric acid	0.0000089
Zinc sulphate	0.0000356
Ammonium molybdate	0.0000178
Manganese sulphate	0.0000356
Ferrous sulphate	0.0002225
Lysine	1.0
Inositol	0.02
Calcium pantothenate	0.002
Aneurine	0.0004
Pyridoxine	0.0004
p-aminobenzoic acid	0.0002
Nicotinic acid	0.0004
Riboflavin	0.0002
Biotin	0.000002
Folic acid	0.000001
Agar	17.8
pH (see directions)

* Adjusted as required to meet performance standards

Directions
Suspend 6.6g in 100ml distilled water containing 1.0ml Potassium lactate SR0037. Bring to the boil to dissolve completely. Agitate frequently to prevent superheating. Cool to 50°C and add 0.1ml of lactic acid 10% SR0021 to adjust to pH 4.8 ± 0.2. Dispense into Petri dishes and remove surface moisture by drying at 37°C.

Description
A complex medium, originally described by Morris & Eddy¹ for the isolation and enumeration of wild yeasts in pitching yeast. Walters and Thiselton² examined 180 species of yeasts in a liquid synthetic medium containing lysine as the sole nitrogen source. They found that no normal cerevisiae or carlsbergensis strains utilised lysine whereas many other yeasts, including wild yeasts, did so. They kept their stock cultures on malt extract agar slopes or on malt extract chalk agar in the case of Brettanomyces species. Later, Morris & Eddy¹ described a solid lysine medium for the isolation and enumeration of wild yeasts in pitching yeast. Oxoid Lysine Agar is made to their published formula.

Technique
Wash and centrifuge the sample of pitching yeast three times with distilled water. Remove 0.2ml of a suspension containing approximately 10⁷ cells per ml and spread with a bent platinum wire, over the surface of a Lysine Medium plate. Incubate at 25°C and examine daily for evidence of growth. Count the number of colonies which develop, and express the degree of contamination as the number of wild yeast cells per million cells of the original inoculum.

The number of cells in the inoculum is important as it has been shown by Morris & Eddy that small numbers of cells (approximately 100 to 1,000) still grow to a limited extent on the medium. Where the number of brewing yeast cells exceeds approximately 10,000, a count of the colonies developing provides a direct measure of the contamination by wild yeasts³.

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: White coloured, free-flowing powder
Prepared medium: Light straw coloured gel

Positive control:	Expected results
Pichia fermentans ATCC® 10651	Good growth; white colonies
Negative control:
Saccharomyces (carlsbergensis) uvarum ATCC® 2700	Slight background film

Precautions
The pitching yeast may grow as a slight background film with the `wild’ yeast appearing as colonies on the film.