Legionella BCYE Agar Base ISO

Feeley et al. described a modification of F-G Agar in which acid hydrolysed casein was replaced by yeast extract as the source of protein, and starch was replaced by activated charcoal. This medium, which they named CYE Agar, has been further supplemented with ACES buffer and alpha-ketoglutarate and is described in the literature as BCYE-alpha Medium. BCYE-alpha Medium has been shown to yield optimal recovery of Legionellaceae from environmental samples in a shorter incubation period.

Yeast extract provides vitamins, particularly of the B-group, and other growth co-factors. L-cysteine provides the required nutritional source. Activated charcoal is a protective agent neutralizing and absorbing toxic metabolites produced by bacterial growth. It decomposes hydrogen peroxide, a toxic metabolic product, and can also collect CO₂ and modify surface tension.

ISO 11731 recommends the following procedure for the isolation of Legionella and its enumeration in water samples. The samples are concentrated by membrane filtration, diluted or inoculated directly on the plate depending on the origin and characteristics of the sample. Independent fractions of the diluted sample should be subjected to heat or acid treatments in case of a high concentration of Legionella and other bacteria. These samples are transferred to the plates with the selective culture medium chosen for Legionella.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 12.5 grams of the medium in 500 mL of distilled water. Heat until boiling and until the medium is completely dissolved. Distribute into appropriate containers and sterilize in autoclave at 121 °C for 15 minutes. Cool to 48 ± 3 °C and aseptically add 1 vial of Legionella BCYE Growth Supplement (LC-6022). Mix well and distribute into appropriate containers.

From this point you can obtain GVPC Agar or MWY Agar:

• To obtain GVPC Agar, also add 1 vial of Legionella GVPC Supplement (LC-6025).
• To obtain MWY Agar, also add 1 vial of Legionella MWY Growth Supplement (LC-6067).

On the other hand, you can obtain BCYE-Cys Agar (BCYE w/o Cysteine) by adding 1 vial of Legionella BCYE w/o Cysteine Supplement (LC-6092) instead of LC-6022.

According to ISO 11731

For the cultivation of Legionella according to ISO 11731.

If the sample contains a high concentration of Legionella and a low concentration of interfering microorganisms:

1. Directly inoculate 0.1-0.5 mL of the sample by distributing it uniformly on a plate of BCYE Agar (this medium + LC-6022) and on a BCYE+AB plate.

If the sample contains a low concentration of Legionella and a low concentration of interfering organisms:

1. Filter the initial sample by membrane.
2. Place the filter on the BCYE plate.
3. Repeat the process for GVPC Agar (this medium + LC-6022 + LC-6025) and/or MWY Agar (this medium + LC-6022 + LC-6067).

If the sample contains a high concentration of interfering microorganisms:

1. It will be inoculated directly, concentrated or diluted.
2. Divide each type of sample into three portions. One of them will be used untreated, the second one will be subjected to a thermal treatment and the third will be subjected to an acid treatment.
3. Inoculate 0.1-0.5 mL on GVPC Agar and MWY Agar plates.

If the sample contains an extremely high amount of interfering microorganisms:

1. It will be inoculated directly and diluted.
2. Each sample is subjected to a combined thermal and acid treatment.
3. Inoculate 0.1-0.5 mL on GVPC Agar and MWY Agar plates.
4. Let the plates rest until the inoculum has been absorbed. Incubate at 36 ± 2 °C for 7-10 days.
5. Confirm presumptive colonies of Legionella on BCYE Agar and BCYE-cys Agar.

The container of this product may suffer deformation due to the high oxygen adsorption capacity of the activated charcoal contained in the formula.

• Feeley J.C., Gorman G.W., Weaver R.E., Mackel D.C.
• International standard ISO 11731 Water quality - Detection and enumeration of Legionella.