Legionella MWY

Description

The addition of antimicrobial agents gives selectivity to the medium. Bromothymol blue and bromocresol purple color the colonies, allowing the identification of the microorganisms.

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.

Technique

Aseptically reconstitute 1 vial with 10 mL of distilled or de-ionized water. Mix gently until complete dissolution and aseptically add to 500 mL of Legionella BCYE Agar ISO medium (LC-1311 + LC-6022) cooled to 50 °C. Mix well and distribute into sterile containers.

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:

• Directly inoculate 0.1-0.5 mL of the sample by distributing it uniformly on a plate of BCYE Agar (LC-1311 + LC-6022) and on a BCYE+AB plate.

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

• Filter the initial sample by membrane.
• Place the filter on the BCYE plate.
• Repeat the process for GVPC Agar (LC-1311 + LC-6025) and/or MWY Agar (LC-1311 + this supplement).

If the sample contains a high concentration of interfering microorganisms:

• It will be inoculated directly, concentrated or diluted.
• 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.
• Inoculate 0.1-0.5 mL on GVPC Agar and MWY Agar plates.

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

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

Reconstitute the original freeze-dried vial by adding 10 mL of sterile distilled water.

Note: each vial is sufficient to supplement 500 mL of Legionella BCYE Agar Base (LC-1311).

Physical/chemical control

Microbiological control

Add 1 vial to 500 mL of medium base. DO NOT HEAT once supplemented.

Inoculate: practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ (selectivity).

Aerobiosis. Incubation at 36 ± 2 °C. Reading 2-5 days.

Microbiological control according to ISO 11133:2014/Amd 1:2018.

Sterility control

100 mL TSB and 100 mL Thioglycollate.

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

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