Pseudomonas CN Agar Base ISO

Pseudomonas aeruginosa is an opportunist pathogen for humans, capable of growing in water with a low concentration of nutrients. This is why natural mineral water and spring water should be checked to be free of Pseudomonas aeruginosa at the time of commercialization. This microorganism can also be found in swimming pool water.

Peptone and casein provide nitrogen, vitamins, minerals and amino acids essential for growth. Cetrimide is added as a selective agent, and nalidixic acid to suppress contaminants of cetrimide media such as Klebsiella, Proteus and Providencia spp. Potassium sulfate and magnesium chloride provide cations to activate pyocyanin production and enhance pigment production. Bacteriological agar is the solidifying agent.

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

Preparation

Suspend 50.6 grams of the medium in one liter of distilled water. Add 10 mL of glycerol. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 118 °C for 15 minutes. Cool to 45-50 °C, mix well and dispense into plates to obtain an agar layer at least 5 mm thick. Do not remelt the medium.

According to ISO 16266

According to ISO 16266 for the detection and enumeration of Pseudomonas aeruginosa:

1. Filter a certain volume of water sample through a filter membrane and place the membrane on a Pseudomonas CN Agar Base plate.
2. Incubate at a temperature of 36 ± 2 °C for 44 ± 4 h.
3. Count the colonies that have a green/blue pigmentation (pyocyanin) as confirmed P. aeruginosa.
4. Examine the membrane under UV light.
5. All colonies that are fluorescence (+) and reddish-brown colonies should be confirmed.
6. Spread all the colonies that should be confirmed on Nutrient Agar plates (LC-1156) to obtain pure cultures. Incubate at 36 ± 2 °C for 22 ± 2 h.
7. Perform the oxidase assay on the reddish-brown colonies.
8. Streak the oxidase (+) colonies on King B Medium (LC-1532) to check the fluorescence production. Incubate at 36 ± 2 °C for up to 5 days. Normally 24 hours are enough.
9. Inoculate all the fluorescence (+) colonies, both in CN agar and in King B Medium, in the Acetamide Broth (LC-1155 or LC-2017) medium and add one or two drops of Nessler reagent to check the ammonia production. Incubate at 36 ± 2 °C for 22 ± 2 h.
10. The colonies that produce pyocyanin in CN agar, the colonies fluorescence (+) in CN agar and ammonia (+) in Acetamide Broth, and the reddish-brown colonies in CN agar, oxidase (+), fluorescence (+) in King B Agar and ammonia (+) in Acetamide Broth, are counted as confirmed P. aeruginosa.

• UNE-EN 12780:2002, Quality of water. Identification and enumeration of Pseudomonas aeruginosa by membrane filtration.
• EN ISO 16266 Water quality - Detection and enumeration of Pseudomonas aeruginosa - Method by membrane filtration