Acetamide Broth Base ISO

Acetamide is the single carbon source. The potassium salt has a high buffering capacity and sodium chloride supplies essential electrolytes for transport and osmotic balance.

It is prepared according to ISO 16266.

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 are Pseudomonas aeruginosa free at the time of their commercialization. This microorganism can also be found in swimming pool water.

Solution B

Preparation

Suspend 3.4 grams of the medium in 900 mL of distilled water. Adjust the pH to 7.0 ± 0.5 at 25 °C. Add one mL of recently prepared Solution B. Whilst agitating, add water to obtain a final volume of one liter. Distribute into tubes in 5 mL aliquots, close and sterilize in autoclave at 121 °C for 15 minutes. Prepared tubes must be stored in a dark place.

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 (LC-1153).
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 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 (this medium or LC-2017) 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.

• ISO 16266 Water quality - Detection and enumeration of Pseudomonas aeruginosa - Method by membrane filtration