Acetamide Broth 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. Magnesium sulfate, sodium molybdate and iron sulfate allow the selective growth of Pseudomonas in the medium.

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.

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

Preparation

Suspend 3.4 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Distribute into tubes in 5 mL aliquots, close and sterilize in autoclave at 121 °C for 15 minutes.

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 (LC-1155 or this 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.

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