Membrane filtration method ISO 16266:2006 for  Pseudomonas aeruginosa in water

This method of the International Organization for Standardization is applicable to bottled water and other types of water with a low background microflora, for example, pool waters and waters intended for human consumption.

1 . Material required for analysis

• Distilled or deionized water sterile (for dilutions if necessary)

• Membrane filtration system

• Sterile membrane filter 0.45μm nominal pore size

• Pseudomonas CN Agar

• Acetamide Broth ISO

• King’s B Medium

• Nutrient Agar (NA)

• Oxidase Kovacs Reagent

• Nessler Reagent

• Laboratory incubator set to 36 ± 2°C

• Ultra violet lamp 360 ± 20 nm

2 . Procedure

A general flowchart for the enumeration of Pseudomonas aeruginosa in water using the membrane filtration method ISO 16266:2006 is shown in Figure.2.

a) Filtration: filter a suitable volume of the water sample through a sterile cellulose ester membrane filter with 0.45  μm pore. The volume depends on the type of sample. For drinking water or bottled water 100 ml is the volume commonly used.

For polluted water the volume should be smaller. When filtering less than 10 ml, mix the sample with 10–100 ml of sterile distilled or deionized water before filtration.

 Note a.1)   The sample volume should be adjusted to obtain plates with well separated colonies. If necessary, pre-pare and filter dilutions of the sample.

b)  Incubation and colony counting:  After filtration, remove the membrane with a sterile forceps and place on the surface of a  Pseudomonas CN Agar plate, avoiding air bubbles. Incubate the plates at 36 ± 2°C/44 ± 4 h and count after 22 ± 2 h and after 44 ± 4 h.

 Note b.1)   The reading after 22 h should be used to calculate the results in case of plates showing overgrowth and merging of colonies after 44 ± 4 h.

Count only the typical colonies which can be of three types:

  Type 1 colonies: Colonies that produce blue/green color (pyocyanin pigment). These colonies are considered P. aeruginosa without additional confirmatory tests.

  Type 2 colonies: Colonies non-pyocianin producing (non-blue/green) but fluorescent under ultra violet light (360 ± 20nm). These colonies are considered presumptive and should be confirmed by the acetamide growth test.

Figure 2   Scheme of analysis for the enumeration of Pseudomonas aeruginosa in water using the membrane filtration method ISO 16266:2006.

Type 3 colonies: Colonies non-pyocianin producing (non-blue/green) and non-fluorescent under ultra violet light (360  ± 20nm) but showing a reddish brown pigment (under visible light). These colonies are considered presumptive and should be confirmed by the oxidase test, the acetamide growth test  and the fluorescence on King’s B medium test.

c) Confirmation: Select as many colonies as possible for confirmation (all colonies if possible). Streak

each colony onto a Nutrient Agar (NA) plate and

incubate at 36 ± 2°C/22 ± 2 h. Select one well isolated colony from each NA plate for the confirmatory tests (check for purity by Gram stain before performing the tests).

 Note c.1)   Five is a number of colonies commonly used for confirmation and if there are fewer than five, all.

c.1)  Oxidase test: Using a platinum/iridium loop or glass rod, take a portion of a well-isolated colony from each NA plate and streak it onto a filter paper moistened with the Oxidase Kovacs Reagent. The appearance of a mauve, violet or deep blue color within 10s indicates a positive reaction. If a commercially avail-able oxidase test kit is used, follow the manufacturer’s instructions.

c.2)  Acetamide test: From the colony on NA inoculate a tube of Acetamide Broth ISO and incubate at 36 ± 2°C/22 ± 2 h. Add one or two drops of Nessler Reagent and observe. Color change varying from yellow to brick red is indicative of ammonia production (alkaline reaction) and considered a positive confirmed test for P. aeruginosa.

c.3)  Fluorescence on King’s B Medium: From the colony on NA inoculate a tube of King’s B Medium and incubate at 36 ± 2°C for five days. Examine daily for growth and fluorescence under ultra violet light (360 ± 20nm).

Consider any fluorescence appearing up to five days as a positive confirmed test for  P.  aeruginosa.

d)  Calculation of the results:  Interpret the results according to Table 3.

Calculate the number of  P. aeruginosa colony forming units per volume of sample examined according the formula below:

P = number of type 1 colonies on Pseudomonas CN Agar

F = number of type 2 colonies on Pseudomonas CN Agar

n_F  = number of type 2 colonies submitted to confirmation

C_F  = number of type 2 colonies confirmed as P. aeruginosa

R = number of type 3 colonies on Pseudomonas CN Agar

n_R  = number of type 3 colonies submitted to confirmation

C_R  = number of type 3 colonies confirmed as P. aeruginosa

Table.3  Guide for the interpretation of Pseudomonas aeruginosa confirmatory tests according the method ISO 16266:2006.

Example

Volume of sample examined = 100 ml

Number of type 1 colonies on  Pseudomonas CN Agar (P) = 20

Number of type 2 colonies on Pseudomonas CN Agar (F) = 12, five submitted to confirmation (c_F = 5), two confirmed (n_F = 2).

Number of type 3 colonies on  Pseudomonas CN Agar (R) = 10, five submitted to confirmation (c_R = 5), one confirmed (n_R = 1).

CFU/100 ml = 20 + 12 × (2/5) + 10 × (1/5) = 27

Alternatively express the result qualitatively by stating that P. aeruginosa was present or absent in the volume of water examined.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .

International Organization for Standardization (2006) ISO 16266:2006. Water quality – Detection and enumeration of Pseudomonas aeruginosa – Method by membrane filtration. Geneva, ISO.