Most probable number (MPN) method APHA/AWWA/WEF 2005 for  Pseudomonas aeruginosa in water

Method of the American Public Health Association (APHA), American Water Works Association (AWWA) & Water Environment Federation (WEF), as described in the Standard Methods for the Examination of Water and Wastewater (Hunt & Rice, 2005).

1. Material required for analysis

Preparation of the sample and serial dilutions

• Magnesium Chloride Phosphate Buffer (Dilution Water)

• Dilution tubes containing 9 ml Magnesium Chloride Phosphate Buffer (Dilution Water)

• Asparagine Broth double strength (10 ml tubes)

• Asparagine Broth single strength (10 ml tubes)

• Acetamide Agar or Acetamide Broth

• Laboratory incubator set to 35–37°C

2. Procedure

A general flowchart for the enumeration of Pseudomonas aeruginosa in water using the Most Probable Number (MPN) method APHA/AWWA/WEF 2005 is shown in Figure 1.

a) Presumptive test: Homogenize the sample by shaking vigorously about 25 times. For potable water apply a MPN single dilution test, inoculating 10 × 10 ml aliquots of the sample onto 10 × 10 ml of double strength  Asparagine Broth (it is also possible to inoculate 5 × 20 ml aliquots of the sample onto 5 × 20 ml of double strength Asparagine Broth or 5 × 10 ml of triple strength Asparagine Broth).

Figure.1   Scheme of analysis for the enumeration of Pseudomonas aeruginosa in water using the Most Probable Number (MPN) method APHA/AWWA/WEF 2005 (Hunt & Rice, 2005).

For non-potable water select three appropriate dilutions and apply a MPN multiple dilution test, inoculating three tubes of Asparagine Broth per dilution. Incubate the samples at 35–37°C/24–48. After 24 h examine the tubes under long-wave ultra-violet light (black light) in a darkened box or room. The production of a green fluorescent pigment constitutes a positive presumptive test. Re-incubate the negative tubes for an additional 24 h and examine and record reactions again at 48 h.

b) Confirmed test:  From each presumptive tube inoculate 0.1 ml of culture into  Acetamide Broth or onto  Acetamide Agar slants. Incubate the tubes at 35–37°C/24–36 h. Development of purple color (alkaline reaction) within 24 to 36 h of incubation is a positive confirmed test for P. aeruginosa.

c)  Calculation of results: Make a note of the number of positive tubes and determine the most probable number (MPN)/ml .

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 .

Hunt, M.E. & Rice, E.W. (2005) Microbiological examination. In: Eaton, A.D., Clesceri, L.S., Rice, E.W. & Greenberg, A.E. (eds). Standard Methods for the Examination of Water & Wastewater. 21st edition. Washington, American Public Health Association (APHA), American Water Works Association (AWWA) & Water Environment Federation (WEF). Part 9213, pp. 9.33–9.34.