A new simple method for the enumeration of nitrifying bacteria in different environments

In this work, a simple, safe and rapid method for enumerating nitrifying bacteria was used as an alternative to traditional harmful chemical methods. The enumeration of nitrifying bacteria was based upon the change of color of the growth media containing pH indicators in response to acid production during nitrification. The oxidation of ammonia to strong acids by nitrifiers leads to pH decrease, which can be detected by pH indicators such as methyl orange (MO), bromocresol green (BCG), methyl red (MR), bromothymol blue (BTB), and phenol red (PhR) using the Most Probable Number (MPN) technique. The use of these pH indicators revealed a higher estimate than the classical chemical methods in all tested samples. Ammonium oxidizer counts always exceeded those of nitrite oxidizers in the surveyed environments. The time required for the detection of growth (positive tubes in MPN) was descending in the following order: MO, BCG, MR, BTB and PhR. The time to detection was shorter for ammonium oxidizers than for nitrite oxidizers. Generally, nitrifier counts were very low in soils compared with farmyard manure or sewage effluent. Incubation periods for both organisms differed from 4 to 8 weeks depended upon the indicator used. Finally, it could be concluded that the use of pH indicators, especially phenol red, as proposed in this study was accurate, sensitive and successfully applicable for the enumeration of nitrifiers in different environments.