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Analytical Methods

Actinomycetes in water by double-agar layer procedure. Updated April 2014

The AIA (Actinomycete Isolation Agar) agar method is a double-agar layer (DAL) method for enumeration of actinomycetes. This method must be done in the laboratory by a trained analyst.

Samples are analyzed in undilute and 10-2 concentrations in duplicate using the DAL method. The bottom agar layer is AIA prepared from dehydrated AIA medium. The top agar layer is prepared by adding 2 mL of the sample concentration being analyzed to 17 mL of AIA agar and 1 mL of cyclohexamide, an antifungal antibiotic. The top agar layer is mixed and then 5 mL is dispensed onto the bottom agar layer. Plates are incubated at 28C for 7 days. Following the incubation period, colonies are identified by microscopy according to colony appearance. Actinomycetes colonies are opaque, dark in the center, and have an irregular, fuzzy edge of hyphal appearance (American Public Health Association, 2010). Results are reported as colonies per 1 mL.

Actinomycetes are filamentous, branching bacteria that are widely disturbed in nature (American Public Health Association, 2010). Actinomycetes are considered nuisance organisms for those in the water industry, as they are one of two types of organisms that impart an earthy-musty odor to waters.
See actinomycetes DAL method directions (Appendix C14).

See preparation instructions for media, reagent solutions, and control culture strains (Appendix C14).


American Public Health Association, American Water Works Association , and Water Environment Federation, 2010, Standard methods for examination of water and wastewater: American Public Health Association, part 9250, Detection of Actinomycetes.

63688 Actinomycetes, colonies per 1 mL