Europium chelate and fluorescent brightener staining of soil propagules and their photomicrographic counting—I. Methods

A method tor direct visual or photomicrographic enumeration of soil propagules in soil-agar films and soil smears, stained with a mixture ofeuropium(III) ttienoyltrifluoroacetonate and the disodium salt of 4,4¹-bis(4-anilino-6-bis(2-hydroxyethyl)amino-S-triazin-2-ylamino)2,2¹-stilbene disulphonic acid, abbreviated to Eu(TTA)₃ and fluorescent brightener (FB), respectively, is described.Soil-agar suspensions at 55–60°C were introduced into haemocytometer sample chambers of 100 μm and 20 μm depth which gave soil-agar film thicknesses of 25 μm and 12 μm, respectively, after air-drying. Soil smears air-dried on 1.25 cm² and 1.0 cm² areas of glass slide were 5.2–6.0 μm and 6.0–8.0 μm thick, respectively.The Eu(TTA)₃ is best formulated in 50% (v/v) ethanol. The best concentrations of Eu(TTA)3 and FB for staining soil propagules were 2 mM and 25 μM. respectively. A staining period of 18 h for soil-agar films and 90–120 min for soil smears was found to be optimal. The optimum volumes of 50% ethanol for rinsing soil-agar films and soil smears were found to be 12.5 and 5.0 ml. respectively.Lack of in-depth focusing on agar films 25 μm thick resulted in 15–17 per cent error in enumerations. The 12 μm agar films and soil smears required little in-depth focusing and were acceptable for photomicrographic recording of numbers of soil propagules. It is concluded that incident u.v. photomicrographic recording of soil propagule numbers in thin agar films or soil smears is a practical solution to operator fatigue likely to occur during direct microscopy.