Estimation of fungal geome size: comparison of image cytometry and photometric cytometry

Besides photometric cytometry (PC), fl uorescence microscopy combined with computerised image analysis, i.e. image cytometry (IC), offers an alternative tool for assessing genome size. These techniques allow direct visualization of hyphae and simultaneous measurement of nuclear fl uorescence intensity. We developed a simple method for quantitative evaluation of nuclear DNA in fungi using DAPI-IC. The intensity of signals from individual nuclei was quantitatively measured in digitized images. In agreement with the results of parallel PC experiments, this simple IC performed on fruitbodies or on pure culture preparations enables to detect the amount of nuclear DNA in fungal cells. This result validates IC as an alternative to PC in such experiments. Kokkuvõte: Seene genoomi suuruse määramine: pildianalüüsi ja fotomeetriise tsütomeetria võrdlus. Genoomi suuruse määramise meetodiks klassikalise tsütofotomeetria (PC) kõrval on fluorestsents-mikroskoopia kombineerituna kompuuter-pildianalüüsiga (IC). See meetod võimaldab mõõta hüüfi tuumade fl uorestsentsi intensiivsust in situ. Töös esitatakse lihtne meetod tuuma DNA-sisalduse kvantitatiivseks määramiseks DAPI-IC. Digitaliseeritud pildil mõõdeti igast tuumast tulevate signaalide intensiivsust kvantitatiivselt. Paralleelsete PC eksperimentide tulemused kinnitasid, et see lihtne IC meetod võimaldab viljakeha või puhaskultuuri preparaatidest kindlaks teha tuuma DNA-sisaldust ja ta on kasutatav alternatiivse meetodina. 44 Folia Cryptog. Estonica review Kullman, 2000) involves measurement of intensity with instruments combining a microscope and a photometer. The photometer can now be replaced with an image analysis system which grabs images from the microscope via a digital camera and calculates intensity or optical density from the grey values of the pixels in the nucleus (for a review see Vidal, 1997; Hardie et al., 2002). The classical method is referred to as photometric cytometry (PC) in contrast to computer-based image cytometry (IC) (Vilhar et al., 2001). Comparison has shown that using Feulgen staining, FC and IC as well as PC and IC, provide a similar effi cacy of DNA quantifi cation (Borgiani et al., 1994; Vilhar et al., 2001). In measurement of plant and animal genome size, the Feulgen image analysis has met an ever wider application. This method is also used in plant pathology (Volgmayr & Greilhuber, 1998; Rincones et al., 2003). Employing PC for measurement of fungal nuclear DNA, mostly fl uorochrome DAPI has been used for estimation of the ploidy level (Bresinsky et al., 1987a, b; Wittmann-Meixner, 1989; Wittmann-Meixner & Bresinsky, 1989; Whittaker et al., 1991; Wittmann-Meixner et al., 1989; Weber & Bresinsky, 1992; Weber, 1992; Bresinsky & Wittmann-Bresinsky, 1995; Bresinsky et al., 1999; Kullman, 2000, 2002 a, b). DAPI is a small water-soluble fl uorescent molecule with extremely high avidity and specifi city for DNA, preferentially binding to the A-T rich regions of DNA. A comparative study of the DAPI dye and the intercalating dyes revealed a higher fl uorescence intensity and resolution of the former (Otto & Tsou, 1985). DAPI staining is less affected by the state of chromatin condensation compared with staining with other fluorochromes (Shapiro, 1995). Comparing the Feulgen image analysis and DAPI image analysis, Volkova (2005) reported higher image resolution in the case of DAPI staining. DAPI is also widely used in current plant FC for ploidy analysis. IC has only rarely been used in mycology (Rincones et al., 2003; Volkova, 2005). Moreover, published data provide no evidence that IC with DAPI staining yields results comparable to the results obtained with PC. Most PC studies report only relative DNA values, while usually no attempt has been made to present the value of absolute DNA content values in fungi. Important steps in measurement of nuclear DNA content in fungi are standardized procedures for DNA staining and measurement, as well as the choice of the standard species. The aim of the present study was to compare PC and IC on the basis of the nuclei stained with the fl uorochrome. MATERIAL AND METHODS