Salamanders (Urodela) possess some of the largest genomes among tetrapods - and the size of these gargantuan genomes profoundly shapes the morphology, metabolism,
ecology, and evolution of this group of amphibians. We present an optimized method for estimating genome size using flow cytometry. The method was optimized on blood
samples of the Iberian ribbed newt (Pleurodeles waltl) and the Mexican axolotl (Ambystoma mexicanum) and tested on the Olm (Proteus anguinus). We demonstrate that A. mexicanum is a suitable internal reference standard for genome size estimation of salamanders by flow cytometry. We evaluated the necessity of using the enzyme
RNase A in sample preparation and compared the suitability of different lysis buffers
and various fluorochromes commonly used in flow cytometry. The optimized protocol was set as follows: 1) cell lysis and nuclear isolation in Galbraith’s buffer, 2) nuclei
staining with 100 µg/mL propidium iodide. The genome size of P. waltl estimated by this method was consistent with existing genome size estimates for this species and with
our estimates using image cytometry of Feulge-stained nuclei. We compared four methods for fixation of salamander blood samples, with fixation times of 7 and 14 days.
Samples fixed in a 3:1 v/v mixture of methanol and acetic acid gave acceptable results when analyzed by flow cytometry. The method presented here is non-destructive and
thus suitable for use with endangered salamander species.
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