Feulgen densitometry: some observations relevant to best practice in quantitative nuclear DNA content determination

Despite of a huge amount of literature on the Feulgen reaction for DNA relatively few in­ vestigations deal with the special needs of plant scientists. As a consequence various mod­ ifications of the method are practised in different laboratories, which may in part be re­ sponsible for contradictory results. In the present work tests are conducted to find out, which steps of the procedure must be stringently controlled and in which steps such a con­ trol is less critical. Test material were mainly root tip meristems of Pisum sativum, which were hydrolysed and further processed in toto. Some tests involved also Zea mays. Glycine max, and Hordeum vulgare. Fixations may be stored at -20 °C in ethanol for at least 5 years without loss of quality. Nothing is known about permitted conditions of stor­ ing fixations at temperatures higher than -20 °C, except that at room temperature a secu­ rity limit for acetic alcohol fixed material is about 3 days. Staining intensity after fixation with formaldehyde is dependent on concentration, time, and temperature of the fixative. Therefore, co-fixation of test and standard material, if to be included, is strongly recom­ mended, while with acetic alcoholic fixation this point is less critical. Hydrolysis is the most critical step of the procedure. Precise control of HC1 molarity, temperature, and opti­ mum time is essential. This is often given not due attention in contemporary publications on plant DNA amounts and Feulgen staining. Hydrolysis curves for methanol acetic acid fixed material with 5 M HC1 at 20 °C, 10 °C, and 0 °C show staining optima after 60 min, 220 min, and 20 h, and a plateau of optimum staining about 5 h long in the latter. Hydroly­ sis optimum (5 M HC1) for formaldehyde fixed material at 20 °C is at 90 min. Washing time after hydrolysis is a surprisingly sensitive step of the procedure and should be kept as short as possible. Time of staining and of all further steps of the procedure, if conducted longer than necessary, lead to a gradual decrease of nuclear dye content. It is suggested to keep these steps as short as possible, because the gradual decay of staining intensity is as­ sumed to proceed not in a stoichiometric fashion. Relatively insensitive steps of the proce­ dure are a final wash in ethanol before drying the slides and storing the slides in the dark.