The resulting brownish solution stained dark black-blue the Russula sporal ornamentations. The formula for what became later known as Melzer’s reagent - and which quickly came into standard use in mycology - is as follows: KI - 1.5 g, iodine - 0.5 g, distilled water - 20 g, chloral hydrate - 22 g). Baral ( 1987a) believed that Melzer was “probably inspired by Arthur Meyer”, a German botanist, renowned for his studies on chloroplasts, who introduced the use of chloral hydrate/iodine in Botany under the name “Chloraljod” (Meyer 1883).

The turning point came when Melzer ( 1924), after studying Russula species for many years, developed a method based on the use of an iodine reagent mixed with chloral hydrate to clear and stain microscopical preparations, which made it possible to study the fine details of the sporal ornamentation of Russula species. As highlighted by Leonard ( 2006), it is worth noting that the following year Patouillard ( 1888) published a paper in which he stressed the importance of spore colour in the classification of fungi, even if he did not mention the use of iodine. At the time, however, such colour changes observed in micro-anatomical elements of Basidiomycetes were not considered to be systematically relevant by the above mentioned authors. The first reports of iodine solutions used in Basidiomycetes are those of Patouillard ( 1887), who described a violet coloration with iodine of the spores of Cyphella vitellina (now Aleurodiscus vitellinus), and of Rolland ( 1887), who signaled a blue coloration in the stipe trama of Mycena tenerrima. Boudier ( 1885, 1905–1910) was the first to describe and illustrate the use of iodine to classify apothecial Ascomycetes.

Then the Tulasne brothers ( 1865), Nylander ( 1865) and Rolland ( 1887) noted and described iodine bluing in lichens and Ascomycetes at ascus and ascospore level. The earliest reference to the use of such a bluing reaction in fungi as a character having a systematic significance is a report of the bluing of a cleistothecial marine ascomycete, Amylocarpus encephaloides by Currey ( 1859). Iodine was used in Mycology in the mid-1800s (as alcoholic solutions) mainly for studying lichens and asci (entire ascus wall or apical apparatus).

2020).Īn overview of the historical use of Melzer’s was provided by Leonard ( 2006). The nature of the starch–iodine interaction is extremely complex and still remains imperfectly known (Bluhm and Zugenmaier 1981 Immel and Lichtenthaler 2000 Shen et al.

The other component, amylopectin, gives a red-purple colour which is much less intense than the amylose stain (Bailey and Whelan 1961 Shen et al. The amylose chain forms a helix shape, and iodine (as triiodide anion I 3 −) can be bound inside the helix channel. The blue colour of the stain is due to the amylose component of plant starch (Takahashi and Ono 1972 Bluhm and Zugenmaier 1981 Moulik and Gupta 1984 Murdoch 1992 Immel and Lichtenthaler 2000).

starch-like), because plant starch gives a similar reaction with iodine (starch-reaction) (Bailey and Whelan 1961 Locquin and Langeron 1978 Immel and Lichtenthaler 2000). Such a staining was termed amyloid reaction, sometimes written as I+ or J+ (the term “amyloid” being derived from the Latin amyloideus, i.e. It has been known for about 150 years that some ascomycete and basidiomycete sporomata may contain elements which stain grey to blue-black with iodine-containing solutions.

Njds spore testing