Conservation of the mycelia of the medicinal mushroom Humphreya coffeata (Berk.) Stey. in sterile distilled water

Graphical abstract


Method details
There are various methods of conservation, some of the most commonly used are repeated subculturing, lyophilization (unsuitable for most basidiomycetes) and cryopreservation [2,[9][10][11]. However, some of these methods are not compatible with all fungi due to the particular characteristics of each species [1]. We used the method described by Castellani [3][4][5] with some modifications for the conservation of the mycelia of the higher basidiomycete Humphreya coffeata (Berk.) Stey., since it has been reported that this method ensures the viability of isolates for 1-20 years depending on the species [5,6,12,13]; however, it has not been used for higher basidiomycetes.

Inoculation of mycelia in malt extract agar
The central area of the Petri dishes with MEA or FZM agar media was inoculated with H. coffeata (Berk.) Stey. (Fig. 1a), and the 5-mm filter paper disks (Whatman 1 No.4) were placed around the inoculum. Thereby, on growing the mycelium of H. coffeata would cover the Petri dish, including the filter paper disks, as seen in Fig. 1b. The Petri dishes were incubated at 30 8C for 4 days (FELISA FE-293A, Mé xico).

Storage in sterile distilled water
After a 4-day period the mycelial growth covered the Petri dish. Then the filter paper disks were carefully removed under sterile conditions, and placed into vials containing 4.0 mL of sterile distilled water. A minimum of 50 vials were prepared. All vials were closed and sealed with 2 cm Parafilm 1 M (Sigma-Aldrich, USA) strips. Finally, all vials were stored at 4 8C (Fig. 2).

Viability of the basidiomycetes in sterile distilled water
The shelf life of H. coffeata (Berk.) Stey. attached to filter paper disks (Whatman 1 No. 4) and stored in sterile distilled water at 4 8C was evaluated at 0, 3, 6, 12, 15 and 18 months. All viability trials were made at least in triplicate. The culture medium for the evaluation was FZM agar. Growth was assessed macroscopically (Fig. 3), viability was determined by measuring the growth diameter formed by the mycelial culture in FZM agar, and the culture was incubated at 30 8C (FELISA FE-293A, Mé xico) for 4 days [4,8].
The conservation of mycelium in filter paper disks of H. coffeata in sterile distilled water assured a high viability of cultivation for 18 months (Fig. 3). There were no visible morphological changes, or contamination by bacteria or other fungi. This suggests that this method, in addition to being easy and economical, is suitable for the conservation of higher basidiomycetes such as H. coffeata. It should be taken into consideration that the time of viability to ensure this method depends on the species of fungus to store [3][4][5][6][7].