STANDARD GROWTH CONDITIONS AND VARIATIONS

ENRIQUE CERDÁ-OLMEDO
Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain
geneco@cica.es

In Phycomyces. E. Cerdá-Olmedo and E. D. Lipson (eds.), 1987, p. 337-339. CSH Laboratory Press, Cold Spring Harbor, NY, USA.

Usual Media

The minimal medium contains, per liter, 20 g of D(+)-glucose, 2 g of L-asparagine·H₂O, 5 g of KH₂PO₄, 500 mg of MgSO₄·7H₂O, 28 mg of CaCl ₂, 1 mg of thiamin·HCl, 2 mg of citric acid·H₂O, 1.5 mg of Fe(NO₃)₃ ·9H₂O, 1 mg of ZnSO₄·7H₂O, 300 µg of MnSO₄·H₂O, 50 µg of CuSO₄·5H₂0, 50 µg of Na₂MoO₄·2H₂O. This medium is based on refinements and modifications introduced by Sutter (1975) to the minimal medium proposed by Ødegård (1952). Glucose should be autoclaved separately. The minor ingredients, including CaCl ₂, may be prepared as a 100-fold concentrate and kept sterile for a great length of time by the addition of a few drops of chloroform. Solid minimal medium is obtained by the use of agar at 15 g/liter.
Additional growth is obtained in minimal medium if the carbon and nitrogen sources are increased to 40 g of glucose and 4 g of asparagine per liter. Exceeding these amounts leads only to minor increases in biomass (Burkholder and McVeigh 1940).
Stout sporangiophores for studies of sensory physiology are produced by cultures grown on potato dextrose agar. This medium may be purchased in a dehydrated form or prepared by boiling 200 g of fresh, diced potatoes in water for 1 hour, filtering the extract, adding 20 g of D(+)-glucose, 1 mg of thiamine-HCl, and 15 g of agar, completing to 1 liter, and autoclaving. Nutrient media are often obtained by addition of various amounts of yeast extract or casein hydrolysate to other media. Many complex media commonly used in mycology support Phycomyces growth.
Discrete colonial growth, needed for the viable counting of spores and many genetic manipulations, is obtained by acidifying the media to pH 3.3 with HCl after autoclaving (Heisenberg and Cerda-Olmedo 1968). For the same purpose, R. Cohen recommends adding the detergent Triton X-100 at 0.1 g / liter.
Good sexual development is observed on potato-dextrose agar or by replacing the 2 g of asparagine in the minimal medium with 1 g of monosodium L-glutamate (Sutter 1975).

Inoculation and Incubation

Spores should be activated (Robbins et al. 1942; Rudolph 1960) either by heat shocking the spore suspension (48°C for 10 min) immediately before inoculation or by adding 10 mM acetate to the medium. The temperature for mycelial growth ranges from near 0°C to 30°C, but standard cultures are obtained at 22-23°C. To ensure the verticality of sporangiophores for behavioral studies, they should be illuminated from above with diffuse light.
Subcultures are obtained by transferring small bits of mycelium (all dimensions under 2 mm) to fresh culture medium. Repeated mycelial transplantations, however, may lead to degeneration, i.e., thinner and slower growth. Another technique to obtain subcultures is to pick up a sporangium between tweezer tips carrying a drop of sterile water, crush it to release the spores, and streak it on fresh medium.
Subculturing of colonies is faster if they are allowed to form microphores (Gutierrez-Corona and Cerdá-Olmedo 1985). Standard 48-hour-old cultures of about 50 spores on 25 ml of acidified potato-dextrose agar are flooded with 12 ml of molten water agar (15 g/liter, 45°C) and incubated for 4 additional days. A colony is subcultured by touching its surface with the tip of a sterile toothpick and then transferring to fresh agar medium.

Spore Collection

Spores are usually collected from standard petri dish cultures by washing the lids (sometimes also the mycelia) with 5-10 ml of sterile, distilled water. The resulting suspension is cleaned by filtration through a sterile tissue or glass fibers and centrifugation in sterile, distilled water in a small clinical centrifuge.
The large amounts of spores needed in many germination studies are obtained from bread cultures, as recommended by A. Van Laere. About 100 g of whole-grain bread is autoclaved in a 1-liter jar with 50 ml of distilled water. About 10⁴ to 10⁵ spores are inoculated on the bread and incubated for 2 weeks in darkness. The sporangiophores are then harvested and mixed with about 100 ml water. The resulting spore suspension is cleaned by glass-fiber filtration and low-velocity centrifugation. The final suspension is filtered through filter paper and dried in an air current. The spores are kept dry at about 4°C.

Strain Conservation

Phycomyces spores remain alive for months or years under refrigeration ( ~ 4°C). Well-sporulated cultures can be kept frozen for several years (Carmichael 1962); deep-freeze temperatures (~-70°C) are now commonly used. Lyophilized spore suspensions in a rich protein solution (e.g., cow plasma) keep well at room temperature or in the refrigerator for many years, as long as the ampules remain intact (Raper and Alexander 1945; Fennell et al. 1950; Hesseltine et al. 1960; Ellis and Robertson 1968; Sarbhoy et al. 1974).
Frequent subculturing of the strains should be avoided, because they may suffer significant, spontaneous genetic changes. Master stocks (lyophilized spore suspensions or frozen cultures) should be distinguished from working stocks (frozen cultures or spores in the refrigerator). New working stocks should be derived from the master stocks periodically, e.g., every 1-2 years.

REFERENCES

Burkholder, P.R. and I. McVeigh. 1940. Growth of Phycomyces blakesleeanus in relation to varied environmental conditions. Am. J. Bot. 27: 634.

Carmichael, J.W. 1962. Viability of mold cultures stored at -20°C. Mycologia 54: 432.

Ellis, J.J. and J.A. Robertson. 1968. Viability of fungus cultures preserved by lyophilization. Mycologia 60: 399.

Fennell, D.I., K.B. Raper, and M.H. Flickinger. 1950. Further investigations on the preservation of mold cultures. Mycologia 42: 135.

Gutiérrez-Corona, F. and E. Cerdá-Olmedo. 1985. Environmental influences in the development of Phycomyces sporangiophores. Exp. Mycol. 9: 56.

Heisenberg, M. and E. Cerdá-Olmedo. 1968. Segregation of heterokaryons in the asexual cycle of Phycomyces. Mol. Gen. Genet. 102: 187.

Hesseltine, C.W., B.J. Bradle, and C.R. Benjamin. 1960. Further investigations on the preservation of molds. Mycologia 52: 762.

Ødegård, K. 1952. On the physiology of Phycomyces blakesleeanus Burgeff. I. Mineral requirements on a glucose-asparagine medium. Physiol. Plant. 5: 583.

Raper, K.B. and D.F. Alexander. 1945. Preservation of molds by the lyophil process. Mycologia 37: 499.

Robbins, W.J., V.W. Kavanagh, and F. Kavanagh. 1942. Growth substances and dormancy of spores of Phycomyces. Bot. Gaz. 104: 224.

Rudolph, H. 1960. Weitere Untersuchungen zur Wärmeaktivierung der Sporangiosporen von Phycomyces blakesleeanus. II. Mitteilung. Planta 55: 424.

Sarbhoy, A.K., S.K. Ghosh, S.P. Lal, and G. Lall. 1974. Investigation of the preservation of fungi by lyophilization technique. Indian Phytopathol. 27: 361.

Sutter, R.P. 1975. Mutations affecting sexual development in Phycomyces blakesleeanus. Proc. Natl. Acad. Sci. 72: 127.