2 MATERIALS AND METHODS

2.1 FUNGAL STRAINS AND MEDIA

Strains of the fungal species Fusarium oxysporum, Alternaria solani, Penicillium notatum, and Chaetomium globosum were obtained from Carolina Biological Supply. F. oxysporum, A. solani, and P. notatum were grown and maintained on either potato dextrose agar or Sabouraud dextrose agar (Difco Laboratories). C. globosum was grown and maintained on V-8 juice agar as recommended by Carolina Biological Supply. Artist's watercolor, laid, medium-weight paper (Saunders Company), readily available at art supply stores, was chosen because it represents features characteristic of paper supports encountered during restoration of paper artworks infested by fungi. It is medium-weight, pliable, rag paper that does not exhibit the application of heavy sizing.

2.2 FUNGAL STAINING OF PAPER

Fungi were cultured on agar plates containing sterilized artist's watercolor paper placed on the agar surface as follows: Sabouraud dextrose agar was rehydrated by suspending 65 g of agar in 1 liter of deionized water. The medium was sterilized by autoclaving for 15 minutes (15 lbs pressure and 121�C). The sterile medium was allowed to cool to 55�C and then poured into approximately 20 100 � 15 mm sterile Petri dishes. Covered agar plates were stored at 4�C until ready for use. Paper cut into 2 in squares and autoclaved for 15 minutes was placed on the agar surface, and either F. oxysporum, P. notatum, or A. solani was aseptically removed from a culture stock and streaked along the interface of the paper and the agar and grown under different conditions (as indicated in text) until staining of paper edges was pronounced. C. globosum was similarly streaked on V-8 juice agar plates containing sterile paper. V-8 juice agar was prepared by adding 200 ml V-8 juice, 3.0 g calcium carbonate, and 20 g Bacto-agar (Difco Laboaraties) to 1 liter deionized water. The medium was autoclaved and poured into plates as described above. It is essential that streaking of fungal strains be done quickly to minimize the length of time that plates are uncovered and exposed to potential contaminants.

2.3 EFFECTS OF LIGHT, TEMPERATURE, AND pH ON FUNGAL GROWTH AND PIGMENT PRODUCTION

Fungi were incubated in a constant temperature growth chamber (4�, 25�, or 37�C), equipped with a timer-controlled fluorescent light source, for several days as indicated. Temperature and light exposure were varied as descibed in the text. The pH of the agar media was varied from 5.0 to 9.0 by the addition of hydrochloric acid or sodium hydroxide before autoclaving. Unless otherwise indicated, strains were grown on media at pH 5.6. The extent of growth and pigment production was evaluated qualitatively by visual inspection, and photographic records were maintained.

2.4 TREATMENT OF STAINED PAPER WITH ORGANIC SOLVENTS

Artists' watercolor paper was stained by fungi during growth over several days (as described above), and fungi were removed by scraping with a scalpel. The stained paper was placed on a porcelain Hirsch funnel, and approximately 5 ml of solvent was applied with a Pasteur pipet. The filtrate was collected. Alternatively, the stained paper was soaked in each solvent for a period of 24 hours prior to filtration. The ability of each solvent to remove stains was assessed in three ways: (1) visual inspection of the paper after treatment; (2) comparison of photographic records from both before and after treatment; and (3) analysis of ultraviolet visible spectra of filtrates.

The solvents used here are hazardous substances that produce harmful vapors and are irritating to the skin. All solvent work was conducted in a ventilated hood.

2.5 SCANNING ELECTRON MICROSCOPY

Paper samples, cut in approximately 0.5 cm squares, were attached to mounting stubs with double-backed tape and sputter coated with gold. Samples were scanned at the indicated magnifications on a Cambridge Stereoscan 100 scanning electron microscope.