2 PRESENT FUMIGATION STUDY

THE FUNGI used in these tests were prepared by simulating the conditions encountered with water-damaged paper. A heavy concentration of spores from mature agar-grown cultures was transferred to filter paper wedges. Some were allowed to dry to simulate airborne or direct contact contamination. Others were kept moist for 48 hours to encourage germination. Theoretically, the most resistant part of the fungus life cycle is the mature spore because its cell wall is the least permeable and its metabolism the lowest. The freshly germinated spore should be the most vulnerable as its metabolism and cell wall permeability are high. Thousands, or perhaps millions, of spores were used in each sample. This large concentration reflects that to be found on moldy paper.

The paper wedges were fumigated and then placed on a fresh nutrient agar plate. Some of the spores were transferred onto the agar, the rest left on the paper which was placed on the agar. In the absence of toxic substance, both sets of spores would germinate and grow well. If the toxin is fungistatic, that is if the fungus remains viable but is prevented from germinating only in the presence of the toxin, then the spores on the agar would germinate and the spores on the toxin-impregnated paper would not germinate until the fumigant disperses. If the fumigant is fungicidal, no germination would take place on either the agar or the paper no matter how long they are incubated. To be an effective fumigant for archival purposes, it must be fungicidal instead of fungistatic.

2.1 Materials and Methods

The fungus strains tested here were obtained from stock cultures maintained at the Mycology Laboratory of New York State Health Department in Albany. Their species and stock numbers are listed in Table 1. They were grown for 7 to 10 days on 2.5% Fisher's Malt extract in 1.5% agar. Whatman 110 mm, #1 filter paper discs were cut into four wedge-shaped segments and steam sterilized. The wedges were pressed lightly onto the mature fungus colony to pick up enough spores to color lightly the surface. The inoculated papers were placed on “V”-shaped glass tubing in sterile plastic petri plates. Sterile water was added to one set of samples while the other was kept dry. Control spores were treated the same way but placed outside the fumigation cabinets during the test. They all germinated and grew. All the paper wedges were transferred to 2.5% malt again after fumigation by pressing them upside down on the agar surface on one side of the petri plate to dislodge some spores and then placing the paper with the spore side up on the other side of the plate. The plates were examined for 15 days after inoculation.

Table 1. Fungus Strains Used for Tests

The chemicals tested were ortho-phenyl phenol flake crystals (as Dowicide 1) obtained from Dow Chemical Company, and thymol crystals purchased from Talas Company. Two identical fumigation chambers were constructed of uncoated poplar wood, 16⅜″ high, 13�″ wide and 10⅜″ deep. At the bottom of the chambers was a 15 watt light bulb immediately above which was a watch glass to hold the fumigant. Two shelves constructed of plexiglass strips so as not to restrict air circulation were placed at 4″ and 8″ above the bulb. One of these chambers was used only for thymol and the other only for OPP.

The lights were operated by means of an automatic timer, set to go on four hours per cycle of twenty-four. Ten grams of Crystalline OPP or thymol was placed in a watch glass and weighed after each experiment (Table 2).

Table 2. Fumigant Use During Tests

To make sure that the lack of penetration of the fumigant was not a problem, the paper wedges were placed on a grate over the fumigant. This led to some cross contamination of the samples but contaminant colonies were not counted in the results.

Samples of the first series of experiments were fumigated in a single, large storage cabinet. When it was discovered that this was not lethal to all the fungi and since there was an uncertainty of how much residual fumigant may be built up in the cabinet, two separate, smaller cabinets were used for the tests reported in the tables. Tests were made in the small cabinets when they were new and again after 30 days of continuous fumigation. In this way the effects of the two fumigants were assessed without chance of cross contamination from residual fumigant. The efficacy of the chamber increased slightly after it became saturated with fumigant. The amount of fumigant used through sublimation from the heated crystals was measurably less in the cabinets after they had been in continuous use than that used when they were new (Table 1).

To make certain the chemicals retained their toxicity, cultures were tested by dipping them in 10% solutions of thymol and OPP in ethyl alcohol. These tests were completely effective for killing the fungi in all cases. Control tests were made in sterile water and ethyl alcohol alone. In fungi both control samples retained viability, although the alcohol visibly retarded the fungus development.

2.2 Results

The fungi were tested in germinated and ungerminated condition with different lengths of fumigation with either OPP or thymol, and the results were interpreted from growth vs. no growth on nutrient agar. In no case was growth of all the test species prevented by either fumigant under all conditions. This indicates that neither of the fumigants is totally fungicidal nor totally fungistatic. Table 3 shows the results of 3 days of fumigation of germinated (wet) and ungerminated (dry) spores with OPP and thymol in small chambers which had been in continuous use for 30 days prior to the test. The growth of fungus colonies on the agar surface and on the paper was recorded separately. Those colonies which were slow to develop are indicated. Table 4 shows the results of 10 days of fumigation.

Table 3 Viability of Fungi After 3 Day Fumigation with Ortho-Phenyl Phenol or Thymol

Table 4 Viability of Fungi After 10 Day Fumigation with Ortho-Phenyl Phenol or Thymol