Proceedings e report - Firenze University Press

WOOD SCIENCE FOR CONSERVATION OF CULTURAL HERITAGE
can be treated. The duration of the treatment is then defined by the velocity of the conveyor belt. In
this work, three times of treatment were studied: 1, 2 and 15 minutes. Moreover, used gases are
mixtures of nitrogen and oxygen, the oxygen percentage being equal to 0, 5 and 20%.
3.4. Characterisation means
Fluorescence microscopy
Fluorescence microscopy observations are performed directly on membrane filters. A double staining
procedure, with two fluorescent dyes: fluorescein diacetate (FDA) and propidium iodide (PI), gives
information about cells viability [4]. FDA stains viable cells because of the enzymatic activity and the
integrity of membrane cells. On the contrary, PI bounds DNA of dead cells emitting red fluorescence ;
this stain cannot cross the membrane of viable cells. In this way, when spores are observed by
fluorescence microscopy, viable and non viable cells respectively fluoresce green and red [5]. Such
characterisation mean is used as a qualitative one.
Quantitative plate count
To measure growth inhibition after afterglow treatment, colonies on plate cultures are counted to
estimate the number of survivors. Filters are rinsed in 10mL sterile water and suspensions are vortexed
for 1 minute in order to recover the spores. Dilutions in sterile water are then streaked on malt agar
plates. The number of CFU (Colonies Forming Unit) is determined after incubation for 5 days at 24°C.
Inhibition percentage of fungal growth is calculated as indicated in (1):
% inhibition = CFUcontrol – CFUafter treatment x 100 (1)
CFUcontrol
4. Results
Three experiments were realised. Results of the first run are presented in Table 1. Gas mixtures
containing 0, 5 and 20% of oxygen were tested and the treatment duration was equal to 15 minutes.
Results show that after 15 minutes of afterglow exposure, spores are inactivated for all gas mixtures.
Table 1: Agar plate cultures after different gas afterglow exposures (first experiment).
Gas Mixture N 2/O 2 control 100/0 95/5 80/20
Agar plate cultures
Percentage inhibition - 100% 100% 100%
Observations by fluorescence microscopy of exposed filters confirm agar plate cultures results: dead
spores could be observed, whereas viable spores were not detected (Fig. 2b). On the contrary, the
presence of viable spores assessed on control filters (Fig. 2a), correlated with the high number of
colonies counted on plates, confirms the decontaminating properties of afterglow on fungal spores.
For the two following runs, the same process and experimental conditions were used. Three exposure
times (1, 2 and 15 minutes) and gas mixtures (N2/O2 80/20, 95/5 and 100/0) were tested. Fig. 3
represents the number of survivors after different treatment duration. Results show a reduction of the
initial spore population as a function of exposure time for all gas mixtures, first significant results are
obtained after 2 minutes of exposition. After 15 minutes of afterglow treatment, inactivation of fungal
spores can be considered as total (inhibition percentage = 100%) and confirmed the first run results.
Results obtained by fluorescence microscopy confirm the evolution revealed by the numeration on
agar plates. However, they were not totally conclusive, since glass slides preparation was not
optimised. Nevertheless, few viable spores were observed on untreated filters and short exposuretimes
treated samples, whereas the number of dead spores increases as a function of exposure time.
103