Identification of Living Fungi on Archives and Library Materials
Malalanirina Rakotonirainy, Jozef Hanus, Maryline Moulin
Identifizierung lebender Pilze auf Materialien in Archiven und Bibliotheken
Der Beitrag beschreibt Schnelltests, die auf Biolumineszenz und dem Nachweis von ATP
(Adenosintriphosphat) beruhen. Die Methode der Messung von ATP, ADP, AMP und EC (energy
charge) zur Bestimmung lebender und abgetb'teter Pilze auf Materialien in Archiven und
Bibliotheken wird zum erstenmal angewendet. Die Resultate der EC-Messungen an verschiedenen
Spezies nach ihrer Behandlung mit Sterilisierungsagenzien und mit Dampf werden vorgestellt.
Various rapid test methods based on bioluminescence and the determination of ATP (adenosine
triphosphate) are described. The techniques for measuring ATP, ADP, AMP and EC (energy
charge) to determine living and dead fungi on materials in archives and libraries are applied for
the first time. Results of the EC measurements for different species after treatment with sterilising
agents and water vapour are presented.
1. Introduction
Microbiological contamination by different fungi represents one of the major problems in archives
and libraries. The presence of these microorganisms often becomes evident via colour spots or dustlike particles. However, besides aesthetic aspects of this problem, the presence of microorganisms is
more than only threatening. If it is not detected in time it can lead to an irreversible paper
degradation.1,2,3 At present, the viability of moulds is evaluated by long and laborious culturing
techniques, which can provide even erroneous results, if the used nutrient broth is not appropriate or
sampled cells are viable but not cultivable. Therefore the application of a simple and fast diagnostic
method to identify and solve the above mentioned problem would be very desirable. There are
several rapid methods, however, which have not been applied in the field of cultural heritage, where
non-destructive analysis is required.4 The determination of ATP via bioluminescence can meet
these requirements.5,6,7 In effect, the ATP characterizes living organisms. However, the dead cells
do not produce ATP any more and the ATP still present in dead cells is rapidly degraded by the
action of ATPases. It means that the ATP is a specific compound for determination of cells
viability. This method, largely used in different fields (medicine, environment, industries ...) for
bacteria and yeast, has been studied only to a certain extent for filamentous fung.,8,910,11,12,13
Therefore the commercially available determination kits do not provide convincing results for
fungi.14,15,16,17,18 This is the reason why the Centre de Recherches sur la Conservation des
Documents Graphiques (CRCDG) has elaborated on a procedure for fungi. This procedure, initially
developed on the spores of Aspergillus niger, may be applied without any problems to other species
of fungi.19 On the contrary, during the assay on spores disinfected by ethylene oxide the quantity of
determined ATP seems to be sometimes comparable to the values obtained from non-treated living
spores despite the classical microbiological analysis reveals their total inhibition.
As for some thirty years all microbiologically contaminated documents in France have been
disinfected by ethylene oxide, we have to face a real problem nowadays. The following question
has to be answered: has disinfecting by ethylene oxide always been effective under the routine
conditions for all treated spores or is the method used for determination of living fungi not adequate
for this purpose? To try to answer this question, we have not only been interested in ATP itself but
in all adenylic nucleotides together in order to be able to calculate energy charge (EC), i.e. the value
of metabolic energy in equilibrium state among the three forms of adenylic nucleotides (ATP, ADP,
AMP). Studies on bacteria and yeast have shown that for living cells the values of energy charge are
stabilized within the range of 0,8-0,9 and for dead cells below 0,5.20'21 We have tested six fungal
strains disinfected by ethylene oxide in different centres and compared obtained values to the same
non-treated strains and also to those treated by vapour, i.e. dead fungi.
2. Materials and methods
2.1. Fungal cultures
Six strains of filamentous fungi from the mycological collection of the CRCDG have been used:
Aspergillus flavus Link ex Gray, Eurotium chevalieri Mangin, Neosartorya fischeri (Welmer)
Malloch & Cain, Penicillium chrysogenum Thorn, Fusarium solaniSaccardo and Chaetomium
globosum Kunze.
They were cultivated on paper disks placed on a malt agar substrate at 26 °C until paper samples
were completely covered with fungi. Contaminated papers were placed in Petri dishes and air dried.
Fungi have been used in the form of suspension of spores either individually or in a mixture with
some other strains.
2.2. Extraction of ATP
The extractions were made in glass vials at 100 °C during 1 minute after adding of 1 ml of spores
suspension in 1 ml of dimethyl sulphoxide extractant diluted in 90 % of Tris-ace-tate EDTA buffer,
pH 7, pre-heated at 100 °C during 2 minutes. Placing the vials on ice immediately stops the
reaction.
2.3. Principal reagents for determination of ATP
- The ATP Monitoring Reagent (AMR) (Labsystem ref. BO1243200) is the principal reagent for
this determination. It contains firefly luciferase reagent (luciferin/luciferase), which provokes a
bioluminescence reaction. It is provided in a lyophilized state and has to be diluted in Tris-acetate
buffer (Labsystem ref. BO1243227) before its usage.
- The ATP standard (SIGMA, ref. FL-AAS) is also provided in a lyophilized form and has to be
diluted in deionized water in order to create concentration of 1 g ATP/ml. The ATP standard is used
for calibration of the assay. In effect, the intensity of measured luminescence may be reduced via
turbidity or light absorption by some samples. The addition of a known quantity of ATP standard
(0,01 g) enables calibration of measuring apparatus and thus eliminates these effects.
- Reagents, necessary for the enzymatic conversion of ADP and AMP, were described by Hysert
and Morrison.22
2.4. Apparatus
Determinations have been performed on Turner Design luminometer TD 20/20. Its sensibility
permits detection up to 0,1 femtogramme (1,10 -16 g) of luciferase.
2.5. Assay procedures
The assays are performed in polystyrene measuring cuvettes into which 200 I of extract and 300 I of
diluted luciferin-luciferase solution (AMR) are added. Use of 3 measuring cuvettes for each assay
allows quantitative determination of ATP, ADP + ATP and the sum of all 3 adenine forms. To be
able to determine the ADP and AMP they have to be converted to ATP in advance. In order to do
that, we have utilized the Hysert and Morrison method.22
2.6. Calculation of nucleotides concentration
The quantity of adenylate nucleotides (A. N.) in each of 3 measuring cuvettes is calculated
according to the following formula:
A. N.: adenylate nucleotides
S: first RLU measurement of sample
T. RLU measurement of blank
I: second RLU measurement of sample after internal
standard addition
ATP quantity is given directly by measurement of Cuvette 1
ADP quantity = Cuvette 2-Cuvette 1
AMP quantity = Cuvette 3-Cuvette 2
2.7. Calculation of the adenylate energy charge EC
The energy charge is calculated according to the following formula23.
Each EC value presented here is the average of at least five assays repeated at least two times.
2.8. Disinfection treatments
Artificially contaminated paper samples by different fungi were inserted into envelopes and treated
by ethylene oxide in two different disinfection centres (EO1 and E02) under the conditions
routinely used for disinfection. Another set of contaminated paper samples - and also samples
treated by ethylene oxide - was sterilized by vapour (20 minutes at 120 °C). The treatment
efficiency was determined by a classical method using malt-agar medium.
3. Results and discussion
3.1. EC values of different strains
The mean values of energy charge measured on different living moulds are within the range of 0,81
-1,00. The EC values for moulds treated by vapour at the CRCDG are within
Tab. 1 EC values of living and dead strains
Fig. 1 Detection limit for determination of energy charge EC
the range of 0,45-0,58 (Table 1). These results confirm that the method is suitable and appropriate
for given purposes as it can very clearly distinguish between living and potentially dead moulds. All
results of EC reach values over 0,8 for living and below 0,6 for dead moulds. The method is also
applicable for identification of mixtures of different moulds. In such cases it is sufficient if only one
living mould occurs among the others in order to get an EC value over 0,8.
3.2. Detection limit
Solutions of Aspergillus flavus have been used in different concentrations in order to determine the
detection limit for measurement of ATP and EC under the given conditions. The results show that
from the concentration of 1,106 spores/ml down to the concentration of 1,103 spores/ml - which
represents a number of 100 spores in one measuring cuvette per one measurement in a luminometer
- energy charge values reliably provide readings within the range of 0,8-0,9 (Fig. 1).
Values for concentration of 50 spores per cuvette give EC readings slightly below 0,8, which is
considered the minimal value for reliable identification of living cells. On the basis of these results
it can be estimated that detection limit lies within the range concentration of 50-100 spores per
cuvette under the given conditions.
3.3. ATP and EC of fungi treated by ethylene oxide
Determination of energy charge for identification of living or dead fungi treated by ethylene oxide
has been pointed out as the most problematic part of the evaluation in previous works at the
CRCDG.24'25 For this reason we have concentrated our attention to fungi, which have been treated
by ethylene oxide disinfection at different places. In order to follow the EC values, the two types of
fungi, which belong to the most resistant ones on archives and library materials - Eurotium
chevalieri and Neosartorya fischeri - have been selected. The results show that E. chevalieri
treated, by ethylene oxide at the places EO1 and EO2 are dead - EC values for both give the same
reading - 0,57 (Table 2). On the other hand, while EC for N. fischeri treated by ethylene oxide at the
place EO1 gives value 0,53 (dead fungi), N. fischeri treated by ethylene oxide at the place EO2
gives value 0,82 - and thus indicates the living cells. Verification of given results by classical
methods
Tab. 2 EC values for ethylene oxide treated strains of E. chevalieri and N. fischeri
Tab. 3 EC values of different strains treated by ethylene oxide
confirms the EC results. Results for C. globosum, F. solani, P. chrysogenum treated by ethylene
oxide at the place EO2 provide EC values within the range of 0,28-0,64 - indicating thus dead fungi,
confirmed also by classical method of the above mentioned verification (Table 3).
The only problematic species used in our test seems to be A. flavus treated by ethylene oxide at the
place EO2. Despite being identified by classical methods as dead, in several tests it has been
provided values of EC 0,8 - and thus indicating living fungi. On the other hand some other tests
provide EC values below 0,65 with indication of dead fungi. As its behaviour is rather curious, this
is the reason why it has been tested in different mixtures with other fungi.
Mixture of A flavus from places E01 and EO2 gives EC value around 0,94 (Table 4). Mixture of E.
chevalieri, A. flavus, N. fischeri and P. chrysogenum treated by ethylene oxide at the place EO1,
prepared as the exact mixture with the concentration of spores of 2,5 105for each of them, gives EC
value around 0,61 while EC for their mixture with routine sampling without calculation of spores
gives value of 0,64-0,67. Dead fungi have been confirmed also by the classical method.
After adding of A. flavus treated by ethylene oxide at the place EO2 to this mixture, EC values has
been determined as 0,84-0,89. These values are similar to those which have been obtain after
addition of living fungi A. flavus from the collection at the CRCDG to the above mentioned mixture
giving EC 0,81 -0,85 - thus real living fungi. On the basis of these results it can be supposed that the
fungi A. flavus treated by ethylene oxide in the place EO2 remains viable but on the other hand also
becomes non-cultivable. It seems that determination and calculation of energy charge provide thus a
suitable tool for inspection of quality of disinfection treatment.
3.4. EC of fungi treated by vapour
Vapour treatment is considered to be the most effective treatment and safely eliminates all kinds of
living fungi. Fungi treated by ethylene oxide at places E01 and EO2 as well as paper samples with
living spores were treated by vapour sterilization at 120 °C for 20 minutes. The results show that
EC values are in general below 0,6. Dead fungi have been confirmed also by the classical method. It
also confirms the viability of the strains N. fischeri and A. flavus after treatment at the centre EO2
and indicates that the disinfection conditions used at this centre may not be adequate in order to
eliminate all microbiological contamination. However, EC values for some mixture give readings
superior to 1,1. As the theoretical range of EC ratios are from 0,0 (all AMP) to 1,0 (all ATP) the EC
values above 1,0 are considered to be errors of measurement.
4. Conclusions
The method for measuring ATP, ADP, AMP and EC in order to determine living and dead fungi on
archives and library materials is applied in this study. Identification of living and dead
microorganisms can be determined within a period of up to 1 hour - it ranks this method as very fast
and at the same time very sensitive. Detection limit has been determined within the range of 50-100
spores per cuvette in luminometer. A minimum of 5 repeats for one assay is recommended.
Experiments with spores disinfected with ethylene oxide confirm our previous results that ATP is
not totally degraded in cells treated with this gas. We suppose that ethylene oxide inhibits spores
activity but possibly pro-:ects the ATP present, preventing it from being degraded. Ethylene oxide
has certainly an effect on enzymes (ATPases) responsible for the degradation of the ATP at the
death of the cells. For this reason, it is necessary to calculate the energy charge values, which are
more representative of the viability of cells.
The average energy charge values measured on different living moulds are within the range of 0,811,00. The EC values for moulds treated by vapour - i. e. dead moulds -are within the range of 0,450,58. The EC values for ethylene oxide treated moulds are below 0,67. These results confirm that
the methods is suitable and appropriate for given purposes as it can distinguish between living and
dead moulds clearly and without problem. The method is also applicable on a mixture of different
moulds. In such case it is sufficient if only one living mould occurs among the others in order to get
EC value over 0,8.
Tab. 4 EC values for mixture of strains treated by ethylene oxid
The only problematic species used in our test seems to be A. flavus treated by ethylene oxide at the
place EO2. Despite being identified by classical methods as dead, in several tests it provided values
of EC about 0,8 - and thus indicating living fungi. On the other hand in some other tests EC values
below 0,65 were provided, indicating dead fungi. As its behaviour is rather curious, it has been
tested in different mixtures with other fungi. On the basis of this results it can be supposed that the
strain A. flavus treated by ethylene oxide in the place EO2 remains viable but becomes noncultivable. It seems that determination and calculation of energy charge provides thus a suitable tool
for inspection of the quality of disinfection treatment.
For further development and practical application of the method it is necessary to continue
experiments involving more model mixtures of different strains with different concentrations as
well as samples taken from really contaminated documents, books and works of art. As the above
mentioned method under the given conditions requires a rather high temperature for extraction (100
°C), one of the further aims of our activity is to develop a kit which would enable a routine
identification of living and dead fungi at ambient or more reasonable conditions than the present
state.
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