D4445 test method. As with the ASTM method, the visual rating
system is subjective and a high number of replicate specimens per
treatment group is important for precision and reproducibility.
Results from the ATP assay corroborated the XTT results by
verifying complete inhibition of metabolic activity after exposure of
mould spores to 50 ppm isothiazolinone. There was no growth of
spores from individual test fungi placed on malt extract agar
following 24-h exposure to 50 ppm isothiazolinone and aseptic
washing with DI water. This result suggests that isothiazolinone
was fungicidal rather than fungistatic against spores from the test
fungi under the conditions used in this study.
4. Discussion and conclusions
Despite micro- to macro- differences in the three test methods
compared in this study, a critical factor for a screening method is
the estimated minimal inhibitory concentration (MIC90) for the
chemical being evaluated. The estimated MIC90 for isothiazolinone
for the XTT assay and D4445 test are in agreement (50 ppm), and
are within one (two-fold) dilution from the estimated MIC90 for
isothiazolinone in the E24 method (100 ppm). Such screening
assays generate relative comparisons of mould inhibitors and are
subject to some degree of biological variability. A higher MIC90 in
the E24 test might also be expected due to specimen exposure to
a greater variety of air and soil microbes in a non-sterile test
apparatus compared to the monocultures utilized in the other two
methods. Higher standard deviations seen at fungistatic concentrations
of biocide would be anticipated due to the sensitivity of the
assay to mitochondrial dehydrogenase activity (Levitz and
Diamond, 1985; Hawser et al., 1998) and inherent metabolic variability
in spore germination rates.
As a screening tool for mould inhibitors, the XTT assay offers
several advantages over the current standardized methods. Quantitative
results can be obtained in just 2 days without the need to
treat and condition wood specimens saving time and resources.
The redeeming feature of the ASTM test is the low cost for
disposable Petri dishes and the polyethylene spacer. It is also
a relatively simple test to conduct and can test either monocultures
or defined mixed cultures, but results are subjective. The AWPA
D4445 test method. As with the ASTM method, the visual rating
system is subjective and a high number of replicate specimens per
treatment group is important for precision and reproducibility.
Results from the ATP assay corroborated the XTT results by
verifying complete inhibition of metabolic activity after exposure of
mould spores to 50 ppm isothiazolinone. There was no growth of
spores from individual test fungi placed on malt extract agar
following 24-h exposure to 50 ppm isothiazolinone and aseptic
washing with DI water. This result suggests that isothiazolinone
was fungicidal rather than fungistatic against spores from the test
fungi under the conditions used in this study.
4. Discussion and conclusions
Despite micro- to macro- differences in the three test methods
compared in this study, a critical factor for a screening method is
the estimated minimal inhibitory concentration (MIC90) for the
chemical being evaluated. The estimated MIC90 for isothiazolinone
for the XTT assay and D4445 test are in agreement (50 ppm), and
are within one (two-fold) dilution from the estimated MIC90 for
isothiazolinone in the E24 method (100 ppm). Such screening
assays generate relative comparisons of mould inhibitors and are
subject to some degree of biological variability. A higher MIC90 in
the E24 test might also be expected due to specimen exposure to
a greater variety of air and soil microbes in a non-sterile test
apparatus compared to the monocultures utilized in the other two
methods. Higher standard deviations seen at fungistatic concentrations
of biocide would be anticipated due to the sensitivity of the
assay to mitochondrial dehydrogenase activity (Levitz and
Diamond, 1985; Hawser et al., 1998) and inherent metabolic variability
in spore germination rates.
As a screening tool for mould inhibitors, the XTT assay offers
several advantages over the current standardized methods. Quantitative
results can be obtained in just 2 days without the need to
treat and condition wood specimens saving time and resources.
The redeeming feature of the ASTM test is the low cost for
disposable Petri dishes and the polyethylene spacer. It is also
a relatively simple test to conduct and can test either monocultures
or defined mixed cultures, but results are subjective. The AWPA
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