yield 1 106 spores ml1. In a preliminary study, 1 106 spores
ml1 of each test fungus was determined to be the optimal inoculum
in the XTT assay (Clausen and Yang, 2011).
For both ASTM and AWPA standardized tests, spores from
individual test fungi were collected in 10 ml of sterile deionized
water (DI) according to ASTM standard D4445-10 (AmericanWood
Protection Association Standards, 2010). Spores were counted and
equal numbers of spores for each test organism were transferred to
a spray bottle. The spore mixture was diluted with DI water to yield
approximately 3 107 spores ml1. The spray bottle was adjusted
to deliver 1 ml inoculum per spray and was shaken frequently
during inoculation to ensure a homogeneous inoculum.
2.3. Wood specimen preparation
Southern pine sapwood specimens either 7 20 mm cross
section 7 cm long or 75 100mm 12.5mmthick were cut from
kiln-dried lumber and conditioned at 27 C and 70% relative
humidity (RH). Typically, mould testing involves dip-treating
unseasoned wood, but to mimic methods for using isothiazolinone
in wood preservatives, vacuum treatment of kilndried
wood was used in this study. Ten random replicates of each
size specimen were vacuum-treated for 40 min at 172 kPa
submerged in each concentration of isothiazolinone test solution.
Treated specimens were air-dried for 1 week prior to conditioning
at 27 C and 70% RH for 21 days.
2.4. XTT assay
For each mould isolate, 1 106 spores were pre-incubated in
RPMI at 27 C for 24 h. In a sterile flat-bottom polystyrene microplate,
80 ml of RPMI and 80 ml of saline were added to each well.
Eighty ml of 2-fold dilutions of isothiazolinone ranging from (0e
800 ppm) were prepared in sterile RPMI and added to each well
(n ¼ 10) with appropriate positive and negative control wells. The
plate was sealed and incubated for 24 h at 37 C. Following exposure
to isothiazolinone, 80 ml XTT-menadione reagent was added to
all wells of the plate which was then wrapped in foil and incubated
for 3 h at 37 C. Absorbance was read with a PowerWave XS 2
microplate reader (BioTek Instruments, Inc., Winooski, VT) at
490 nm.
2.5. ASTM D4445-10
Treated wood specimens (7 20 mm 7 cm) (n ¼ 10) were
placed on top of a polyethylene mesh spacer in Petri dishes
(150 25 mm) (B-D Falcon, Los Angeles, CA) containing 4 layers of
blotting paper that was saturated with 30 ml DI water. Specimens
were sprayed with 1 ml of mixed mould spore inocula, sealed in
polyethylene bags to retain 100% RH, and incubated at 27 C and
70% RH. Individual specimens were visually evaluated for mould
growth after 4, 8, and 12 weeks of incubation on a scale of 0e5 with
0 indicating no mould growth and 5 indicating heavy mould
growth on all sides of the specimen.
2.6. AWPA E24
Environmental chambers fabricated according to standard
method AWPA E24 specifications were used to suspend treated
wood specimens (75 100 mm 12.5 mm) (n ¼ 10) over nonsterile
soil pre-inoculated with spores from the three test fungi.
The test chambers were placed in a conditioning room at 27 C.
Treated specimens were vertically suspended across the width of
the chamber over inoculated soil and sprayed with spores of the
test fungi. Individual specimens were rated for mould growth on
yield 1 106 spores ml1. In a preliminary study, 1 106 sporesml1 of each test fungus was determined to be the optimal inoculumin the XTT assay (Clausen and Yang, 2011).For both ASTM and AWPA standardized tests, spores fromindividual test fungi were collected in 10 ml of sterile deionizedwater (DI) according to ASTM standard D4445-10 (AmericanWoodProtection Association Standards, 2010). Spores were counted andequal numbers of spores for each test organism were transferred toa spray bottle. The spore mixture was diluted with DI water to yieldapproximately 3 107 spores ml1. The spray bottle was adjustedto deliver 1 ml inoculum per spray and was shaken frequentlyduring inoculation to ensure a homogeneous inoculum.2.3. Wood specimen preparationSouthern pine sapwood specimens either 7 20 mm crosssection 7 cm long or 75 100mm 12.5mmthick were cut fromkiln-dried lumber and conditioned at 27 C and 70% relativehumidity (RH). Typically, mould testing involves dip-treatingunseasoned wood, but to mimic methods for using isothiazolinonein wood preservatives, vacuum treatment of kilndriedwood was used in this study. Ten random replicates of eachsize specimen were vacuum-treated for 40 min at 172 kPasubmerged in each concentration of isothiazolinone test solution.Treated specimens were air-dried for 1 week prior to conditioningat 27 C and 70% RH for 21 days.2.4. XTT assayFor each mould isolate, 1 106 spores were pre-incubated inRPMI at 27 C for 24 h. In a sterile flat-bottom polystyrene microplate,80 ml of RPMI and 80 ml of saline were added to each well.Eighty ml of 2-fold dilutions of isothiazolinone ranging from (0e800 ppm) were prepared in sterile RPMI and added to each well(n ¼ 10) with appropriate positive and negative control wells. Theplate was sealed and incubated for 24 h at 37 C. Following exposureto isothiazolinone, 80 ml XTT-menadione reagent was added toall wells of the plate which was then wrapped in foil and incubatedfor 3 h at 37 C. Absorbance was read with a PowerWave XS 2microplate reader (BioTek Instruments, Inc., Winooski, VT) at490 nm.2.5. ASTM D4445-10Treated wood specimens (7 20 mm 7 cm) (n ¼ 10) wereplaced on top of a polyethylene mesh spacer in Petri dishes(150 25 mm) (B-D Falcon, Los Angeles, CA) containing 4 layers ofblotting paper that was saturated with 30 ml DI water. Specimenswere sprayed with 1 ml of mixed mould spore inocula, sealed inpolyethylene bags to retain 100% RH, and incubated at 27 C and70% RH. Individual specimens were visually evaluated for mouldgrowth after 4, 8, and 12 weeks of incubation on a scale of 0e5 with0 indicating no mould growth and 5 indicating heavy mouldgrowth on all sides of the specimen.2.6. AWPA E24Environmental chambers fabricated according to standardmethod AWPA E24 specifications were used to suspend treatedwood specimens (75 100 mm 12.5 mm) (n ¼ 10) over nonsterilesoil pre-inoculated with spores from the three test fungi.The test chambers were placed in a conditioning room at 27 C.Treated specimens were vertically suspended across the width ofthe chamber over inoculated soil and sprayed with spores of thetest fungi. Individual specimens were rated for mould growth on
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yield 1 106 spores ml1. In a preliminary study, 1 106 spores
ml1 of each test fungus was determined to be the optimal inoculum
in the XTT assay (Clausen and Yang, 2011).
For both ASTM and AWPA standardized tests, spores from
individual test fungi were collected in 10 ml of sterile deionized
water (DI) according to ASTM standard D4445-10 (AmericanWood
Protection Association Standards, 2010). Spores were counted and
equal numbers of spores for each test organism were transferred to
a spray bottle. The spore mixture was diluted with DI water to yield
approximately 3 107 spores ml1. The spray bottle was adjusted
to deliver 1 ml inoculum per spray and was shaken frequently
during inoculation to ensure a homogeneous inoculum.
2.3. Wood specimen preparation
Southern pine sapwood specimens either 7 20 mm cross
section 7 cm long or 75 100mm 12.5mmthick were cut from
kiln-dried lumber and conditioned at 27 C and 70% relative
humidity (RH). Typically, mould testing involves dip-treating
unseasoned wood, but to mimic methods for using isothiazolinone
in wood preservatives, vacuum treatment of kilndried
wood was used in this study. Ten random replicates of each
size specimen were vacuum-treated for 40 min at 172 kPa
submerged in each concentration of isothiazolinone test solution.
Treated specimens were air-dried for 1 week prior to conditioning
at 27 C and 70% RH for 21 days.
2.4. XTT assay
For each mould isolate, 1 106 spores were pre-incubated in
RPMI at 27 C for 24 h. In a sterile flat-bottom polystyrene microplate,
80 ml of RPMI and 80 ml of saline were added to each well.
Eighty ml of 2-fold dilutions of isothiazolinone ranging from (0e
800 ppm) were prepared in sterile RPMI and added to each well
(n ¼ 10) with appropriate positive and negative control wells. The
plate was sealed and incubated for 24 h at 37 C. Following exposure
to isothiazolinone, 80 ml XTT-menadione reagent was added to
all wells of the plate which was then wrapped in foil and incubated
for 3 h at 37 C. Absorbance was read with a PowerWave XS 2
microplate reader (BioTek Instruments, Inc., Winooski, VT) at
490 nm.
2.5. ASTM D4445-10
Treated wood specimens (7 20 mm 7 cm) (n ¼ 10) were
placed on top of a polyethylene mesh spacer in Petri dishes
(150 25 mm) (B-D Falcon, Los Angeles, CA) containing 4 layers of
blotting paper that was saturated with 30 ml DI water. Specimens
were sprayed with 1 ml of mixed mould spore inocula, sealed in
polyethylene bags to retain 100% RH, and incubated at 27 C and
70% RH. Individual specimens were visually evaluated for mould
growth after 4, 8, and 12 weeks of incubation on a scale of 0e5 with
0 indicating no mould growth and 5 indicating heavy mould
growth on all sides of the specimen.
2.6. AWPA E24
Environmental chambers fabricated according to standard
method AWPA E24 specifications were used to suspend treated
wood specimens (75 100 mm 12.5 mm) (n ¼ 10) over nonsterile
soil pre-inoculated with spores from the three test fungi.
The test chambers were placed in a conditioning room at 27 C.
Treated specimens were vertically suspended across the width of
the chamber over inoculated soil and sprayed with spores of the
test fungi. Individual specimens were rated for mould growth on
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