May 2017 Volume 2 JEMI+ 1
Improvement of protocols for the screening of biological
control agents against white-nose syndrome
Robyn L McArthur, Soumya Ghosh and Naowarat Cheeptham*
Department of Biological Sciences, Thompson Rivers University, Kamloops, British Columbia
White-nose syndrome, caused by the fungus Pseudogymnoascus destructans (Pd), has become increasingly prevalent
in North America since 2006 and has caused mass mortality in many bat populations. This study focuses on
standardizing protocols for efficient Pd cultivation, which includes harvesting Pd spores from agar plates, culturing
Pd on both normal and modified culture media and finally, testing chemical agents and cave bacterial isolates
against Pd for antimicrobial activities. Pd cultivation was performed on Sabouraud Dextrose Agar (SDA), Rose
Bengal Agar (RBA) and modified RBA-bat tissue media. Only Pd grown on RBA supplemented with 0.25% bat
tissue had a final colony diameter that was significantly different from Pd grown on SDA and could be attributed to
the difference in the growth rates. Pd lawns grown using both pour- and spread-plate techniques were observed on
SDA media. Although the former technique led to slower fungal lawn growth in comparison to the latter, the more
uniform lawn produced with the pour-plate technique allowed the size of inhibitory zones to be determined
quantitatively. Further, the cultured Pd lawns were tested against the chemical antimicrobial agents Nystatin,
peroxigard and bleach (10%, 50%, 100%) and cave bacterial isolates using a Kirby Bauer and agar plug diffusion
assay. Both bleach (10%) and peroxigard (1.5%) created zones of inhibition in Pd lawns, with diameters measuring
25.5 mm and 12.7 mm, respectively, after 24 days of incubation. No antimicrobial activities were observed for the
cave bacteria tested against Pd. Our findings will allow more efficient and reliable screening of cave bacteria against
Pd, elucidating biological control agents against white-nose syndrome.
White-nose syndrome (WNS) emerged in North America
in 2006, and has now been confirmed in 29 US states and 5
Eastern Canadian provinces
(https://www.whitenosesyndrome.org). This bat disease
has been devastating for many bat populations, killing
over 5.7 million bats and causing 90-100% mortality in
some hibernacula. WNS is named for the appearance of
bats infected with Pseudogymnoascus destructans (Pd), the
causative fungus, which leads to white growth on bat ears,
tails, wings, and muzzles (1, 2). Although the origin of Pd
remains unknown, it is speculated that it may have been
introduced into North America from Europe (3). Pd is a
psychrophilic fungus that thrives at the temperatures
found in bat hibernacula and can degrade collagen, which
is believed to allow tissue invasion in the bat host (2, 4).
Infection with Pd causes more frequent arousal from
torpor, leading to the loss of crucial fat reserves and often
death of the bat (5). The rapid decline in bats due to WNS
has not just ecological, but also economic impacts (6). For
example, the role bats play in pest control is estimated to
save the agricultural sector at least $3.7 billion/year in
North America (7).
Due to the importance of bats, the development of a
treatment or control agent for WNS is imperative and the
recent case of WNS in Washington, the first confirmed case
in Western North America, adds a renewed sense of
urgency to the situation (8).
Studies to find control agents for WNS are ongoing, but
there are still no treatments that are being used in the field
on a large scale. Chemical agents, such as antifungals,
fungicides, biocides, and volatile organic compounds
originating from bacteria have been tested against Pd, with
many able to inhibit the growth of spores and mycelia (9,
10). However, most of the research has focussed on
biological control agents, which are less likely to disrupt
the cave ecosystem than the harsh physical and chemical
treatments that have been used in caves in the past (11).
Some of these potential biological control agents include
volatile compounds produced by strain DAP96253 of
Rhodococcus rhodochrous, extract produced by the fungus
The Journal of Experimental
Microbiology & Immunology+
May 2017 Volume 2 JEMI+ 2
Trichoderma polysporum WPM 39143 isolated from a cave air
sample, and cold-pressed, terpeneless orange oil, all of
which strongly inhibited the growth of Pd in vitro (12-14).
The ability to produce secondary metabolites with
antimicrobial properties is a well-established feature of
many bacteria, and cave bacteria, especially actinomycetes,
are known for this ability (15, 16). Therefore, it would be
novel to screen bacteria from the cave environment for
antagonistic activities against Pd. However, growing Pd
and testing it against diverse antimicrobial agents under
laboratory conditions is a challenging process since Pd has
a very slow growth rate and colonies must be mature to
produce adequate numbers of spores.
Our laboratory has continuously worked to elucidate
novel antimicrobial agents against bacterial pathogens
(Multi Drug Resistant strains) and Pd, preferably of
biological origin. Therefore, this project intended to
optimize a protocol to enhance the cultivation of Pd and to
test agents, both biological (cave bacterial isolates) and
chemical (bleach, peroxiguard, Nystatin) for their anti-Pd
activities. These findings will not only aid in our own
ongoing experiments, but also will benefit other
researchers who are working in this field.
MATERIALS AND METHODS
Bat tissue supplemented media preparation. A bat, which had
died of natural causes, was obtained frozen from Dr. Cori Lausen,
a bat ecologist. This frozen little brown bat (Myotis californicus)
was sliced in liquid nitrogen and ground into fine powder.
Following this, 0.25 g, 0.5 g and 1 g of bat tissue was added to 100
mL of Rose Bengal Agar (RBA) solution to obtain 0.25%, 0.5%, and
1% RBA-bat tissue media respectively, along with the control
where no bat tissue was added (0% RBA-bat tissue media). A
second control of Sabouraud Dextrose Agar (SDA) media plates
were also prepared as per the manufacturer’s instructions. All of
these media were prepared in triplicates. All the media were
autoclaved at 121°C for 15 mins before pouring into Petri plates.
Growth and analysis of Pd on bat supplemented media. A
piece of SDA-agar media (approximately 7 square mm)
containing P. destructans M3906-2 culture grown for 2 months at
15°C was excised out and placed into the centre of each media
plate (mentioned above). The P. destructans M3906-2 strain was
procured from Dr. J.P. Xu, McMaster University, Canada. The
plates were incubated at 15°C for a period of 27 days. The Pd
colony growth was noted on the 5th day, since no outgrowth of the
Pd was visualized for the initial 4 days. After which, the Pd
colonies were examined every 2-4 days. The growth of Pd on each
of the plates was quantified by measuring the diameter of the
colony both horizontally and vertically with a ruler and taking the
average of these two measurements (Fig 1). The colony diameter
measurement for each of the media types was considered as the
mean of triplicates. The mean colony diameters obtained for each
media type were compared by plotting them on an XY-scatter,
with standard error shown as error bars. The standard error was
calculated for each media type on each day measurements were
taken using the descriptive statistics function on Minitab 17.
Furthermore, the Pd growth rates on each of the media types were
calculated by using the formulae given below (Fig 1). To assess
whether mean colony diameters for the various media types were
significantly different from one another, Minitab®17
(http://www.minitab.com/en-us/products/minitab/) was used
to conduct a One-way ANOVA (Analysis of Variance), followed
by a Tukey Pairwise Comparison (α = 0.05 for both tests). This
was done for the means calculated for the initial day (day 5) and
the final day (day 27).
Pd spore isolation and preparation of fungal lawns. Cultures
of Pd strain M3906-2 were maintained on SDA plates at 15°C.
Spores were isolated from Pd cultures using mechanical scraping
and filtration through glass wool as previously described (12).
The concentration of spores in solution was quantified with a
haemocytometer and spore solutions were stored at 4°C. To grow
a fungal lawn, 200 μl of spore suspension was plated onto each 90
mm agar plate and spread with a sterile bent glass rod until the
solution was evenly spread out over the plate surface (17). The
plate was allowed to dry, inverted, and placed at 15°C for
Kirby Bauer diffusion assay for chemical antimicrobial
agents. Ninety microliters of a spore suspension containing 2 x 106
spores/mL was spread on each 60 mm SDA plate and allowed to
dry. Eight mm Kirby-Bauer discs (Toyo Roshi Kaisha Ltd., Japan)
were soaked in the chemical agents, air dried, and laid down in
the centre of each SDA plate containing the spread Pd spores.
Three chemicals; Nystatin (150 μg/ml) (EMD chemicals, Inc. San
Diego, USA), Concentrated bleach (100%, 50% and 10%) (London
Drugs, Richmond, Canada), and Peroxigard (1.5%) (Bayer,
Toronto, Canada), were used in quadruplets for this study. The
negative control of sterile water was prepared in duplicates. All
of the plates were inverted and incubated at 15°C. The
macroscopic morphology of Pd growth was recorded and any
zones of inhibition were measured with an electronic Vernier
calliper (Guangxi China, Mainland) every 2-6 days.
Figure 1. Measurement methods and formulae used to analyse Pd
colony growth on SDA and RBA media. For the two concentric
circles, the smaller one represents the ‘colony’ while the larger one is
the ‘Petri-dish’. A1 is the horizontal colony diameter, A2 is the
vertical colony diameter, B# (number refers to replicate) is the
average colony diameter for the plate, C is the colony diameter for
the media type, C27 is the colony diameter for that media type on day
27, C5 is the colony diameter for that media type on day 5, X is the
growth rate of Pd on that media type. The denominator of equation
3 gives the number of days over which the diameter was measured.
May 2017 Volume 2 JEMI+ 3
Kirby Bauer and agar plug diffusion assay for antimicrobial
testing of bacteria. Previously isolated bacteria from New
Brunswick caves (Gomes et al., unpublished data) were randomly
chosen to screen for anti-Pd activities. Both pour- and spread-plate
techniques were employed to seed the SDA media with Pd spores.
555 μL of Pd spore suspension (approximately 7 x 106 spores/mL)
was mixed with 120 mL of molten SDA media and was poured
into 150 mm plates. Concurrently, the same volume of the spore
suspension was spread-plated on SDA media plates. Three plates
of each spore-seeding technique were prepared and the same cave
bacterial isolates were tested once using each seeding technique.
Kirby Bauer discs were soaked with bleach (10%), Peroxigard
(1.5%), or sterile water and laid down on the SDA media plates
containing Pd spores. Simultaneously, agar plugs containing the
cave bacteria isolates were excised out from the media plates on
which they were cultured, and the agar plugs were laid down in
a similar way on the SDA media plates containing Pd spores with
a bent pick (agar plug diffusion assay). Bleach (10%), Peroxiguard
(1.5%) and sterile water were used as positive and negative
controls respectively. All of the plates were incubated at 15°C and
the macroscopic morphology of P. destructans growth was
recorded every 1-3 days. Any zones of inhibition observed were
measured with an electronic Vernier calliper.
Pd growth on bat-supplemented media. All of the media
plates (SDA, RBA, RBA-Bat tissue) exhibited growth of
raised white-grey Pd colonies and the production of liquid
exudate was observed on the surface of the colonies (Fig 2).
Pigment secreted into the agar was observed when Pd was
grown on SDA, but could not be seen in the other media
types. Characteristic curved spores attached to the mycelia
ends were visible when using light microscopy. The initial
(day 5) mean colony diameters measured for 1% 0.5%,
0.25%, and 0% bat tissue-RBA and SDA were 9.5 mm, 8.2
mm, 8.3 mm, 7.8 mm, and 8.0 mm, respectively. When the
initial mean colony diameters were compared using a one-
way ANOVA, they were found to be significantly different
from one another (F = 5.23, P = 0.015). Using a Tukey
pairwise comparison indicated that the differences were
between 1% bat tissue-RBA and 0% bat tissue-RBA (T =
4.08, P = 0.015), as well as SDA and 1% bat tissue-RBA (T =
-3.67, P = 0.028). The mean growth rates for P. destructans
grown on SDA and RBA were 0.65 mm/day and 0.75
mm/day respectively; whereas, P. destructans exhibited
mean growth rates of 0.83 mm/day, 0.75 mm/day, and
0.80 mm/day on 0.25%, 0.5%, and 1% bat tissue-RBA
respectively (Fig 3). The final mean colony diameters (day
27) measured for 1% 0.5%, and 0.25% bat tissue-RBA were
27.2 mm, 24.7 mm, and 26.5 mm respectively, followed by
RBA (24.3 mm) and SDA (22.3 mm). When these means
were compared using a one-way ANOVA, they were
found to be significantly different from one another (F =
5.06, P = 0.017). A Tukey pairwise comparison indicated
that the differences were between SDA and 0.25% bat
tissue-RBA (T = -3.47, P = 0.038), as well as SDA and 1% bat
tissue-RBA (T = -4.02, P = 0.016).
Pd lawn growth from a spore suspension.
Concentrations of spores up to 1.7 x 107 spores/mL were
obtained using the chosen spore isolation method. Plating
of isolated spores on SDA media led to the growth of
consistent fungal lawns after an average of 10 days (Fig 4).
However, plating higher concentrations (1.7 x 107
spores/mL) of spores led to fungal lawn growth faster (5
days), in comparison to lower concentrations (9.4 x 105
spores/ml) that produced a fungal lawn in 12-14 days.
Kirby Bauer diffusion assay for chemical antimicrobial
agents. Four plates exposed to Nystatin exhibited fungal
lawn growth covering the entire plate surface after 10 days
of incubation, with no zones of inhibition (Fig 5). Plates
with 10% bleach had very little P. destructans growth for the
first 12 days, with zones of inhibition visible from the 18th
day of incubation onwards. The average diameters of these
zones were approximately 30 mm and decreased to 25.5
mm after the 24th day of growth (Table 1). Pd spores
exposed to 50% bleach showed minimal growth along the
plate periphery after 18 days, exhibiting an inhibitory zone
almost the diameter of the plate (60 mm). Pd spores
exposed to 100% bleach did not produce any growth on the
agar plates, indicating that an inhibitory zone could be
equal to or larger than the plate diameter. Two of the 4
plates containing Pd spores exposed to Peroxigard showed
very minimal zones of inhibition after 12 days, but these
zones were no longer present after the 19th day of
incubation. The remaining 2 plates exhibited zones of
Figure 2. Colony morphology of Pd strain M3906-2 was visualized
on the SDA and RBA-Bat tissue media for a range of days.
May 2017 Volume 2 JEMI+ 4
inhibition with diameters of approximately 20 mm after
the 12th day of growth. However, the zone of inhibition was
not retained for one of the plates after the 18th day, and the
other plate had a zone diameter of 13.25 mm on the 19th
day, which decreased to 12.71 mm after 24 days of
incubation. Although the negative control plate exposed to
sterile water exhibited no Pd growth until the 18th day, it
did develop some patchy lawn growth, and the plate was
almost completely covered with a fungal lawn on the 19th
day of incubation.
Kirby Bauer and agar plug diffusion assay for
antimicrobial testing of bacteria. The pour- and spread-
plate techniques were employed to seed the spores on the
SDA plates. Implementation of the former technique
created minimal growth on the plate surface after 6 days,
with a consistent lawn visible after 9 days of growth, while
the latter displayed uneven lawn growth after 5 days, with
the lawn becoming more consistent on the 7th day of
growth. The spread-plates had areas with visibly thicker
growth than others and a few bare patches were also
observed. The agar plug diffusion assay with the cave
bacteria did not show any inhibitory activities against Pd.
The Kirby Bauer inhibition assay with 10% bleach created
inhibitory zones with average diameters measuring 13.19
mm with the pour-plated spores, while the spread-plated
spores exhibited zones measuring 33.85 mm in diameter
after 7 days of incubation. These zones decreased to 12.18
mm and 30.45 mm, respectively, after the 9th day. The
average inhibitory zone diameter observed for peroxigard
using the pour-plate technique was 21.67 mm after the 7th
day of incubation, which decreased to 19.33 mm after the
9th day of growth. Zones created by peroxigard could not
be measured on the plates with spores spread on the agar
surface due to fungal contamination (Table 2).
The present study provides insight into some of the
challenges associated with cultivation of Pd and
improvements that can be made to methods for
screening bacteria from cave soil samples against Pd.
The initial goal of the study was to determine if the
growth rate of Pd could be improved to augment the
entire screening process. Supplementing the media with
different concentrations (0.25-1%) of bat tissue was
implemented since a similar study showed that
incorporation of bird feathers in the growth media
induced the keratinolytic protease activities of B.
pumilus and B. cilius isolated from poultry feathers (18).
Our study found that Pd grown on SDA and 0.25% bat
tissue-RBA began with a similar colony diameter, but
had colony diameters that were significantly different
from one another after 27 days. This can be attributed to
a difference in the growth rate of 0.18 mm/day for Pd
grown on RBA media supplemented with 0.25% bat
tissue in comparison to Pd grown on SDA media. Pd
grown on 1% bat tissue-RBA and SDA also had a
significantly different colony diameter after 27 days of
Figure 3. Comparative analysis of Pd growth (colony diameter in millimetres) on SDA and Bat tissue (0%-1%) RBA media at various time
points (days). Error bars represent the standard error.
May 2017 Volume 2 JEMI+ 5
growth; however, Pd grown on these two media types
also had initial colony diameters that were significantly
different. This indicates that the difference in the final
diameters may not be due to a difference in the growth
rate, but could have been due to a difference in the
initial inoculum size. Although the addition of bat
tissue led to a slight increase in the growth rate of Pd
compared to the non-supplemented plates, the
concentration of bat tissue did not correspond to a
proportional increase in the growth rate. Moreover,
grinding of bat tissue is a time consuming and laborious
process; therefore, this process cannot be recommended
for antimicrobial screening of cave bacteria.
Creation of fungal lawns on agar media from Pd
spores is a common process and therefore, we adopted
a technique from a previous study (12) to isolate Pd
spores. Our study has shown that filtering of the spores
through glass wool to prevent unwanted mycelial
contamination in the desired spore suspension was very
effective for isolating spores of high concentrations.
Furthermore, the plating of these spores created
consistent fungal lawns that are useful in screening
assays, although production of sufficient spore numbers
for screening many cave bacteria remains a bottleneck
in this study.
The Kirby Bauer diffusion assay for chemical
antimicrobial agents had notable results. Nystatin did
not produce a zone of inhibition in the Pd fungal lawn.
Nystatin is an antifungal drug, but it is more commonly
used against yeast than against filamentous fungi,
which may be why it was ineffective against Pd (19).
However, a previous study showed that some
antifungal drugs, such as amphotericin B, could be used
effectively against Pd (13). Amphotericin B and Nystatin
belong to the same class of antifungal drugs, however,
the mode of action of Amphotericin B differs from
Nystatin (20), which could explain the difference in
activity observed when tested against Pd. Pd spores
exposed to 50% and 100% bleach produced no growth
or minimal growth on the plate edge. The lack of growth
is likely because these concentrations created zones of
inhibition larger than the plate diameter, although this
was never confirmed by using larger plates (Nunc®
Bioassay Dish, 245 mm x 245 mm x 25 mm) for testing.
Exposing Pd spores to 10% bleach created zones of
inhibition with diameters that decreased from 30 mm
after 18 days of growth to 25.5 mm after 24 days of
growth. Similar results were observed in another study:
Geomyces pannorum spores exposed to 10% bleach in a
Kirby-Bauer assay produced zones of inhibition with a
diameter of 67 mm after 7 days, which decreased to 45
mm in diameter after 30 days (21). Also in this study, Pd
spores exposed to 10% bleach in a Kirby-Bauer assay
did not produce any growth on the plate, suggesting
that Pd is more susceptible to treatment with 10% bleach
than G. pannorum (21). All plates exposed to peroxigard
produced zones of inhibition after 12 days of growth,
but only one of the four plates still had a zone of
inhibition after 24 days, which measured 12.7 mm in
diameter. In another study, exposing G. pannorum
spores to 0.3% Hydrogen peroxide created a zone of
inhibition with a 12 mm diameter (21). Based on the
inhibitory activity, 10% bleach and peroxigard were
used in subsequent experiments as positive controls.
Figure 4. Growth of Pd on SDA media from a Pd spore suspension.
(A) The colonies started to form as indicated by the black arrowhead
as observed under a dissecting microscope. (B-F) The formation of
the Pd lawn (white-grey) as the days progressed.
Figure 5. Chemical antimicrobial agents used during the
experiment. Incubation time in each of the cases is specified in the
right top corner. The arrowheads indicate the zone of inhibition. (A)
Growth of the Pd lawn from a range of concentrations of Pd spore
suspension. (B) Incubation of the Pd lawn with Nystatin. (C-F)
Cultivation of Pd spores with 10% Bleach for a range of time points
(days). (G-J) Cultivation of Pd spores with Peroxigard for a range of
time points (days).
May 2017 Volume 2 JEMI+ 6
However, 10% bleach seems to cause more prolonged
inhibitory activities in comparison to peroxigard, but
both may be used for experiments lasting 10-15 days.
Previous studies have employed the spread-plate
technique to seed agar plates with spores for the
creation of Pd lawns and it has been reported that this
technique produces distinct results when performing a
Kirby-Bauer disc diffusion assay (13, 21). However, the
pour-plate technique is used for seeding agar plates
when screening cave bacteria against both bacteria and
yeast (15); therefore, in this study we wanted to
determine if the pour-plate technique is a better option
for creating Pd lawns. Although the pour-plate
technique led to slower growth of Pd lawns in
comparison to the spread-plate method, more
consistent fungal lawn growth was observed in the case
of the former. Spreading Pd spores on SDA also
produced a fungal lawn, but led to patchy and uneven
growth due to the uneven spreading of spores. Since our
future experiments for screening cave bacteria will
include larger plates, the pour-plate technique will be
more convenient and consistent for creating uniform
Moreover, in our study we observed that the agar plug
diffusion assay to screen bacteria for inhibitory
activities against Pd is a simple and effective technique.
This technique is used extensively in our lab to screen
cave bacteria for antagonistic activities against
pathogenic microorganisms, such as bacteria and
single-celled and filamentous fungi, giving consistent
and reliable results (15). However, in our study, none of
the cave bacteria tested showed inhibitory activity
against P. destructans, which is not uncommon when
screening such a small sample of bacteria. Both
peroxigard and bleach created zones of inhibition, as
expected, but inhibitory zones were smaller on the
pour-plate SDA media in comparison to the spread-
plate SDA media.
The main limitation of the media supplementation
experiment is the lack of replicates, making it difficult
to definitively conclude if bat tissue supplementation
has an impact on the growth rate of Pd. Also, as
indicated by statistical testing and as can be seen in
Figure 3, the initial inoculum size on 1% bat tissue-RBA
appears to have been larger than those on the other
media types, so a more consistent inoculating method
should be used in the future. Additionally, in future
studies, a more accurate method of measuring Pd
colony growth, such as photographing the colonies and
using software to convert pixels to millimetres could be
used for greater reproducibility and reliability (10).
Future studies should also include pipetting a set
amount of chemical agents onto the Kirby Bauer discs
instead of disc-dipping in the Kirby Bauer disc diffusion
assays. This will allow us to spot a known volume and
concentration of the chemical used, enhancing the
reproducibility of the results. To circumvent the
problem of bacterial overgrowth in the Kirby-Bauer
assay, bacterial broth cultures can be prepared,
centrifuged down to collect the supernatant and this can
be spotted onto paper discs for testing. This technique
has been used previously for antimicrobial activity
screening from environmental samples where bacterial
secondary metabolites obtained in the supernatant were
In conclusion, despite the difficulties of growing Pd,
we have improved techniques that could be
implemented for our future antimicrobial screening of
cave bacteria against Pd.
We would like to thank Dr. Cori Lausen for providing us with the
bat, Dr. Nancy Flood for her statistics advice, Joanna Urban for
her help with prepping the bat for the bat media, Karen
Vanderwolf for providing us the New Brunswick cave sediment
Table 1. Diameters of inhibitory zones created when Pd was
cultured with chemical antimicrobial agents.
Table 2. Diameters of Pd inhibitory zones from the Kirby Bauer
and agar plug diffusion assay for screening cave bacteria.
May 2017 Volume 2 JEMI+ 7
samples, Sarah Gomes for her help with isolating the cave bacteria
from the New Brunswick cave sediment samples, and Dr. JP Xu
of McMaster University for his guidance and Pd to use in this
study. We would also like to thank the TRU Department of
Biological Sciences for some of the supplies used in this project.
Thank-you to the TRU Office of Research and Graduate Studies
for the funding provided by McArthur's Undergraduate
Apprenticeship Award. We would also like to acknowledge the
funding provided by Dr. Cheeptham’s and Dr. Lausen’s U.S. Fish
and Wildlife Service Grant (F15AS00188).
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