An inexpensive moist chamber culture technique for finding microbiota on live tree bark

Abstract

Premise
Traditional moist chamber cultures (MCs) prepared in aseptic laboratory environments using sterile Petri dishes are commonly used to quantify the microbiota of rough‐bark tree species and woody vines. MCs are typically expensive and may be difficult to make, so a less expensive option made from easily available supplies was developed. These cost‐friendly MCs were compared with standard laboratory methods to demonstrate their efficacy.

Methods and Results
Modified MCs were made using inexpensive, store‐bought supplies; compared to a standard laboratory setting, the modified MCs are shown to be less expensive with a faster setup time and larger size that facilitates a variety of tree and woody vine species. MC use resulted in the discovery of new species of fungi and myxomycetes with associated locality records. We provide detailed instructions for creating modified MCs, as well as a list of myxomycete species and their associated bark characteristics, pH values, and water‐holding capacity.

Conclusions
This new, low‐cost MC technique makes the study of microbiota more inclusive and accessible for those in research laboratories, classrooms, and homes, including both amateurs and professionals. MCs are easy to prepare, versatile, and applicable for many areas of botany and the biological sciences, potentially allowing exploration into unexplored areas in urban ecosystems.

Full text

Received: 29 June 2023
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Accepted: 7 March 2024
DOI: 10.1002/aps3.11578
PROTOCOL NOTE
An inexpensive moist chamber culture technique for finding
microbiota on live tree bark
Ashley P. Bordelon
1
|Harold W. Keller
1
|Angela R. Scarborough
2
1
Botanical Research Institute of Texas, 1700
University Drive, Fort Worth, Texas 76107, USA
2
Independent researcher, Kailua‐Kona,
Hawaii, USA
Correspondence
Harold W. Keller, Botanical Research Institute of
Texas, 1700 University Drive, Fort Worth, Texas
76107, USA.
Email: haroldkeller@hotmail.com
This article is part of the special issue “Resilient
botany: Innovation in the face of limited mobility
and resources.”
Abstract
Premise: Traditional moist chamber cultures (MCs) prepared in aseptic laboratory
environments using sterile Petri dishes are commonly used to quantify the microbiota
of rough‐bark tree species and woody vines. MCs are typically expensive and may be
difficult to make, so a less expensive option made from easily available supplies was
developed. These cost‐friendly MCs were compared with standard laboratory
methods to demonstrate their efficacy.
Methods and Results: Modified MCs were made using inexpensive, store‐bought
supplies; compared to a standard laboratory setting, the modified MCs are shown to
be less expensive with a faster setup time and larger size that facilitates a variety of tree
and woody vine species. MC use resulted in the discovery of new species of fungi and
myxomycetes with associated locality records. We provide detailed instructions for
creating modified MCs, as well as a list of myxomycete species and their associated
bark characteristics, pH values, and water‐holding capacity.
Conclusions: This new, low‐cost MC technique makes the study of microbiota more
inclusive and accessible for those in research laboratories, classrooms, and homes,
including both amateurs and professionals. MCs are easy to prepare, versatile, and
applicable for many areas of botany and the biological sciences, potentially allowing
exploration into unexplored areas in urban ecosystems.
KEYWORDS
biodiversity, fungi, myxomycetes, slime molds, urban ecology, video
The first general use of moist chamber cultures (MCs) to
isolate myxomycetes (plasmodial slime molds) from live
tree trunk bark is attributed to G. W. Martin and H. C.
Gilbert, working in the Mycological Laboratory of the
Department of Botany at the University of Iowa (Gilbert
and Martin, 1933; Gilbert, 1934). Rare and newly described
species were included in their original list of myxomycetes,
and more recently, additional new and rare species of
myxomycetes have been found using the MC technique
(Keller and Brooks, 1976,1977; Keller, 2004; Keller
et al., 2008,2009; Keller and Marshall, 2019) as well as
fungi (Perry et al., 2020) (Figure 1). Some of these species
were discovered using a rope‐climbing technique to collect
bark samples for MC cultures in the tree canopy (Parker
and Keller, 2003; Snell and Keller, 2003; Keller et al., 2004;
Everhart et al., 2009; Kilgore et al., 2009; Scarborough
et al., 2009). Collection techniques to obtain bark samples
from trunks of live trees and woody vines for MC cultures
are described in Keller and Braun (1999), Keller and
Everhart (2010), and Keller et al. (2009). Examples of
myxomycete species associated with the two tree species
focused on here, Juniperus virginiana L. (eastern red cedar)
and Ulmus americana L. (American elm), are provided in
Tables 1and 2.
As described by Stephenson (1982,1985), laboratory
preparation of MCs involved field collection of live tree
trunk bark and ground samples of decayed wood and leaves.
This methodology included sterile plastic Petri dishes,
boiled tap water, and costly laboratory equipment, but the
studies did not report detailed bark characteristics. In these
Appl. Plant Sci. 2024;e11578. wileyonlinelibrary.com/journal/AppsPlantSci
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https://doi.org/10.1002/aps3.11578
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studies, only myxomycete species were targeted; moreover,
little information was provided for trees (e.g., tree species
designations, a complete list of rough‐bark tree species,
examples of smooth‐bark unproductive tree species, pH of
bark, myxomycete species associations, water‐holding
capacity of bark) and supply costs were not included.
In contrast, the new MC culture technique described here
can recover a broader spectrum of species at a much lower
cost. This MC technique found diverse organisms on live tree
bark, including green algae, cyanobacteria (blue‐green algae),
myxobacteria, fungi (molds and fleshy mushrooms), lichens
(crustose and foliose types), liverworts, mosses, nematodes,
tardigrades, and insects; the presence of fungi, myxomycetes,
nematodes, and tardigrades demonstrates that this technique
has broad use for the botanical research community and
biologists. By using low‐cost store‐bought supplies available
from local sources and a low‐tech approach, this MC
technique is inclusive of a broader spectrum of users,
including research and amateur botanists, classroom teach-
ers, and community scientists. The design of this modified
MC still serves the same purpose as the traditional MC—to
create ideal conditions in a microenvironment to facilitate the
formation of fungi and myxomycetes, while allowing safe
observation over a longer time.
There is a growing need for more cost‐effective
accessibility in research laboratories and increased involve-
ment of community scientists in scientific research. Many
researchers may be in academic or private institutions
where funding is lacking or inadequate, physical access to
research laboratories may not be possible, or informed
FIGURE 1 Fleshy mushroom Mycena ulmi growing on the surface of
live Ulmus americana tree bark in a moist chamber culture. (Photo by
Brian A. Perry, reproduced with permission from the Journal of the
Botanical Research Institute of Texas; Perry et al., 2020).
TABLE 1 Myxomycete species found on live Juniperus virginiana tree bark, arranged by taxonomic order, genus, and species. Nomenclature generally
follows Martin and Alexopoulos (1969).
a
Order
Echinosteliales Liceales Physarales Stemonitales Trichiales
Field collections Clastoderma debaryanum
var. emperatorium
C. microcarpum
C. pachypus
Echinostelium arboreum
E. coelocephalum
E. elachiston
E. fragile
E. minutum
Cribraria minutissima
C. violacea
Dictydiaethalium
plumbeum
Licea biforis
L. denudescens
L. inconspicua
L. kleistobolus
L. operculata
L. parasitica
L. pedicellata
L. pseudoconica
L. scyphoides
Badhamia affinis
B. rugulosa
Badhamiopsis ainoae
Diachea arboricola
Diderma corrugatum
D. chondrioderma
Didymium clavus
D. orthonemata
D. synsporon
Physarum
auriscalpium
P. crateriforme
P. nutans
Trabrooksia
applanata
Comatricha laxa
Macbrideola
cornea
M. declinata
M. decapillata
M. scintillans
Stemonitis
flavogenita
Arcyria cinerea
Calomyxa metallica
Minakatella longifila
Perichaena
chrysosperma
P. depressa
P. minor
P. minor var. pardina
Moist chamber cultures
(Traditional method)
Clastoderma debaryanum
var. emperatorium
C. microcarpum
C. pachypus
Echinostelium arboreum
E. coelocephalum
E. elachiston
E. fragile
E. minutum
Cribraria minutissima
C. violacea
Licea biforis
L. denudescens
L. inconspicua
L. kleistobolus
L. nannengae
L. operculata
L. parasitica
L. pedicellata
L. pseudoconica
L. scyphoides
Badhamia affinis
B. rugulosa
Badhamiopsis ainoae
Diachea arboricola
Didymium clavus
Physarum
auriscalpium
P. crateriforme
P. nutans
Comatricha laxa
Macbrideola
cornea
M. declinata
M. decapillata
M. scintillans
Stemonitis
flavogenita
Arcyria cinerea
Calomyxa metallica
Minakatella longifila
Perichaena
chrysosperma
P. depressa
P. minor
P. minor var. pardina
a
Total number of different myxomycete taxa from bark of living Juniperus virginiana trees = 54. Author citations can be found in Appendix S3.
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expertise to identify taxa is not available. This updated do‐
it‐yourself moist chamber culture was designed to be done
with inexpensive, cost‐effective, readily available, and time‐
saving supplies that are available in both research laborato-
ries and home settings to facilitate the observation,
collection, and publication of microbiota data.
METHODS
Cost comparisons and overview of moist
chamber culture preparations
Researchers studying fungi and myxomycetes in the early 1930s
used standard (and more expensive) laboratory equipment, such
as sterile glass Petri dishes, to make moist chamber cultures.
More recently, sterile disposable plastic Petri dishes have
become more common in modern laboratories. These Petri
dishes and sterile deionized water have contributed in part to
repeatability within the controlled environmental parameters of
MCs (Figure 2A). However, there are disadvantages to using
this standard laboratory equipment. These supplies are typically
available only in larger bulk lots, leading to overall higher costs
and larger space requirements for storage; furthermore, Petri
dishes have size limitations of about 150 mm in diameter and
15 mm in depth, which does not allow the use of larger or
greater numbers of tree bark pieces (size often limited to 4–5
smaller bark pieces). A standard reusable aluminum foil pie tin
was chosen to replace the traditional Petri dish due to its wide
availability, lower cost, and larger size (Figure 2B). This pie tin,
about 25 cm in diameter and >2.5 cm in depth, permits the use
of more pieces (8–12 larger bark pieces without overlapping) as
well as thicker tree bark sizes, thus increasing the possibility for
multiple microbiota in shorter setup times. This modified
methodology resulted in cost and time savings (Table 3);
furthermore, pie tin culture containers can be washed with soap
andwater,sterilizedwithalcohol,dried,andreused,thus
reducing the cost even more. One disadvantage of this MC
protocol is that it is more susceptible to airborne contaminants
because of less sterile conditions; however, this can be
minimized by scanning observations through clear plastic wrap
and limiting the amount of uncovered exposure time to external
surroundings.
These batch cultures have resulted in harvesting many
tiny myxomycete species (100–200 µm), especially in the
genera Echinostelium and Licea, or slightly larger sizes for
Macbrideola species (Figure 3), enabling many more
myxomycete fruiting bodies per collection deposited in
the herbarium of the Botanical Research Institute of Texas
(BRIT). This modified MC culture was developed at BRIT
and has been used in published papers (Keller and
Marshall, 2019; Perry et al., 2020). Proper curation and
labeling become extremely important, especially when
describing species that are new to science (Figure 4).
Here, we provide a brief summary of the MC
preparation instructions; the detailed protocol is provided
TABLE 2 Myxomycete species found on live Ulmus americana tree bark, arranged by taxonomic order, genus, and species. Nomenclature generally
follows Martin and Alexopoulos (1969).
a
Order
Echinosteliales Liceales Physarales Stemonitales Trichiales
Field collections Clastoderma debaryanum
var. emperatorium
C. microcarpum
Cribraria confusa
C. minutissima
C. violacea
Licea denudescens
L. inconspicua
L. kleistobolus
L.marginata
L.nannengae
L. operculata
L. parasitica
L. pedicellata
L. scyphoides
Badhamia affinis
Badhamiopsis ainoae
Diderma
chondrioderma
D. corrugatum
Didymium clavus
D. orthonemata
Physarum
crateriforme
Trabrooksia
applanata
Comatricha cornea
C. laxa
Macbrideola cornea
M. decapillata
Stemonitis
flavogenita
Arcyria cinerea
Calomyxa metallica
Dianema (clustered
spores)
Minakatella longifila
Perichaena
chrysosperma
P. depressa
P.quadrata
Moist chamber cultures
(Traditional method)
Clastoderma debaryanum
var. emperatorium
Echinostelium arboreum
E. coelocephalum
E. minutum
Cribraria confusa
C. minutissima
C. violacea
Licea denudescens
L. inconspicua
L.iridescens
L. kleistobolus
L.nannengae
L. operculata
L. parasitica
L. pedicellata
L. pseudoconica
L. scyphoides
Badhamiopsis ainoae
Didymium clavus
Physarum
crateriforme
Comatricha cornea
C. fimbriata
C. laxa
Enerthenema
papillatum
Macbrideola cornea
M. decapillata
M. scintillans
Stemonitis
flavogenita
Arcyria cinerea
Calomyxa metallica
Minakatella longifila
Perichaena
chrysosperma
P. depressa
P.quadrata
P. minor
a
Total myxomycete species on Ulmus americana = 42. Author citations can be found in Appendix S3.
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in Appendix S1. Using rough bark samples from living trees
and woody vines (Table 4) increases the chances of
harvesting myxomycetes and fungi. Juniperus virginiana
tree trunk bark, shown as an example in Video S1, can often
be removed using fingers to strip the bark. Low‐cost
materials used for the batch MCs can be purchased at local
stores; these include reusable pie tins, paper towels, mineral
spring or distilled water, and clear plastic wrap for quick
preparation and wetting bark samples. Periodic examination
of MC cultures over several weeks using a dissecting
microscope at magnifications of 20–100× reveals an
assortment of life forms. Proper curation and deposition
of specimens is essential; therefore, as an example, we
provide here instructions from BRIT for harvesting, boxing,
and labeling specimens for transport (Appendix S2). A link
to a Google Form is also available on the BRIT website for
the public to submit questions and their own MC images to
the program staffto answer and review.
Visual and instructional aids
Materials and methods to make traditional MCs are available
in publications that may or may not be freely accessible online,
and there has never been an accompanying online visual aid
for the process of creating any MC. To make this new MC
method more useful and accessible, we have created a step‐by‐
step instructional video (Video S1); this is also available on
YouTube (https://youtu.be/aiwUkbz947M)andontheFungi,
Myxomycetes, and Trees Research Program webpage of the
Fort Worth Botanic Garden‐BRIT website (https://fwbg.org/
research-projects/fungi-myxomycetes-and-trees-program/diy-
moistchamber/). Detailed instructions accompanying this
video are provided in Appendix S1 and are also available as
a PDF download at https://fwbg.org/wp-content/uploads/
2021/06/FINAL-DIY-Moist-Chamber-Culture.pdf.
The four‐minute video begins by highlighting the
materials needed to create the MC, as well as additional
materials needed for collecting a tree bark sample. The
video then shows how to collect bark from a live tree, gives
examples of the data to be collected, and explains each step
of constructing a MC. The video concludes with compound
microscope images showing examples of the early life‐cycle
stages of myxomycetes that begin to emerge within the first
days following the first wetting of the MC (Figure 3). Next,
examples of other microbiota that may be seen in a MC,
such as mosses, insects, and nematodes, are shown; the
video concludes with images of common contaminants of
FIGURE 2 Traditional and modified moist chamber culture (MC)
techniques. (A) Traditional MC technique using sterile oversized plastic
disposable Petri dishes. (Photo by Courtney M. Kilgore, used with
permission.) (B) Uncovered and reusable foil pie tin used for batch MC
technique. (Photo by Bob O'Kennon, used with permission.)
TABLE 3 Cost comparison of moist chamber cultures (MC).
a
Traditional MC Modified MC
Materials Price (USD)/unit Materials Price (USD)/unit
Plastic, sterile, disposable Petri dish bottom with top lid $2.24 to $4.55 Reusable aluminum foil pie tin $0.40 to $1.25
Plastic sheet PVC cling film wrap $0.013 per sq. ft.
Deionized and sterilized water (30 mL) $5.40 Non‐sterile natural spring water (30 mL) $0.08
Sterile Whatman P8‐creped filter paper (150 × 25 mm) $0.13 Non‐sterile white paper towels $0.08
Fine‐point permanent marker $1.00 Fine‐point permanent marker $1.00
Total cost $8.77 to $11.08 $1.58 to $2.42
a
Material unit costs were based on estimates from scientific supply sources and local convenient hardware and grocery stores available in urban areas.
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MCs, such as types of green mold (species of Trichoderma)
that may occur from long exposure to the air. There have
been more than 800 views of this demonstration video since
it was made public on 5 June 2021.
RESULTS
Examples of microbiota found on rough bark
of live trees and woody vines
Live Malus domestica (Suckow) Borkh. (apple trees) planted
in apple orchards, especially older abandoned orchards, are
productive habitat sources for myxomycetes (Keller and
Braun, 1999). Bark surfaces of mature, larger apple trees are
roughened, irregularly fissured, sometimes thin, scaly, and
peeling with myxomycete fruiting bodies often found on the
underside of sloughing thin bark sections. Abundant sporan-
gia of Licea biforis,Perichaena depressa,andP.quadrata
(Figure 5) were found on large trunks of live apple trees in
apple orchards and also developed in moist chamber cultures
(Keller and Eliasson, 1992; Keller and Braun, 1999); additional
rough bark tree species are listed in Table 4. These rough
bark tree and woody vine species have in common bark
characteristics that are mostly thicker, fissured, ridged, and
sometimes cracking into squares or smaller scales that often
create flowage waterways, with highly water‐absorptive bark
(Table 5) and a pH nearer a neutral 7.0 (Table 4).
Smaller, younger, and less mature trees are not as
productiveformicrobiotaasolder,muchlargertrees
(taller than 15 m and approximately 1.8 m in diameter).
Bigger and older live trees are better for greater microbiota
species diversity (Snell and Keller, 2003; Keller et al., 2009).
Bark samples taken from about 2 m high on the tree trunk
and on any side of the tree—especially along flowage
waterways, deep fissures, and crevices—stay wetter longer
and are more productive for myxomycete species. Lichens,
mosses, or liverworts may be visible on bark surfaces;
therefore, bark should be collected in these areas as well as
in different locations on the tree to increase the probability
for recovery of myxomycetes. Although tree bark is
considered nonliving, the underlying tissues are living, so
great care must be taken to avoid injuring trees. The size of
bark samples should also be considered; pie‐tin batch MC
containers allow the use of bark pieces 15–23 cm,
increasing opportunities for species abundance, as
opposed to using plastic Petri dishes, which limit the size
of bark pieces to 2.5–5cm.
FIGURE 3 Examples of myxomycete species found in moist chamber cultures (MCs). (A) The stipitate sporangium of Macbrideola cornea (0.6–2.5 mm
tall) is frequently found in MCs of live Juniperus virginiana and Ulmus americana tree bark. Note the translucent stalk, peridial collar, and branching
capillitium. (Photo by Edward D. Forrester, used with permission.) (B) Stalked sporangium of Echinostelium arboreum (100–150 µm tall) showing persistent,
shiny peridium on moss phyllid from the bark of a live Ulmus americana tree. (Photo by Edward D. Forrester, used with permission.) (C) Sessile sporangia
of immature (right, orangish) and mature (left, blackish) Licea parasitica with operculum showing the circumscissile line of dehiscence (50–200 µm in
diameter). This species is often found on J. virginiana and U. americana bark surfaces of live trees in association with crustose lichens. (Photo by Sydney E.
Everhart, used with permission.)
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Herbicolous myxomycetes discovered on
prairie plants using MC cultures
Herbicolous myxomycetes also have been discovered on
vascular prairie plants. Samples of mature fruits and stems
of herbaceous prairie plants harbor diverse and abundant
myxomycete species (Table 6) (Kilgore et al., 2009). Prairie
forbs like Echinacea species (prairie cone flower), Yucca
species (Spanish bayonet), and Asclepias syriaca L. (com-
mon milkweed) can be found in native prairies, planted as
ornamentals, and along roadside right of ways.
DISCUSSION
Environmental factors affecting microbiota on
live tree bark
Bark pH is one factor that influences the presence, growth,
and development of corticolous myxomycetes and fungi in
MCs (Snell and Keller, 2003; Everhart et al., 2008;
Scarborough et al., 2009; Kilgore et al., 2009; Perry
et al., 2020). Therefore, during live tree bark collection,
collectors should be aware that pH ranges and assemblages
of acidophilic, neutrophilic, and basophilic myxomycete
species associated with live tree species bark and woody
vines increase the chances of finding a more diverse group
of myxomycete species (Everhart et al., 2008,2009; Kilgore
et al., 2009; Scarborough et al., 2009). For example, Cercis
canadensis L. (redbud) is a smaller tree with a higher pH
value that also had a different composition of myxomycete
species than the other prairie plants studied (Table 4)
(Kilgore et al., 2009). This pH was measured by saturating
the bark with neutral sterile water for 24 hours, then
decanting the water and measuring the pH using a standard
laboratory pH meter. More details regarding this method
are available in Snell and Keller (2003).
It should also be noted that water‐holding capacity
(WHC) and retention enhance the presence, growth, and
development of myxomycetes and fungi in MCs (Snell and
Keller, 2003). The WHC of living tree bark has been
determined for only five tree species: Fraxinus americana L.,
Quercus alba L., Liriodendron tulipifera L., Acer rubrum L.,
and Pinus strobus L. (Snell and Keller, 2003). The only
significant difference in WHC was between the species that
held the most (L.tulipifera) and the least (P. strobus) water
(Table 5). This difference can be determined in part by sight
because L.tulipifera bark initially absorbs more water in the
MC culture dish and has a water‐soaked appearance. In
contrast, P. strobus has resiniferous bark, with beaded water
droplets appearing on bark surfaces, so that more free water
remains in the bottom of the Petri dish. Bark WHC was
determined by measuring the difference in weight between
the water‐saturated bark and the oven‐dried bark. More
details can be found in Snell and Keller (2003).
Corticolous myxomycetes‐associated trees
and vines
Unfortunately, live tree bark examples of Juniperus
virginiana and Ulmus americana were not part of the tree
species test group for WHC (Snell and Keller, 2003).
However, direct observation of MC cultures clearly showed
both of these tree species have bark that acts like a wick and
absorbs water quickly, resulting in a water‐soaked
FIGURE 4 A stalked Diachea arboricola sporangium harvested from a
MC. Note silvery iridescent peridial surface; additional developmental
stages and scanning electron microscope images of this species are available
in its original publication (Keller et al., 2004). This species was found on
trunk bark of live Quercus alba and Juniperus virginiana trees. (Photo by
Kenneth L. Snell, used with permission.)
FIGURE 5 Perichaena quadrata sessile sporangia growing in MC
culture. (Photo by Sydney E. Everhart, used with permission.)
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appearance. Rough bark surfaces, bark with higher WHC,
and bark with a near‐neutral pH clearly account for the
higher species diversity of myxomycetes (Tables 4and 5)
(Snell and Keller, 2003; Scarborough et al., 2009).
Juniperus virginiana bark is thin, fibrous, flattened, with
slight grooves or vertical crevices (Figure 6A). This shreddy
bark can be peeled using fingers into long, thin strips
sometimes 60–122 cm in length. The bark is often an ashy
TABLE 4 List of rough bark live tree and woody vine species, with pH and number of myxomycete species found.
a,b
Species name and authority Common name pH ± SE
No. of myxomycete species
present
Malus domestica (Suckow) Borkh. Domestic apple 10
Picea rubens Sarg. Red spruce 3.7 ± 0.05 10
Pinus strobus L. White pine 3.8 ± 0.16 24
Pinus echinata Mill. Shortleaf pine 3.9 ± 0.88 14
Abies fraseri (Pursh) Poir. Fraser fir 4.1 ± 0.06 none
Tsuga canadensis (L.) Carrière Eastern hemlock 4.1 ± 0.08 17
Acer rubrum L. Red maple 4.7 ± 0.14 49
Liriodendron tulipifera L. Yellow poplar 4.9 ± 0.22 39
Taxodium distichum (L.) Rich. Bald cypress 4.9 ± 0.24 20
Platanus occidentalis L. American sycamore 5.1 ± 0.04 10
Vitis aestivalis Michx. Summer grapevine 5.2 ± 0.03 15
Vitis vulpina L. Fox grapevine 5.5 ± 0.08 12
Acer saccharum Marshall Silver maple 5.5 ± 0.05 17
Quercus alba L. White oak 5.7 ± 0.46 41
Liquidambar styraciflua L. Sweet gum 5.8 ± 0.04 10
Cercis canadensis L. Eastern redbud 6.3 ± 0.51 11
Tilia americana L. American basswood 6.6 ± 0.08 10
Fraxinus americana L. White ash 6.9 ± 0.16 31
Ulmus americana L.
c
American elm 7.0 ± 0.12 42
Juniperus virginiana L.
c
Eastern red cedar 7.0 ± 0.09 54
a
Tree and woody vine species ordered from acidophilic pH (3.7–4.1), to mesophilic pH (4.7–5.8), to neutrophilic pH (6.3–7.0).
b
Data adapted in part from the following publications: Keller and Braun (1999), Snell and Keller (2003), Keller (2004), Everhart et al. (2008), Keller et al. (2009), Kilgore
et al. (2009).
c
Species compiled from field‐collected myxomycetes on the bark surface of living Juniperus virginiana and Ulmus americana trees and fruiting bodies harvested from moist
chamber bark cultures. These myxomycete species were collected over a period of 35 years from approximately 250 trees. Bark was collected from these two tree species at a height
of 1.5–2m.
TABLE 5 Percent water‐holding capacity (WHC) by tree and tree species.
a
Tree species
Tree no. F. americana Q. alba L. tulipifera A. rubrum P. strobus
1 123 65 107 105 84
2 120 49 141 132 53
3 51 114 146 103 60
4 60 80 117 83 80
5 82 90 68 118 55
Mean 87 80 116 108 66
SD ±33 ±25 ±31 ±18 ±15
a
Data adapted from Snell and Keller (2003).
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gray color on exposed surfaces and may have foliose or
crustose lichens, leafy liverworts, mosses, or be void of
epiphytes and bare. This bark acts like a wick with a higher
WHC, which likely accounts in part for its higher
myxomycete species diversity, often with hundreds of
myxomycete fruiting bodies and sometimes up to six or
eight species on a single tree. This bark makes ideal MC
cultures because the thinner bark will fit evenly into the
bottom of a culture pie tin. Many more generalist
myxomycete species with pH values near or at pH 7 are
found in great abundance on live J. virginiana trees (see
Table 1) (Scarborough et al., 2009).
A species list of myxomycete species was compiled from
field‐collected myxomycete species on the bark surface of
living Juniperus virginiana trees and fruiting bodies
harvested from traditional moist chamber bark cultures
(Table 1); this list was made over a period of at least 35 years
from approximately 250 trees. Bark from most of these trees
was collected at a height of 1.5–2 m. Two notable tree
species included in our studies are widely available to
collectors in our sample areas: J. virginiana, which was
found in cemeteries, around rural homesteads, in shelter
belts, along road right of ways and fence lines, and in open
field habitats, and Ulmus americana, which is an ornamen-
tal shade tree found in residential areas and along streets
and avenues in urban areas. Users may wish to search rough
bark tree species in their home areas to discover myxomy-
cete taxa anywhere and at any time.
Species of woody vines characterized by the genus Vitis,
especially V. aestivalis Michx. and V.vulpina L., have
grooved bark often peeling in long shredded strips that is
notably water absorptive and productive for myxomycete
species (Table 4).
Collecting bark from the trees and woody vines discussed
above will maximize the chances for discovery and recording
of myxomycete species throughout the midwestern, eastern,
and southern USA. Many of the smaller myxomycete species
in the genera Echinostelium and Licea are found on the trunk
bark of living trees (Keller and Brooks, 1976,1977;Kellerand
Braun, 1999; Keller and Marshall, 2019), and all five
myxomycete orders are found on live tree and woody vine
species. The following species listed in Tables 1and 2can be
picture‐keyed and easily recognized based on macromor-
phological features: Arcyria cinerea,Cribraria violacea,Licea
TABLE 6 Prairie plant species, with pH and number of myxomycete species found.
a
Species name Common name pH No. of myxomycete species present
Yucca spp. Great plains yuccas 7.1 ± 1.0 11
Echinacea spp. Prairie coneflowers 7.3 ± 1.0 7
Asclepias syriaca Common milkweed 7.9 ± 1.1 5
a
Alkaliphilic pH rank ordered (7.1–7.9).
FIGURE 6 Rough bark surface characteristics of live (A) Juniperus virginiana and (B) Ulmus americana trees. (Photos by Bob O'Kennon, used with
permission.)
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operculata,L.parasitica,L. pedicellata,L. pseudoconica,
Macbrideola cornea,M.decapillata,Perichaena chrysosperma,
P.depressa,Physarum crateriforme,Stemonitis flavogenita.
They occur frequently on live tree bark samples in moist
chamber cultures.
Smooth bark live trees unproductive for
myxomycetes
Trees with smooth bark surfaces usually have thinner bark
that adheres tightly to the underlying tissues. This bark is
difficult to remove safely without injury to underlying tree
tissues. Species of Celtis (hackberry) are good examples of
this, with a bark surface that is smooth, thin, warty, and
tight (e.g., C. occidentalis L. and C. laevigata Willd.). Species
of birch can have peeling bark (e.g., Betula cordifolia Regel
[heart‐leaf paper birch]) and should be avoided. Populus
tremuloides Michx. (quaking aspen) has smooth bark, often
flaking or peeling from the woody core. Fagus grandifolia
Ehrh. (American beech) also has smooth bark that is dense,
tight, and difficult to sample without injuring the tree. Bark
from older, larger Platanus occidentalis L. (American
sycamore or planetree) trees originating from about 3–6m
into the upper canopy is often found peeled and broken into
plate‐like scales in sections on the ground. Samples collected
nearer to ground level are more productive, whereas those
collected at higher levels are unproductive. Quercus species
(oaks) are usually productive for myxomycete fruiting
bodies, but large red oaks (Quercus rubra L.) have dense,
iron‐like tight bark that is difficult to remove without
injuring the tree. Individual trees may be exceptions,
especially with abundant epiphytes of lichens, mosses, and
liverworts, but in general this group of smooth bark tree
species should be avoided for bark sampling.
CONCLUSIONS
The low‐cost, low‐tech, modified moist chamber culture
technique described here (Appendix S1,VideoS1)willmake
the study of microbiota accessible to researchers in science
laboratories; community scientists; classroom teachers in
elementary, middle, and high schools; and naturalists inter-
ested in exploring new life forms on the trunk bark of urban
tree species. The cost comparison outlined here clearly shows
that the modified batch MC method using reusable pie tins
versus standard laboratory disposable plastic Petri dishes saves
time and money. Myxomycete‐productive tree species can be
easily found in urban and residential environments. A list of
18 species of rough bark trees and two species of woody vines
(Table 4) is provided here as possible collection sites for moist
chamber cultures. The physical and chemical properties of
these species are also provided to increase the chances of
recovery of myxomycetes and fungi in moist chamber
cultures. This new batch MC technique will allow professional
and amateur researchers to discover many myxomycete and
fungi taxa from their immediate environments.
AUTHOR CONTRIBUTIONS
A.P.B conceived of this research project by preparing the video
and posting it on the BRIT website; all authors contributed to
the video content and instructions. A.R.S. conducted the
research using moist chamber cultures and collected and
analyzed data with the support of H.W.K. A.P.B and H.W.K.
prepared the original manuscript draft, and H.W.K. prepared
the figures. A.R.S. and H.W.K. revised and edited manuscript
drafts according to reviewers' suggestions. All authors approved
the final version of the manuscript.
ACKNOWLEDGMENTS
The authors thank the Botanical Research Institute of Texas for
use of research space and equipment, and the herbarium
support staffwho assisted with curation and deposit of
collections. Grants from the National Science Foundation,
Discover Life in America, National Geographic Society, Sigma
Xi, Missouri Department of Natural Resources, and U.S.
Department of Education McNair Scholars Program provided
financial assistance in part for this research project.
DATA AVAILABILITY STATEMENT
All data used in this study are found within this article and
supplementary materials.
REFERENCES
Everhart, S. E., H. W. Keller, and J. S. Ely. 2008. Influence of bark pH on
the occurrence and distribution of tree canopy myxomycete species.
Mycologia 100: 191–204.
Everhart, S. E., J. S. Ely, and H. W. Keller. 2009. Evaluation of tree canopy
epiphytes and bark characteristics associated with the presence of
corticolous myxomycetes. Botany 87(5): 509–517.
Gilbert, H. C. 1934. Three new species of myxomycetes. University of Iowa
Studies in Natural History. Contributions from the Botanical
Laboratories 16: 153–159.
Gilbert, H. C., and G. W. Martin. 1933. Myxomycetes found on the bark of
living trees. University of Iowa Studies in Natural History, Papers on
Iowa Fungi IV 15(3): 3–8.
Keller, H. W. 2004. Tree canopy biodiversity: Student research experiences
in Great Smoky Mountains National Park. Systematics and Geography
of Plants 74: 47–65.
Keller, H. W., and T. E. Brooks. 1976. Corticolous Myxomycetes V:
Observations on the genus Echinostelium. Mycologia 68: 1204–1220.
Keller, H. W., and T. E. Brooks. 1977. Corticolous Myxomycetes VII:
Contribution toward a monograph of Licea,five new species.
Mycologia 69: 667–684.
Keller, H. W., and U. H. Eliasson. 1992. Taxonomic evaluation of
Perichaena depressa and Perichaena quadrata (Myxomycetes) based
on controlled cultivation, with additional observations on the genus.
Mycological Research 96: 1085–1097.
Keller, H. W., and K. L. Braun. 1999. Myxomycetes of Ohio: Their
systematics, biology and use in teaching. Ohio Biological Survey
Bulletin New Series, Vol. 13. The Ohio State University, Columbus,
Ohio, USA.
Keller, H. W., and S. E. Everhart. 2010. Importance of Myxomycetes in
biological research and teaching. Fungi 3(1): 13–27.
Keller, H. W., and V. M. Marshall. 2019. A new iridescent corticolous
myxomycete species (Licea: Liceaceae: Liceales) and crystals on
AN INEXPENSIVE MOIST CHAMBER CULTURE TECHNIQUE
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9of10

American elm tree bark in Texas, U.S.A. Journal of the Botanical
Research Institute of Texas 13(2): 367–386.
Keller, H. W., M. Skrabal, U. H. Eliasson, and T. W. Gaither. 2004. Tree
canopy biodiversity in the Great Smoky Mountains National Park:
Ecological and developmental observations of a new myxomycete
species of Diachea.Mycologia 96: 537–547.
Keller, H. W., C. M. Kilgore, S. E. Everhart, G. J. Carmack, C. D. Crabtree,
and A. R. Scarborough. 2008. Myxomycete plasmodia and fruiting
bodies: Unusual occurrences and user‐friendly study techniques.
Fungi 1(1): 24–37.
Keller, H. W., S. E. Everhart, M. Skrabal, and C. M. Kilgore. 2009. Tree
canopy biodiversity in temperate forests: Exploring islands in the sky.
Southeastern Biology 56(1): 52–74.
Kilgore, C. M., H. W. Keller, and J. S. Ely. 2009. Aerial reproductive
structures on vascular plants as a microhabitat for myxomycetes.
Mycologia 101: 303–317.
Martin, G. W., and C. J. Alexopoulos. 1969. The Myxomycetes. University
of Iowa Press, Iowa City, Iowa, USA.
Parker, E. E., and H. W. Keller. 2003. Correlation of pH with assemblages
of corticolous myxomycetes in Big Oak Tree State Park. Journal of the
McNair Central Achievers Program 12(Issue 1): 4–8.
Perry, B. A., H. W. Keller, E. D. Forrester, and B. G. Stone. 2020. A new
corticolous species of Mycena section viscipelles (Basidiomycota, Agar-
icales) from the bark of a living American elm tree in Texas, U.S.A.
Journal of the Botanical Research Institute of Texas 14(2): 167–185.
Scarborough, A. R., H. W. Keller, and J. S. Ely. 2009. Species assemblages of
tree canopy myxomycetes related to pH. Castanea 74(2): 93–104.
Snell, K. L., and H. W. Keller. 2003. Vertical distribution and assemblages
of corticolous myxomycetes on five tree species in the Great Smoky
Mountains National Park. Mycologia 95: 565–576.
Stephenson, S. L. 1982. Slime molds in the laboratory. The American
Biology Teacher 44(2): 119–127.
Stephenson, S. L. 1985. Slime molds in the laboratory II: Moist chamber
cultures. The American Biology Teacher 47(8): 487–489.
SUPPORTING INFORMATION
Additional supporting information can be found online in
the Supporting Information section at the end of this article.
Appendix S1. Detailed instructions to create a moist
chamber culture and prepare collection data, to accompany
Video S1.
Appendix S2. Information needed for myxomycete and
fungal specimen labels for deposit in BRIT herbarium.
Appendix S3. Binomial names and author citations of the
myxomycete species discussed. (+) represents laboratory
observations made in moist chamber cultures. (x) represents
field observations made on trunk bark of living trees.
Appendix S4. Myxomycete life cycle stages.
Video S1. How to create a moist chamber culture to view
the biodiversity growing on live tree bark.
How to cite this article: Bordelon,A.P.,H.W.
Keller, and A. R. Scarborough. 2024. An
inexpensive moist chamber culture technique for
finding microbiota on live tree bark. Applications in
Plant Sciences 12(2): e11578.
https://doi.org/10.1002/aps3.11578
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