Up-dating the Cholodny method using PET films to sample microbial communities in soil
The aim of this work was to investigate the use of PET (polyethylene terephtalate) films as a modern development of Cholodny’s glass slides, to enable microscopy and molecular-based analysis of soil communities where spatial detail at the scale of microbial habitatsis essential to understand microb...
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Інститут молекулярної біології і генетики НАН України
2011
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irk-123456789-1563992019-06-19T01:25:28Z Up-dating the Cholodny method using PET films to sample microbial communities in soil Moshynets, O.V. Koza, A. Dello Sterpaio, P. Kordium, V.A. Spiers, A.J. Molecular and Cell Biotechnologies The aim of this work was to investigate the use of PET (polyethylene terephtalate) films as a modern development of Cholodny’s glass slides, to enable microscopy and molecular-based analysis of soil communities where spatial detail at the scale of microbial habitatsis essential to understand microbial associations and interactions in this complex environment. Methods. Classical microbiological methods; attachment assay; surface tension measurements; moleculartechniques: DNA extraction, PCR; confocal laserscanning microscopy (CLSM); micro-focus X-ray computed tomography (µCT). Results. We first show, using the model soil and rhizosphere bacteria Pseudomonas fluorescens SBW25 and P. putida KT2440, that bacteria are able to attach and detach from PET films, and that pre-conditioning with a filtered soil suspension improved the levels of attachment. Bacteria attached to the films were viable and could develop substantial biofilms. PET films buried in soil were rapidly colonised by microorganisms which could be investigated by CLSM and recovered onto agar plates. Secondly, we demonstrate that µCT can be used to non-destructively visualise soil aggregate contact points and pore spaces across the surface of PET films buried in soil. Conclusions. PET films are a successful development of Cholodny’s glass slides and can be used to sample soil communities in which bacterial adherence, growth, biofilm and community development can be investigated. The use of these films with µCT imaging in soil will enable a better understanding of soil micro-habitats and the spatially-explicit nature of microbial interactions in this complex environment. Keywords: Pseudomonas, soil, buried slide, PET film. Мета цієї роботи полягала у дослідженні можливості використання плівок, виготовлених із ПЕТ (поліетилентетрафталат), як модифікації методу скелець обростання Холодного для мікроскопічного і молекулярно-генетичного аналізу ґрунтових спільнот із збереженням їхньої просторової архітектури на мікрорівні. Таке збереження деталей просторового розташування об’єктів дозволило б глибше вивчити їх у подібних складних середовищах проживання. Методи. Класичні мікробіологічні методи; аналіз прикріплення; вимірювання поверхневого натягнення; молекулярно-генетичні методи: екстракція ДНК, ПЛР; конфокальна лазерна скануюча мікроскопія (КЛСМ); мікрофокусна рентгенівська комп’ютерна томографія (мікроКТ). Результати. По-перше, використовуючи модельні ґрунтові і ризосферні бактерії Pseudomonas fluorescens SBW25 і P. putida KT2440, ми показали, що бактерії здатні до прикріплення і відкріплення від ПЕТ плівок, а прекультивація за умов відфільтрованої ґрунтової суспензії покращує рівень прикріплення. Бактерії, які прикріпилися до плівок, зберігають свою життєздатність і спроможні до формування повноцінної біоплівки. ПЕТ плівки, занурені в ґрунт, колонізуються мікроорганізмами, що спостерігали як за допомогою КЛСМ, так і методом культивування ПЕТ плівок, видалених з ґрунту, на агаризованому поживному середовищі. По-друге, ми продемонстрували, що мікроКТ можна використовувати для неруйнівного спостереження сайтів зв’язування ґрунтових агрегатів і ґрунтових пор з поверхнею плівки, що перебуває в ґрунті. Висновки. Застосування ПЕТ плівок виявилося вдалою модифікацією методу скелець обростання Холодного та може бути корисним для відбору ґрунтових мікробних спільнот, дослідження бактерійного прикріплення, росту і розвитку як біоплівок, так і спільноти. Використання цих плівок при аналізі ґрунтів за допомогою мікроКТ дозволить краще визначити ґрунтові мікроеконіші і природу архітектури мікробних взаємодій за таких складних екологічних умов. Ключові слова: Pseudomonas, ґрунт, скельця обростання, ПЕТ плівки. Цель данной работы состояла в исследовании возможности использования плeнок, изготовленных из ПЭТ (полиэтилентерефталат), как современной модификации метода стeкол обрастаний Холодного для микроскопического и молекулярно-генетического анализа почвенных сообществ с сохранением их пространственной архитектуры на микроуровне. Такая сохранность деталей пространственного расположения объектов позволила бы глубже изучить их в подобных сложных условиях обитания. Методы. Классические микробиологические методы; анализ прикрепления; измерение поверхностного натяжения; молекулярногенетические методы: экстракция ДНК, ПЦР; конфокальная лазерная сканирующая микроскопия (КЛСМ); микрофокусная рентгеновская компьютерная томография (микроКТ). Результаты. Во-первых, используя модельные почвенные и ризосферные бактерии Pseudomonas fluorescens SBW25 и P. putida KT2440, мы показали, что бактерии способны к прикреплению и откреплению от ПЭТ плeнок, а прекультивирование в условиях отфильтрованной почвенной суспензии улучшает уровень прикрепления. Бактерии, прикреплeнные к плeнкам, сохраняют жизнеспособность и могут формировать полноценную биоплeнку. ПЕТ плeнки, погружeнные в почву, колонизируются микроорганизмами, что наблюдали как с применением КЛСМ, так и методом культивирования извлечe нных из почвы плeнок на агаризованной питательной среде. Во-вторых, мы продемонстрировали, что микроКТ можно использоватьа для недеструктивного наблюдения за сайтами связывания почвенных агрегатов и почвенных пор с поверхностью ПЕТ плeнки, находящейся в почве. Выводы. Применение ПЕТ плeнок оказалось удачной модификацией метода стeкол обрастаний Холодного и может стать полезным для отбора почвенных микробных сообществ, изучения бактериального прикрепления, роста, развития как биоплeнок, так и сообщества. Использование этих плeнок при анализе почвы с помощью микроКТ позволит лучше определять почвенные микроэкониши и природу архитектуры микробных взаимодействий в таких сложных экологических условиях. Ключевые слова: Pseudomonas, почва, стeкла обрастания, ПЭТ плeнки. 2011 Article Up-dating the Cholodny method using PET films to sample microbial communities in soil / O.V. Moshynets, A. Koza, P.Dello Sterpaio, V.A. Kordium, A.J. Spiers // Вiopolymers and Cell. — 2011. — Т. 27, № 3. — С. 199-205. — Бібліогр.: 29 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0000BA http://dspace.nbuv.gov.ua/handle/123456789/156399 579.262 + 57.083.1 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies |
| spellingShingle |
Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies Moshynets, O.V. Koza, A. Dello Sterpaio, P. Kordium, V.A. Spiers, A.J. Up-dating the Cholodny method using PET films to sample microbial communities in soil Вiopolymers and Cell |
| description |
The aim of this work was to investigate the use of PET (polyethylene terephtalate) films as a modern development of Cholodny’s glass slides, to enable microscopy and molecular-based analysis of soil communities where
spatial detail at the scale of microbial habitatsis essential to understand microbial associations and interactions
in this complex environment. Methods. Classical microbiological methods; attachment assay; surface tension
measurements; moleculartechniques: DNA extraction, PCR; confocal laserscanning microscopy (CLSM); micro-focus X-ray computed tomography (µCT). Results. We first show, using the model soil and rhizosphere bacteria Pseudomonas fluorescens SBW25 and P. putida KT2440, that bacteria are able to attach and detach from
PET films, and that pre-conditioning with a filtered soil suspension improved the levels of attachment. Bacteria
attached to the films were viable and could develop substantial biofilms. PET films buried in soil were rapidly
colonised by microorganisms which could be investigated by CLSM and recovered onto agar plates. Secondly,
we demonstrate that µCT can be used to non-destructively visualise soil aggregate contact points and pore spaces across the surface of PET films buried in soil. Conclusions. PET films are a successful development of Cholodny’s glass slides and can be used to sample soil communities in which bacterial adherence, growth, biofilm
and community development can be investigated. The use of these films with µCT imaging in soil will enable a
better understanding of soil micro-habitats and the spatially-explicit nature of microbial interactions in this
complex environment.
Keywords: Pseudomonas, soil, buried slide, PET film. |
| format |
Article |
| author |
Moshynets, O.V. Koza, A. Dello Sterpaio, P. Kordium, V.A. Spiers, A.J. |
| author_facet |
Moshynets, O.V. Koza, A. Dello Sterpaio, P. Kordium, V.A. Spiers, A.J. |
| author_sort |
Moshynets, O.V. |
| title |
Up-dating the Cholodny method using PET films to sample microbial communities in soil |
| title_short |
Up-dating the Cholodny method using PET films to sample microbial communities in soil |
| title_full |
Up-dating the Cholodny method using PET films to sample microbial communities in soil |
| title_fullStr |
Up-dating the Cholodny method using PET films to sample microbial communities in soil |
| title_full_unstemmed |
Up-dating the Cholodny method using PET films to sample microbial communities in soil |
| title_sort |
up-dating the cholodny method using pet films to sample microbial communities in soil |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2011 |
| topic_facet |
Molecular and Cell Biotechnologies |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/156399 |
| citation_txt |
Up-dating the Cholodny method using PET films to sample microbial communities in soil / O.V. Moshynets, A. Koza, P.Dello Sterpaio, V.A. Kordium, A.J. Spiers // Вiopolymers and Cell. — 2011. — Т. 27, № 3. — С. 199-205. — Бібліогр.: 29 назв. — англ. |
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Вiopolymers and Cell |
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| fulltext |
MOLECULAR AND CELL BIOTECHNOLOGIES
Up-dating the Cholodny method using PET films to sample
microbial communities in soil
O. V. Moshynets, A. Koza1, P. Dello Sterpaio1, V. A. Kordium, A. J. Spiers1, 2
Institute of Molecular Biology and Genetics NAS of Ukraine
150, Akademika Zabolotnoho St., Kyiv, Ukraine, 03680
1The SIMBIOS Centre, University of Abertay Dundee
Bell St., Dundee DD1 1HG, UK
2Division of Forensics and Bio Sciences, School of Contemporary Sciences, University of Abertay Dundee
Bell St., Dundee DD1 1HG, UK
moshynets@gmail.com
The aim of this work was to investigate the use of PET (polyethylene terephtalate) films as a modern develop-
ment of Cholodny’s glass slides, to enable microscopy and molecular-based analysis of soil communities where
spatial detail at the scale of microbial habitats is essential to understand microbial associations and interactions
in this complex environment. Methods. Classical microbiological methods; attachment assay; surface tension
measurements; molecular techniques: DNA extraction, PCR; confocal laser scanning microscopy (CLSM); mic-
ro-focus X-ray computed tomography (µCT). Results. We first show, using the model soil and rhizosphere bac-
teria Pseudomonas fluorescens SBW25 and P. putida KT2440, that bacteria are able to attach and detach from
PET films, and that pre-conditioning with a filtered soil suspension improved the levels of attachment. Bacteria
attached to the films were viable and could develop substantial biofilms. PET films buried in soil were rapidly
colonised by microorganisms which could be investigated by CLSM and recovered onto agar plates. Secondly,
we demonstrate that µCT can be used to non-destructively visualise soil aggregate contact points and pore spa-
ces across the surface of PET films buried in soil. Conclusions. PET films are a successful development of Cho-
lodny’s glass slides and can be used to sample soil communities in which bacterial adherence, growth, biofilm
and community development can be investigated. The use of these films with µCT imaging in soil will enable a
better understanding of soil micro-habitats and the spatially-explicit nature of microbial interactions in this
complex environment.
Keywords: Pseudomonas, soil, buried slide, PET film.
Introduction. The complex interactions between mic-
roorganisms and their role in soil processes is funda-
mental to soil and plant health and productivity [1, 2].
The last two decades have seen a significant develop-
ment of molecular methods allowing the investigation
of microbial diversity and activity in soils and the rhi-
zosphere [3]. However, sampling methods largely ig-
nore the spatial distribution of organisms within the
complex 3D structure of soil pores and aggregates. Al-
though some techniques, including thin sectioning [4,
5], the physical isolation of individual soil aggregates
[6], the use of sampling tubes [7] and micro-sampling
rods [8] allow investigation of microbial communities
at the micro-scale, we suggest that a modern develop-
ment of the classical buried slide method [9] could pro-
vide a means of sampling soil communities in a manner
in which spatial distributions are retained, and is also
compatible with modern molecular methods.
Nikolay Cholodny famously developed the buried
glass slide method to sample soil microorganisms al-
most a century ago in Kiev. This and similar methods
are largely forgotten today as focus shifted to the use of
199
ISSN 0233–7657. Biopolymers and Cell. 2011. Vol. 27. N 3. P. 199–205
Institute of Molecular Biology and Genetics, NAS of Ukraine, 2011
molecular techniques to investigate microbial diversity
and activity. He buried slides for extended periods be-
fore recovery and microscopic examination of the ad-
hered soil particles and associated microflora. Both
fungi and bacteria were seen to colonize the glass surfa-
ce from soil particles, and regions of poor colonization
corresponding to hollows (i. e. pores) where «the par-
ticles of soil do not lie close to the slide surface» (pg.
150 in [10]).
Cholodny recognized that the glass surface which,
due to its hydrophilic nature, was covered in a thin film
of water that would allow the movement of bacteria and
the diffusion of nutrients. The «surface effect» of arti-
ficial structures such as sampling devices and contai-
ners is often regarded as problematic since it may bias
the development of fungal hyphae and plant roots.
However, this problem is ameliorated as the size of the
structure is reduced: soil microcosms are often produ-
ced using sieved soil with aggregate and particle sizes
of < 2 mm, which suggests that samplers of this di-
mension should be employed rather than the 70 ×
× 35 mm glass slides used by Cholodny. Unlike glass,
plastic films, e. g. PET (polyethylene terephtalate), are
readily cut to size and are also suitable for microscopy.
The adherence of some bacterial pathogens to the hyd-
rophobic PET film surface has been investigated [11–
13], but as yet the attachment of soil bacteria to this ma-
terial has not been examined.
Advances in micro-focus X-ray computed tomo-
graphy (µCT) allow the internal 3D structure of soil to
be non-destructively imaged at a resolution of ~10 µm
[14]. µCT-determined pore networks can then be used
as the basis for modelling water distribution, nutrient
and oxygen gradients etc., in a way that describes mic-
robial habitats at the micro-scale and predicts com-
munity activity and soil function [14]. Although the
threshholding (binary segmentation) of µCT images
into pore space and solids is problematic [15], it may be
possible to identify PET films in situ in soil micro-
cosms using this technique, and then to map the points
of soil contact across the surface of the film to visualise
Cholodny’s «hollows» and other spatial features. Such
3D information could then be combined with film-ba-
sed microscopical analyses of microbial distribution,
diversity and activity to produce a better understanding
of soil micro-habitats.
In this report, we investigate whether PET films
can be used as a modern development of Cholod-
ny’s buried slides, and whether they can be visualised
in soil with respect to local 3D pore structure by µCT
imaging.
Materials and methods. Bacteria and culturing
conditions. Wild-type Pseudomonas fluorescens
SBW25 [16] and P. putida KT2440 [19] were used in
this work, as well as the two P. fluorescens SBW25
mutants, ∆viscA [17] and WS-GFP [18]. Pseudomo-
nads were cultured using KB (King’s B) medium [20]
at 18–20 °C.
WS-GFP biofilms were produced in statically (i. e.
vibration-free) incubated KB cultures [21]. Inocula for
experiments were provided by cells from over-night
cultures re-suspended in PBS.
PET films and assays. Pieces of PET film were cut
from 40 µm- or 310 µm-thick sheets and sterilized by
autoclaving (for the thin films) or with ethanol (thick
films). Aliquots of bacterial suspension were placed
onto 1 cm2 pieces of PET film incubated for 1–4 h. Un-
attached bacteria were removed by rinsing twice in ste-
rile deionised water (hereafter «water»). Films were
stained with 0.05 % (w/v) Crystal violet (CV) for 3 min
before rinsing twice in water. CV was then eluted in
1 ml 96 % ethanol for 1 h before absorbance (OD570)
was measured to determine the level of attachment (or
detachment). A Kruss K100 Mk2 Tensiometer was
used to measure liquid surface tension of cell-free
culture supernatants, produced after the centrifugation
of stationary phase (18 h) KB cultures at 3,220 g for
10 min, as described previously [18]. Data is reported
as the mean ± standard error (SE). Differences between
means was determined by Student’s t-test assuming
unequal variances.
PCR. Pieces of PET film (0.25 cm2) were cut into
4–5 fragments and added to 25 µl PCR reactions mix-
tures (Taq PCR Kit, «New England Biolabs») contai-
ning universal 16S primers (uni-for: 5'-TGC CAG
CAG CCG CGG TA-3' and uni-rev: 5'-GAC GGG
CGG TGT GTA CAA-3') [22]. These were amplified
after 6 min at 95 °C by 28 cycles of 30 s at 95 °C, 30 s at
57.1 °C and 30 s at 70 °C. Purified genomic DNA was
used as a positive control. The PCR products were vi-
sualised by 1.2 % agarose-TBE gel electrophoresis af-
ter EtBr staining.
200
MOSHYNETS O. ET AL.
Soil microcosms and filtered soil suspension. Bul-
lion field soil from the Scottish Crop Research Institute
(Invergowrie, UK) [23] was air-dried and sieved to ob-
tain <2 mm sized aggregates. Soil microcosms were
produced by packing soil with PET films into plastic
rings to a density of 1.3 g/cm3 (microcosms are artifi-
cial or simplified environments used to investigate as-
pects of ecology; typically they are small and easily
manipulated for experimentation). These were satura-
ted with water and then equilibrated to –8 kPa on a ten-
sion table before incubation in a plastic box to reduce
evaporation for 7 days. Aliquots of soil, and PET films
recovered from microcosms washed twice in water,
were shaken in 2 ml PBS for 2 h before dilution and
spreading onto KB plates.
A filtered soil suspension was prepared by shaking
0.5 g soil in 6 ml water for 24 h before filtration through
a 0.55 µm membrane.
Microscopy. PET films were fixed by exposure to
formalin or gluteraldehyde vapour for 30 min (except
the P. fluorescens SBW25 WS-GFP biofilms which
were not fixed), then stained with 5 µg/ml Acridine
orange (AO), 5 µg/ml Calcofluor, 2 µg/ml Ethidium
bromide (EtBr), 5 µg/ml Hoechst 33342, and/or 5 µg/
ml Propidium iodide (PI) for 3–5 min (90 min for
Hoechst) before washing with water and the addition of
antibleach reagent (para-phenylendiamine) [24]. Films
were then placed onto conventional microscope slides
and covered with a slip before CLSM (confocal laser
scanning microscopy).
µCT imaging. A representative soil microcosm con-
taining PET films was imaged using a Nikon Metrology
micro-focus X-ray µCT system at 90 kV, 138 µA, with
3,000 angular projections at 1 frame per second and a
detector resolution giving a sample voxel resolution of
24.9 µm. Radiographs were reconstructed as a 3D volume
using CTAgent/CTPro («Nikon», Japan), imported into
VGStudio Max (http://www.volumegraphics.com/) for
inspection and exported as JPEG files.
Results and discussion. In order to examine the
utility of PET film samplers for soil and rhizosphere
studies, we first investigated the ability of two model
soil and plant-associated pseudomonads, P. fluores-
cens SBW25 [16] and P. putida KT2440 [19] to attach
and detach from PET films. Preliminary CV staining
experiments showed that both bacteria clearly adhered
to this novel hydrophobic substrate (P. fluorescens
SBW25 has already been reported to attach to hydro-
philic surfaces such as glass, e. g. [18]; P. putida
KT2440 is known to attach to a range of surfaces inclu-
ding glass [25]). Bacterial attachment could be quanti-
fied by CV measurements as shown in Fig. 1, demonst-
rating that P. putida KT2440 attachment to PET films
increased with incubation time and cell numbers (simi-
lar results were observed using P. fluorescens SBW25,
data not shown). P. putida KT2440 attachment levels
were ~3× higher when cells were re-suspended in KB
rather than PBS, suggesting that bacterial attachment to
201
UP-DATING THE CHOLODNY METHOD USING PET FILMS TO SAMPLE MICROBIAL COMMUNITIES IN SOIL
0.30
0.20
0.10
0.35
0.25
0.15
0.05
0.0
Time, h
0.50
0.30
0.20
0.60
0.40
0.10
0.0
Relative numbers
0 1 2 3 4 01 0.1 0.010.001
a b
c
1 2 3 4 5
Fig. 1. P. putida KT2440 cells are able to attach to PET films. Bac-
terial attachment levels, determined by Crystal violet (CV) staining
(OD570), increases with incubation time and with cell numbers: a –
attachment levels after 4 h incubation were ~ 6× greater than that seen
after only 1 h incubation (the bacterial suspension was a 0.01× dilution
of P. putida KT2440 cells in PBS; the inset shows two pieces of film
stained with CV after incubation with PBS (left) and P. putida KT2440
suspension (right)); b – attachment levels decrease with increasing
dilution of the bacterial suspension (1 – 0.001× dilutions of P. putida
KT2440 suspensions were allowed to attach for 2 h before assay); c –
DNA sequences can be directly amplified from P. putida KT2440 atta-
ched to PET films. Shown are the results of PCR amplifications of ge-
nomic DNA control (1); a sample of sterile film (2); a sample with atta-
ched bacteria (3); a sample with attached bacteria after drying (4); and a
sample with attached bacteria after fixation (5)
PET film surfaces was sensitive to chemical conditi-
ons. This attachment was clearly reversible, as P. puti-
da KT2440 cells detached from PET films when sha-
ken in PBS, with a 10 % decrease in CV levels obser-
ved after 1 h and a 30 % decrease after 4 hr incubation.
These observations suggest that other soil and plant-as-
sociated bacteria could be expected to attach to PET
films, as is the case for a variety of medically-important
pathogens.
As a simple demonstration that PET films are likely
to be compatible with modern molecular techniques,
we have shown the results of PCR amplifications of
DNA from bacteria attached to PET films in Fig. 1, c.
Bacterial surface attachment involves a range of in-
teractions dependent on cell and substrate surface che-
mistry [26]. For example, Campylobacter jejuni and
Mycobacterium avium have different cell surface pro-
perties which effect attachment to PET [12], whilst
modification of the surface chemistry of PET reduces
the attachment of Staphylococcus epidermis [11, 13].
Similar studies have not been undertaken with soil and
plant-associated bacteria which might be expected to
bind to a wider range of substrates under different con-
ditions than these pathogens.
Modification or pre-treatment of surfaces to produ-
ce a «conditioning film» with altered physicochemical
properties can have a significant impact on bacterial
attachment and colonisation [26]. To examine whether
P. fluorescens SBW25 and P. putida KT2440 attach-
ment was sensitive to altered PET surface chemistry,
we compared attachment to clean PET film and samp-
les that had been pre-treated with PBS, cell-free culture
supernatants or filtered soil suspension.
Pre-treatment with PBS had no significant impact
on attachment (P = 0.8732), suggesting that a phos-
phate and saline (pH 7.4) solution did little to modify
the surface chemistry of the PET film. Similarly, pre-
treatment with a mixture of bacterially-derived com-
ponents present in a cell-free P. fluorescens SBW25
culture supernatant, resulted in a small increase in at-
tachment (2×; P = 0.0078), suggesting that some of the-
se components might interact with the film surface to
provide a better attachment site for bacteria. Interes-
tingly, pre-treatment with a cell-free culture superna-
tant derived from the P. fluorescens SBW25 ∆viscA
mutant unable to produce the surfactant viscosin, resul-
ted in significantly higher levels of attachment (8×; P =
= 0.0001). The presence of viscosin was confirmed in
the wild-type supernatant and not in the mutant super-
natant, by surface tension measurements (25.61 ± 0.14
and 49.19 ± 0.14 mN⋅m–1, respectively; P < 0.0001).
This suggests that viscosin interacts with the PET film
surface and reduces bacterial attachment. Finally, pre-
treatment with a filtered soil suspension resulted in
significantly higher levels of attachment (9×; P =
= 0.0001), indicating that soil-soluble components
such as salts, clay, organic material and small particu-
late matter interact with the film to provide a surface
environment more suitable for bacterial attachment.
The impact of pre-treatment with a filtered soil suspen-
sion seen here suggests that artificial surfaces with a
variety of surface chemistries may be rapidly impro-
ved for bacterial adhesion once covered by appropria-
te environmental chemicals and particulate matter.
If PET films are to be used to sample soil and rhizo-
sphere microbial communities, bacteria must be able to
colonise the PET film after initial attachment. Prelimi-
nary experiments suggest that this is possible, as P. pu-
tida KT2440 cells detached from PET films by shaking
in PBS were still viable and produced colonies when
spread onto KB plates. Colonies could also develop
from PET films directly placed onto or embedded in the
surface of agar plates.
In order to determine whether the growth of atta-
ched bacteria was inhibited by the physicochemical
properties of PET, films were tested as a substrate for
P. fluorescens SBW25 WS-GFP biofilm formation.
The WS mutant of P. fluorescens SBW25 produces a
cellulose-matrix-based air-liquid (A-L) interface bio-
film which develops from bacteria attached to the glass
walls of static liquid microcosms at the meniscus, and
ultimately extends out to cover the entire A-L interface
[21, 27]. CLSM of PET films recovered from static
microcosms showed clear evidence of P. fluorescens
SBW25 WS-GFP biofilm development (Fig. 2, see in-
set), indicating that growth was not inhibited by the
PET film surface. The growth of attached bacteria is
generally influenced more by the physicochemical sur-
face properties of the substrate than the initial attach-
ment levels [26, 28].
The fact that bacteria such as P. fluorescens
SBW25 and P. putida KT2440 can attach to, colonise
202
MOSHYNETS O. ET AL.
the surface and detach from PET films under artificial
conditions suggests that this novel substrate may prove
to be a successful means of sampling microbial com-
munities from soil and rhizosphere environments.
Preliminary CV-staining experiments suggested
that soil bacteria colonise PET films with a ~4 × increa-
se in CV staining after 3 days incubation in soil micro-
cosms. However, this observation may be confounded
by CV binding to other organic components adhering
to the film in addition to at tached bacterial cells.
Colonisation of PET films in soil by resident bacteria
was subsequently demonstrated by recovering Pseudo-
monas spp. from soil microcosms by plating onto KB
agar. These contained ~7⋅106 CFU (colony forming
units) per gram of soil, and between 1– 2⋅ 106 CFU co-
uld be recovered using PET films of 1 cm2. Pseudomo-
nas cold also be recovered from 1–2 mm wide PET film
strips by embedding in KB agar. Colonization of PET
films by soil microorganisms was also examined by di-
rect visualisation of PET films by fluorescent micro-
scopy and CLSM; four examples of CLSM images are
shown in Fig. 3 (see inset). Clumps of material, pos-
sibly consisting of fungal hyphae and soil detritus with
associated bacteria, could be observed after staining
with EtBr and AO. Bacterial colonization of the PET
film, hyphae and detritus surfaces could also be ob-
served using a combination of natural fluorescence,
AO and Hoechst. The appearance of chains of cocci
and arrays of bacillus suggest replication had occurred
during the incubation of the films in the soil micro-
cosms. The visualisation of both cocci and bacillus
forms within aggregations also indicates that some spa-
tial associations were retained at the scale of microbial
habitats.
In order to determine whether the contacts between
soil aggregates and the surface of PET film could be vi-
sualised in situ, we used µCT to investigate soil contai-
ning films. Typically, X-ray energies, filters and image
capture settings are balanced to obtain the best diffe-
rentiation of X-ray dense (e. g. mineral, sand and stone
particles), intermediate (soil aggregates and water) and
light (air-filled pores) materials. For this work, we used
preliminary scans of a microcosm with a protruding
piece of PET film to decide on an X-ray energy of
90 kV and the use of a 0.25 mm aluminium filter. We
have shown two sagittal views of a soil microcosm in
Fig. 4 in which films are clearly identified (though such
images are normally inspected as 3D structures). From
the modified image in Fig. 4, c, it is clear that the surfa-
ce of the film is intimately connected to the pore space
of the surrounding soil, and gives an indication of how
this technique could be extended to map the contact
points between the film surface and soil aggregates.
Conclusions. The introduction of the buried slide
technique by Cholodny in 1930 heralded the investi-
gation of soil microbiology by microscopy. Eight de-
cades later, there is a growing need to examine soils at
the microbial scale, using techniques which retain spa-
tial information that can be used to assess microbial in-
teractions in a highly heterogeneous physical environ-
ment. In this report, we demonstrate that PET films are
a successful development of Cholodony’s microscope
slides, allowing the sampling of natural microbial com-
munities from soils. Fixing films for microscopy and
203
UP-DATING THE CHOLODNY METHOD USING PET FILMS TO SAMPLE MICROBIAL COMMUNITIES IN SOIL
a
b
c
Fig 4. Buried PET films can be imaged in situ by µCT. Non-
destructive imaging of soil microcosms by µCT can locate the posi-
tion of buried PET films with respect to local soil aggregate and po-
re structures. Shown are sagittal views of a soil microcosm contai-
ning two small (a) and one long (b) pieces of film. The grey-scale
values of the image provided in b have been arbitrarily adjusted to
show how the pore spaces (black) are in intimate connection with
the buried film in (c). The scale bar indicates 1 cm
preliminary PCR experiments suggest that these may
be compatible with FISH (fluorescent in situ hybrdiza-
tion) and other molecular techniques which may allow
taxonomic identification and gene-expression analysis
of soil communities, while parallel tests have already
demonstrated that PET films can be used to recover
bacteria from the rapeseed rhizosphere [29]. In the
future, µCT-derived maps of soil aggregate contact po-
ints and pore spaces across the surface of PET films
might be combined with post-recovery microscopical
analyses to enable a better understanding of soil micro-
habitats and the spatially-explicit nature of microbial
interactions in this complex environment.
Acknowledgments. Some of the work presented
here was undertaken by OE during a visit to the SIM-
BIOS Centre. We thank W. Otten for his help with the
µCT. AS is funded by the University of Abertay Dun-
dee and is a member of the Scottish Alliance for Geo-
science Environment and Society (SAGES). AK is a
SAGES-associated PhD student. The University of
Abertay Dundee is a charity registered in Scotland, No:
SC016040.
О. В. Мо ши нець, А. Коза, П. Дело Стер па йо, В. А. Кор дюм,
Е. Д. Спайрс
Мо дифікація ме то ду Хо лод но го із за сто су ван ням ПЕТ плівок для
відбо ру зразків мікроб них це нозів у ґрунті
Ре зю ме
Мета цієї ро бо ти по ля га ла у дослідженні мож ли вості ви ко ри-
стан ня плівок, ви го тов ле них із ПЕТ (поліети лен тет раф та лат),
як мо дифікації ме то ду ске лець об рос тан ня Хо лод но го для мікро-
скопічно го і мо ле ку ляр но-ге не тич но го аналізу ґрун то вих спіль-
нот із збе ре жен ням їхньої про сто ро вої архітек ту ри на мікро-
рівні. Таке збе ре жен ня де та лей про сто ро во го роз та шу ван ня
об’єктів доз во ли ло б глиб ше вив чи ти їх у подібних склад них се ре -
до ви щах про жи ван ня. Ме то ди. Кла сичні мікробіологічні ме то -
ди; аналіз при кріплен ня; вимірю ван ня по вер хне во го на тяг нен ня;
мо ле ку ляр но-ге не тичні ме то ди: екстракція ДНК, ПЛР; кон фо -
каль на ла зер на ска ну ю ча мікрос копія (КЛСМ); мікро фо кус на рен -
тгенівська ком п’ю тер на то мог рафія (мікроКТ). Ре зуль та ти.
По-пер ше, ви ко рис то ву ю чи мо дельні ґрун тові і ри зос ферні бак -
терії Pseudomonas fluorescens SBW25 і P. putida KT2440, ми по ка -
за ли, що бак терії здатні до при кріплен ня і відкріплен ня від ПЕТ
плівок, а пре куль ти вація за умов відфільтро ва ної ґрун то вої сус -
пензії по кра щує рівень при кріплен ня. Бак терії, які при кріпи ли ся
до плівок, зберіга ють свою життєздатність і спро можні до
форму ван ня по вноцінної біоплівки. ПЕТ плівки, за ну рені в ґрунт,
ко лонізу ють ся мікро ор ганізма ми, що спос терігали як за до по мо -
гою КЛСМ, так і ме то дом куль ти ву ван ня ПЕТ плівок, ви да ле них
з ґрун ту, на ага ри зо ва но му по жив но му се ре до вищі. По-дру ге, ми
про де мо нстру ва ли, що мікроКТ мож на ви ко рис то ву ва ти для не -
руйнівно го спос те ре жен ня сайтів зв’я зу ван ня ґрун то вих аг ре -
гатів і ґрун то вих пор з по вер хнею плівки, що пе ре бу ває в ґрунті.
Вис нов ки. Зас то су ван ня ПЕТ плівок ви я ви ло ся вда лою мо дифіка-
цією ме то ду ске лець об рос тан ня Хо лод но го та може бути ко -
рис ним для відбо ру ґрун то вих мікроб них спільнот, досліджен ня
бак терійно го при кріплен ня, рос ту і роз вит ку як біоплівок, так і
спільно ти. Ви ко рис тан ня цих плівок при аналізі ґрунтів за до по -
мо гою мікроКТ доз во лить кра ще виз на чи ти ґрун тові мікро е ко-
ніші і при ро ду архітек ту ри мікроб них взаємодій за та ких склад -
них еко логічних умов.
Клю чові сло ва: Pseudomonas, ґрунт, скель ця об рос тан ня, ПЕТ
плівки.
Е. В. Мо ши нец, А. Коза, П. Дел ло Стер па йо, В. А. Кор дюм,
Э. Д. Спайрс
Мо ди фи ка ция ме то да Хо лод но го с при ме не ни ем ПЭТ плeнок для
от бо ра об раз цов мик роб ных це но зов в по чве.
Ре зю ме
Цель дан ной ра бо ты со сто я ла в ис сле до ва нии воз мож нос ти ис -
поль зо ва ния плeнок, из го тов лен ных из ПЭТ (по ли э ти лен те реф -
та лат), как со вре мен ной мо ди фи ка ции ме то да стeкол обра-
ста ний Хо лод но го для мик рос ко пи чес ко го и мо ле ку ляр но-ге не ти -
чес ко го ана ли за по чвен ных со об ществ с со хра не ни ем их про ст-
ра нствен ной ар хи тек ту ры на мик ро у ров не. Та кая со хран ность
де та лей про стра нствен но го рас по ло же ния об ъ ек тов по зво ли ла
бы глуб же из учить их в по до бных слож ных усло ви ях об и та ния.
Ме то ды. Клас си чес кие мик ро би о ло ги чес кие ме то ды; ана лиз при -
креп ле ния; из ме ре ние по вер хнос тно го на тя же ния; мо ле ку ляр но-
ге не ти чес кие ме то ды: экс трак ция ДНК, ПЦР; кон фо каль ная ла -
зер ная ска ни ру ю щая мик рос ко пия (КЛСМ); мик ро фо кус ная рен -
тге нов ская ком пью тер ная то мог ра фия (мик роКТ). Ре зуль та-
ты. Во-пер вых, ис поль зуя мо дель ные по чвен ные и ри зос фер ные
бак те рии Pseudomonas fluorescens SBW25 и P. putida KT2440, мы
по ка за ли, что бак те рии спо соб ны к при креп ле нию и от креп ле нию
от ПЭТ плeнок, а пре куль ти ви ро ва ние в усло ви ях от фи льтро ван -
ной по чвен ной сус пен зии улуч ша ет уро вень при креп ле ния. Бак те -
рии, при креп- лeнные к плeнкам, со хра ня ют жиз нес по соб ность и
мо гут фор ми ро вать по лно цен ную би оплeнку. ПЕТ плeнки, по гру-
жeнные в по чву, ко ло ни зи ру ют ся мик ро ор га низ ма ми, что на блю -
да ли как с при ме не ни ем КЛСМ, так и ме то дом куль ти ви ро ва ния
из влечe нных из по чвы плeнок на ага ри зо ван ной пи та тель ной сре -
де. Во-вто рых, мы про де мо нстри ро ва ли, что мик роКТ мож но
ис поль зо ватьа для не дес трук тив но го на блю де ния за сай та ми
свя зы ва ния по чвен ных аг ре га тов и по чвен ных пор с по вер хно-
стью ПЕТ плeнки, на хо дя щей ся в по чве. Вы во ды. При ме не ние
ПЕТ плeнок ока за лось удач ной мо ди фи ка ци ей ме то да стeкол об-
рас та ний Хо лод но го и мо жет стать по лез ным для от бо ра по -
чвен ных мик роб ных со об ществ, из уче ния бак те ри аль но го при -
креп ле ния, рос та, раз ви тия как би о плeнок, так и со об щес тва.
Исполь зо ва ние этих плeнок при ана ли зе по чвы с по мощью мик -
роКТ по зво лит луч ше опре де лять по чвен ные мик ро э ко ни ши и
при ро ду ар хи тек ту ры мик роб ных вза и мо де йствий в та ких слож -
ных эко ло ги чес ких усло ви ях.
Клю че вые сло ва: Pseudomonas, по чва, стeкла об рас та ния,
ПЭТ плeнки.
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UDC 579.262 + 57.083.1
Received 26.11.10
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UP-DATING THE CHOLODNY METHOD USING PET FILMS TO SAMPLE MICROBIAL COMMUNITIES IN SOIL
Figures to article by O. V. Moshynets et al.
ISSN 0233-7657. Biopolymers and Cell. 2011. Vol. 27. N 3
Fig. 2. PET films supported the development of WS-GFP biofilms. Film
pieces positioned to pierce the A-L interface of static liquid microcosms
were colonised by WS-GFP and developed biofilms. Shown here is a mic-
ro-colony with associated cellulose imaged by CLSM where active WS-
GFP cells are green, PI-stained dead cells are red, and Calcofluor-stained
cellulose is blue. The scale bar indicates 100 µm
d
a b
c
Fig. 3. Soil microbial communities on
PET films are readily imaged by fluo-
rescent microscopy. Films recovered
from soil microcosms can be imaged by
CLSM after staining with (a) EtBr and
(b) AO to reveal microbial colonisation
of the PET film surface and adhering soil
particles. More complex images are
shown in (c) and (d) using a combination
of natural fluorescence, AO and Hoechst.
The scale bars indicate 10 µm
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