PLANT MICRO-ORGANISMS
Microscopy Methods for MicroOrganisms on Plant Surfaces
Mike Edwards, Campden & Chorleywood Food Research Association, Chipping Campden, UK
INTRODUCTION
BIOGRAPHY
Mike Edwards has
been Head of the
Microscopy Section
at CCFRA for the past
fourteen
years,
working on Foreign
Body Identification,
Food Structure and
Texture, Microscopy
of Food Packaging, Food Hygiene and
Microbes on Food Surfaces. This followed a
period of research at Aberdeen University
looking at Plant Surfaces. Mike holds a first
degree in Plant Sciences and a PhD in Plant
Pathology.
ABSTRACT
Micro-organisms on plant surfaces have
been intensively studied for many years
because of their significance in plant pathology. A wide range of microscopical techniques is now available for the study of
microbes on plant surfaces. The more established techniques such as epifluorescence
microscopy and scanning electron
microscopy are now being joined by a range
of new tools to enable microscopists to study
the metabolic activity of micro-organisms on
plant surfaces, and to track individual
species amongst the general microbial population. These include fluorescent antibody
techniques, confocal microscopy and a
range of marker technologies.
KEYWORDS
Plants,
microbiology,
fluorescence
microscopy, fluorescence antibody technique, confocal microscopy, scanning electron microscopy, marker technologies
ACKNOWLEDGEMENTS
The author would like to acknowledge the
help of his colleagues Liz Biddlecombe, Liz
Sheasby and Ian Seymour in the preparation
of this article.
A U T H O R D E TA I L S
Dr Mike Edwards, Microscopy Section, Dept.
of Chemistry & Biochemistry, Campden &
Chorleywood Food Research Association,
Chipping Campden, Glos. GL55 6LD, UK
Tel: +44(0)1386 842017 (UK).
Fax: +44(0)1386 842100 (UK).
Email: [email protected]
Micro-organisms on plant surfaces have been
intensively studied for many years because of
their significance in plant pathology, and
much of the work is summarised in a series of
symposium proceedings. In 1981 Baker [1]
reviewed the methods available for observation and enumeration of microbes on plant
surfaces by light microscopy, and emphasised
the advantages of fluorescence microscopy for
use at the high magnifications required in the
study of bacteria. Later, Andrews [2] reviewed
methods of tracking epiphytic micro-organisms, but most of the methods available then
were, and still are, applicable to micro-organisms washed off plant surfaces and subsequently cultured. Relatively few methods are
available to study micro-organisms in situ. A
number of marker systems are also available
to label particular strains, some of which are
appropriate for direct use on plant surfaces.
Figure 1.
Bacteria on a leaf surface, stained with acridine orange and observed by
epifluorescence microscopy (I3 filter set). Bacteria are concentrated
along the anticlinal walls between the epidermal cells. Image dimensions
625 x 427 µm. Taken on a Leica DM LB microscope.
FLUORESCENCE MICROSCOPY
Epifluorescence microscopy has been used for
many years for the study of micro-organisms on
plant surfaces, and is ideal for studies of the general distribution of populations of bacteria and
yeasts on natural surfaces of all kinds. Sample
preparation is very simple: a drop of a suitable fluorescent stain is added to the surface and rinsed
off after a minute or two. The sample is then
examined directly using a fluorescence microscope fitted with filters appropriate to the stain
being used. Bacteria and yeasts appear as brightly
coloured spots against a dark background (Figs 1
and 2). Magnifications of 400 to 1,000 times may
be used. Microbial cells can be distinguished on
the basis of shape and size. Some stains can be
used to differentiate between live and dead cells
(e.g. fluorescein diacetate, only taken up and
cleaved to produce the fluorescent fluorescein
moiety by live cells). However, Babuik and Paul
[3], working with fluorescein isothiocyanate
(FITC), which works in a similar way, concluded
that the amount of stain taken up and
metabolised by bacteria was too small in other
than very young cells for the method to be an
effective staining technique. Propidium iodide
stains dead cells red [4]. SYTOX nucleic acid stains
can also be used to localise dead cells or in cell viability studies. The epifluorescent detection of INT
formazan in respiring cells has been used to distinguish actively-metabolising cells in a bacterial
population. However, the more recent development of CTC formazan appears to offer some
advantages over INT formazan. Autoradiography has been used in conjunction with epifluorescent microscopy to detect bacterial cells taking
up 14C-labelled sugars or amino acids [5]. It was
MICROSCOPY
AND
Figure 2:
Bacteria on a leaf surface, as in Fig 1. Note some autofluorescence from
vascular bundles within the leaf. Image dimensions 625 x 427 µm. Taken
on a Lecia DM LB microscope .
earlier thought that the stain acridine orange was
able to differentiate between live and dead cells
by fluorescing different colours. However, this
has been shown to be false [6]. When used in a
buffer solution at pH 2.1, acridine orange can be
used to differentiate between nucleic acids, RNA
fluorescing bright yellow and DNA fluorescing
flame red [7]. Fluorescent brighteners such as
Calcofluor White M2R are also useful fluorescent
stains for such work: see the review by Paton [8].
F L U O R E S C E N T A N T I B O DY
TECHNIQUE
The advantages of staining with fluorochromes
can be combined with the specificity of serological techniques to provide a powerful tool of
staining with fluorescent antibodies [1]. The fluorescent antibody technique enables specific
organisms to be identified and seen in situ on the
plant surface. Antigens against the micro-organism of interest are raised in suitable animals such
as mice, and the fluorochrome (usually fluorescein isothiocyanate or FITC) is conjugated to an
A N A LY S I S • S E P T E M B E R 2 0 0 1
9
a
b
Figure 3.
(a) and (b) SEM views of bacteria around a stoma on the lower surface of a parsley leaf. Taken on a Leica Cambridge 360.
anti-mouse antibody, normally available combled by computer into a single three-dimenmercially. The plant surface is first treated with
sional image, so overcoming the major disadthe specific antibody against the micro-organism
vantage of the conventional fluorescence
being studied, so that antibodies bind with the
microscope, that of a limited depth of field at
microbial cells. After rinsing, the surface is then
high magnifications. An excellent introductreated with the labelled antibodies, resulting in
tion to CLSM is given by Sheppard and Shotton
the first antibody, bound to the microbial cells,
[11]. For the examination of micro-organisms
being labelled with the second antibody carrying
on plant surfaces, the method is claimed to
the stain, which can be visualised using a fluohave the advantage over SEM that it avoids
rescence microscope. The major disadvantage of
the distortion of morphology and fixation artithis technique is the difficulty of raising antibodfacts which may be inherent in that technique
ies to the micro-organisms being studied, and
[4].
relatively few immunofluorescent studies have
Kim et al.[12] studied attachment of Salmobeen carried out on plant surface micro-organnella to poultry skin using CSLM, staining with
isms as a result. However, antibodies to a numPyronin-Y to image both bacteria and skin.
ber of bacteria are now available commercially
They showed that Salmonella cells remaining
from Kirkegaard & Perry Laboratories, Gaithersafter rinsing were mostly located in crevices
burg, MD, USA. Seo and Frank [4] used a direct
and feather follicles. Salmonella cells could be
antibody staining technique with FITC-labelled
observed in any depth of the feather follicles,
antibodies to E. coli O157:H7. Huang et al [9]
indicating that even unattached, floating Saldeveloped a system to enumerate Salmonella
monella cells in entrapped water cannot be
cells on sample slides of poultry carcass wash
easily washed out. This demonstrates two parwater after immunolabelling with fluorescein.
ticular advantages of CSLM over SEM: (1) the
An image analysis method was developed to
ability to look within an enclosed structure
recognise Salmonella cells on the basis of area,
rather than just at the surface; and (2) the abilaspect ratio, diameter, major and minor axes,
ity to examine fresh, hydrated material with
maximum and minimum radii, perimeter, radius
cells floating in water.
ratio, length, width and intensity.
Seo and Frank [4] used CSLM to observe the
An impressive recent application of the fluolocation of E. coli O157:H7 on and within letrescent antibody technique was by Itoh et al.
tuce leaves, using FITC-labelled antibody to
[10], who examined the distribution of E. coli
visualise the attached bacteria. They demonO157:H7 in radish sprouts. Interestingly, they
strated the presence of bacteria on the leaf
also used an immunolabelling technique to
surfaces, trichomes, stomata and cut edges.
detect the same bacterium using scanning elecThree-dimensional volume reconstruction of
tron microscopy (see section below).
interior portions of leaves showed that E. coli
O157:H7 was entrapped 20 to 100 µm below
CONFOCAL MICROSCOPY
the surface in stomata and cut edges. Agar
Confocal laser scanning microscopy (CLSM)
plate culturing and microscopic observation
first became available commercially about
indicated that E. coli O157:H7 preferentially
eleven years ago and has many potential
attached to cut edges, as opposed to the intact
applications in food research. There are many
leaf surface. Dual staining with FITC-labelled
applications in research on food structure. It
antibody and propidium iodide was used to
has been used in recent years to study microdetermine cell viability on artificially contamiorganisms on plant surfaces. Samples are prenated leaves after treatment with chlorine
pared in exactly the same way as for convensolution.
tional fluorescence microscopy. The microscope scans the sample with a focused laser
SCANNING ELECTRON MICROSCOPY
SEM has also been used for the study of microbeam at pre-determined depths through the
organisms on plant surfaces ever since its
sample, and so a series of ‘optical sections’ can
invention, and has the great advantage of
be accumulated. These can later be re-assem-
10
MICROSCOPY
AND
A N A LY S I S • S E P T E M B E R 2 0 0 1
producing an immediate three-dimensional
image without the need for further computer
processing required with confocal microscopy.
The tendency, however, is to use it at a higher
magnification than one would use a light
microscope, with the result that a much
smaller area of the plant surface is examined.
Not only does this mean that fewer microorganisms are studied, it dramatically
increases the detection limit for these microorganisms [1]; many more bacteria must be
present before they are reliably detected. A
further disadvantage is that the SEM merely
images the three-dimensional topography of
the surface: it may be impossible in some cases
to differentiate bacteria from small pieces of
debris, and it most cases it will be impossible to
identify individual bacteria to species or even
to genus level without the aid of additional
methods of identification such as immunolabelling techniques.
SEM has shown that the common sites for
microbial aggregation are the veins, the trichomes, the stomata (Fig 3) and the cell wall
junctions.
An interesting application of the combination of immunolabelling techniques and SEM
was the work of Itoh et al. [10], who used SEM
to study E. coli O157:H7 on the surfaces of
radish sprouts. The bacteria were chemically
fixed, labelled with primary antibody raised in
rabbits, and then treated with a secondary
gold-labelled anti-rabbit antibody. These
workers studied both the external plant surfaces and also the internal tissues using this
method, with a good deal of success (Fig 4).
SEM gives useful information on the threedimensional structure of the sample, particularly useful in interpreting the relationship
between micro-organisms and their substrates. An illustration of this is a study of the
water-repellent, ‘self-cleaning’ nature of some
plant surfaces carried out by Neinhuis and
Barthlott [13]. These authors used unfixed airdried material to avoid the possibility of introducing changes in the surface waxes of plant
surfaces during specimen preparation. They
found that leaves that are permanently waterrepellent can be differentiated by distinctively
convex to papillose epidermal cells and a very
PLANT MICRO-ORGANISMS
dense layer of epicuticular waxes. Rough,
waxy leaves are not only water-repellent but
also anti-adhesive with respect to particulate
contamination [14].
Figure 4.
Secondary electron (a) and
backscatter (b) SEM images of Salmonella cells on a Nuclepore membrane. Bacteria were fixed with glutaraldehyde and treated successively with a commercial anti-Salmonella antibody, a secondary antibody carrying a gold label, followed
by silver enhancement. The silver is
clearly visible in the backscattered
picture, but appears to surround
the cells, suggesting that the primary antibody may react with an
extracellular bacterial metabolite
rather than the microbial cells
themselves. Taken on a Leica Cambridge 360.
MARKER TECHNOLOGY
Marker technology involves the introduction
of one or more specific genes (either chromosomal or plasmid-borne) into the target bacterium [15]. Current marker technology often
links specific metabolic activity with the production of a colorimetric or light-induced phenotype, or with specific degradative capability,
or resistance to, for example, heavy metals.
The use of a specific marker gene is determined by the uniqueness of the phenotype,
both in the bacterial strain and within the
environment to be examined. Marker technology has greatly improved the understanding
of microbial interactions in the plant-soil system by providing methods which allow microbial populations to be studied without perturbing the environment, and there is potential to extend some of these methods for use
on plant surfaces. The success and future use
of a particular marker system will be determined by: (a) the ease with which strains can
be successfully marked; (b) the degree of
expression of a particular marker phenotype;
(c) the sensitivity of available detection methods; and (d) performance under specific environmental conditions [16]. Several efficient
marker systems now exist, each with their own
potential and ability in solving specific problems.
Marker technologies in general have certain
specific disadvantages [16]. The relative cost of
marking each strain and the cost of specialised
equipment for tracking the marked strain
must be taken into account. Legislation and
licensing procedures for the use of genetically
modified organisms are currently the most
important factors limiting the use of marker
technology. If there is any possibility of release
of the modified organism to the environment,
the fact that gene transfer to members of the
indigenous microbial community has been
demonstrated must be taken into consideration. The fitness of the strain or the potential
metabolic burden caused by the introduction
of foreign genes has also to be considered,
although Kozdrój [17] found that the insertion
of lux genes into E. coli and B. subtilis did not
affect their competitiveness and survival in the
rhizosphere and bulk soil.
LUX GENE TECHNOLOGY
Lux gene technology is based on the transformation of bacteria with genes of the lux
operon from the marine bacteria Vibrio fischeri and V. harveyri. Transformed strains are
bioluminescent and thus provide a rapid and
very accurate tool for the study of population
dynamics, metabolic activity and spatial distribution of specific bacteria in environmental
samples [16]. A major benefit of the luxmarker system is the opportunity for in situ
visualisation of microbial colonisation of the
plant surface without the need to use extrac-
tive and culturable techniques. Lux-marked
bacteria have been demonstrated as single
cells and microcolonies in soil slurries and on
the rhizoplane respectively, and in plant tissue.
lacZY and lacZ
lacZY has been used in studies of biocontrol
[18] and lacZ in studies of interactions
between tomato roots and invading
Pseudomonas solanacearum bacteria [19].
Bacteria can be localised in plant tissue by light
microscopy after staining of the bacterial bgalactosidase activity which forms a dark blue
precipitate in the presence of the chromogenic substrate X-Gal (5-bromo-4-chloro-3indolyl -b- galactopyranoside) [20]. The principal disadvantage of this approach appears
to be that the staining system shows areas
where bacteria are present rather than
labelling individual bacteria, which may be a
problem in plant surface studies. However, this
may well still be adequate to demonstrate the
presence of target bacteria within plant tissues
of salad vegetables.
G R E E N F L U O R E S C E N T P R OT E I N
Dumas et al. [21] introduced a gene encoding
a modified version of the Aequoria victoria
green fluorescent protein (SGFP-TYG) into
Colletotrichum lindemuthianum, the fungus
causing anthracnose of bean. By fusing this
gene to one coding for the endopolygalacturonase enzyme, they were able to demonstrate
the point during infection when the
endopolygalacturonase gene was switched
on, visualising the fluorescent infection structures using fluorescence microscopy. While this
technology is clearly at an early stage, there
may be future applications for incorporating
fluorescent genes into other micro-organisms.
OT H E R M A R K E R T E C H N O L O G I E S
ß-glucuronidase has been used for studies of
microbe-plant interaction [22]. xylE has been
used to study environmental perturbation
using introduced inocula [23].
REFERENCES
1. Baker J.H. Direct observation and enumeration of
microbes on plant surfaces by light microscopy. In
"Microbial ecology of the phylloplane", (Blakeman,
J.P., ed.). London: Academic Press, pp. 3-14, 1981.
2. Andrews J.H. How to track a microbe. In "Microbiology
of the phyllosphere", (Fokkema, N.J. and van den Heuvel,
J., eds). Cambridge University Press, pp. 14-34, 1986.
3. Babuik, L.A. and Paul E.A. Canadian Journal of Microbiology
16, 57-62, 1970.
4. Seo K.H. and Frank J.F. Journal of Food Protection 62, 3-9,
1999.
5. Edwards M.C. and Blakeman J.P. The use of autoradiography and other isotope techniques in phyllosphere
studies. In "Microbiology of the phyllosphere", (Fokkema
N.J. and van den Heuvel J. eds). Cambridge: Cambridge
University Press, pp. 35-49,1986
6. Jones J.G. and Simon B.M. Journal of Applied Bacteriology
39, 317-329, 1975.
7. Gahan P.B. Plant histochemistry and cytochemistry: an
introduction. London: Academic Press, 1984.
8. Paton A.M. Light microscope techniques for the
microbiological examination of plant materials. In
"Bacteria and Plants" (F.A. Skinner & M.E. Rhodes-Roberts
eds). London: Academic Press, 1981.
9. Huang J. et al. Transactions of the American Society of
Agricultural Engineers 42 ,267-273, 1999.
10. Itoh Y. et al. Applied and Environmental Microbiology 64,
1532-1535, 1998.
11. Sheppard C.J.R. and Shotton D.M. Confocal Laser
Scanning Microscopy. Microscopy Handbooks No. 38.
Oxford: Bios Scientific Publishers, 1997.
12. Kim K.Y. et al.. Letters in Applied Microbiology 22,
280-282,1996.
13. Neinhuis C. and Barthlott W. Annals of Botany 79, 667-677
1997.
14. Barthlott W. and Neinhuis C. Planta 202 , 1-8, 1997.
15. van Elsas J.D. and Waalwijk C. Agriculture, Ecosystems and
Environment 34, 97-105, 1991.
16. White D. et al. New Phytologist 133, 173-181, 1996.
17. Kozdrój J. World Journal of Microbiology and Biotechnology
12, 261-265, 1996.
18. Ryder M. . FEMS Microbiology Ecology 15, 139-146, 1994.
19. Vasse J. et al. Plant Journal 4 555-566, 1993.
20. Boivin C. et al. Plant Cell 2, 1157-1170, 1990.
21. Dumas B. et al. Applied and Environmental Microbiology 65,
1769-1771,1999.
22. Wilson K.L. Soil Biology and Biochemistry 27, 501-514, 1995.
23. De Leij F. et al. FEMS Microbiology Ecology 13, 249-258, 1994.
DIDYOUENJOYTHISARTICLE
? ODYOUHAVE T
AOPIC
YOUCOULDWRITE
ABOUT
? IRCLE
C READER
ENQUIRY
NO. 336
ORVISIT OUR
WEBSITE
: www.microscopy-analy
MICROSCOPY
AND
A N A LY S I S • S E P T E M B E R 2 0 0 1
11