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Scanning Electron Microscopy in the Study of Terrestrial Microbial EcologyAuthor(s): R. L. Todd, K. Cromack Tr., R. M. KnutsonSource: Bulletins from the Ecological Research Committee, No. 17, Modern Methods in theStudy of Microbial Ecology (1973), pp. 109-118Published by: Oikos Editorial Office Stable URL: http://wwwjstor.org/stable/20111548Accessed: 01/09/2011 17:30

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Bull. Ecol. Res. Comm. (Stockholm) 17: 109-118 (1973).

Scanning Electron Microscopy in the Study ofTerrestrial Microbial Ecology*

R. L. Todd, K. Cromack, Jr. & R. M. Knutson

IntroductionThe increasing importance being attributed to terrestrial microflora

in decomposition and nutrient cycling demands improved assay systems toprovide an accurate assessment of their ecological role. Numerous meth-ods have been employed or proposed for enumerating microbial popula-tions and for estimating their production and turnover. Parkinson (1970),in a review of existing techniques, stressed the need to perform thesequantifications of bacterial and fungal biomass within the microhabitatsthey occupy. Inability to examine these microhabitats is the major limi-tation of existing techniques. In this paper we present a technique foruse in terrestrial ecological research that enables the microbiologist toexamine microorganisms in situ.

Operating PrincipleThe interception of a high energy electron beam by a solid specimen

results in a number of physical phenomena. Their assessment can revealinformation characteristic of the specimen. Four of these phenomena areillustrated in Fig. 1: cathodoluminescence, X-radiation and the emissionof backscattered and secondary electrons. Scanning electron microscopyand microprobe analysis are based on the latter three phenomena.

In scanning electron microscopy a focused electron beam scans asmall area of a specimen in a "raster" fashion. The secondary or back-scattered electrons emitted from each individual point are then displayedas a spot on the face of a cathode ray tube which is scanned in synchro-ny with the specimen. The brightness of each spot formed on the cathoderay tube is proportional to the quantity of electrons from a correspondingspot on the specimen. The resultant image can be recorded on photo-graphic film, videotape or linked directly to a suitable computer processunit. Application of this technique allows for the microscopic examination,at 100-200 angstrom resolution, of a specimen which can be several mmin thickness. Kimoto & Russ (1969) provide a detailed discussion of thescanning electron microscope.

Scanning electron microscopy has been used for observation of axenicbacterial cultures (Klainer & Betsch, 1970; Todd & Kerr, 1972). Obser-vations have been recorded of microbes in soil (Gray, 1967; Hagen et al. ,1968) and in estuarine environments (Todd, Humphreys & Odum, 1973).

Analysis of the X-ray radiation from the specimen is made by elec-tron microprobe analyzer. A focused electron beam of approximately onemicron in diameter is located on the specimen. The emitted X-ray spec-trum, which is characteristic for the elemental content of the specimen,

* Contribution No. 47 from the Eastern Deciduous Forest Biome, US-IBP.

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Fig. 1 Schematic diagram of resultant emission when a specimen isbombarded with an incident electron beam. (Courtesy of D.J.Evins, Material Analysis Company, Palo Alto, California)

is analyzed with a crystal spectrometer. Quantitative and qualitative in-formation is obtained by comparing both wave length and intensity withstandards of known composition. The principle of X-ray microprobe anal-ysis is discussed in detail by Birks (1963). This analytical procedure hasfound wide application in geological and metallurgical engineering re-search. Its application to analysis of biological systems has only recentlybecome apparent (Sawhney & Zelitch, 1969; Humble & Raschke, 1971;Kaufman et al., 1972). To our knowledge the preliminary observationssummarized here comprise the first reported application to microbialchemical analysis.

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Observations and ApplicationsThe application of scanning electron microscopy to terrestrial micro-

bial decomposition and nutrient cycling proved very productive. Repre-sentative electron micrographs of microfauna and microflora observed insamples of soil and forest litter taken from under a variety of vegetationcover are shown in the following series of figures.

Figs. 2-19 were made from a secondary electron image produced bya Cambridge Steroscan Electron Microscope (Mark IIA, Cambridge In-struments Company, Ltd., London, England) at an accelerating voltageof 10 kV. Prior to examination specimens were coated (approximatethickness of 200-400 A) with a gold-palladium alloy by vacuum evapora-tion. The images were recorded with Type 55 P/N Polaroid sheet film(Polaroid Corporation, Cambridge, Massachusetts).

Figs. 20-23 were made from images produced by X-ray and second-ary electron emission following electron bombardment of the specimen atan accelerating voltage of 10 kV in an electron microprobe analyzer(Model 400-S, Materials Analysis Company, Palo Alto, California). Spec-imens were photographed with Type 55 P/N Polaroid sheet film. Metalcoated (gold-palladium alloy) specimens were affixed by double adhesivecellulose tape to the surface of 2 1/2 inch diameter aluminium "plugs"of which four could be accommodated simultaneously in the standard spec-imen holder of the analyzer. Each of three spectrometers can be stand-ardized for a different element, thus allowing for three different yet si-multaneous analyses of the identical site on the specimen. In the preli-minary observations reported here a KAP crystal and flow detector(10 % methane — 90 % argon, wave length of 1495 was used for theanalysis of magnesium, a PET crystal and flow detector (1548 4) forcalcium analysis and a PET crystal with xenon filled proportioned count-er (1725 /2) for potassium analysis. Images resulting from the output ofan individual detector are built up by scanning the surface of a long per-sistence phosphor cathode ray tube. The display can be recorded photo-graphically from a storage display system.

Conclusions and RecommendationsScanning electron microscopy can be used to observe microbes in

terrestrial microhabitats. This technique can be refined to estimate mi-crobial biomass by application of existing techniques for the quantifica-tion of filamentous microorganisms using light microscopy (Olson, 1950;Melhuish & Lang, 1968). The technique of microprobe analysis makespossible for the first time in situ assessment of microbial chemical com-position. This technique has been used to estimate calcium, magnesiumand potassium content of microbial biomass and to compare these contentswith those of the substrate used to produce the cells. Elemental nutrientpools can be estimated by combining scanning electron microscopy andmicroprobe analysis by selecting subsamples from specimens observedinitially using scanning electron microscopy.

AcknowledgementsWe thank W .J . Humphreys , Director, Electron Microscopy Labora-

tory, University of Georgia for his cooperation with the scanning electronmicroscopy. We are indebted to J. Stormer, Department of Geology,University of Georgia, for his assistance with the electron microprobeanalysis.

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Research supported by the Eastern Deciduous Forest Biome, U.S.International Biological Program, funded by the National Science Founda-tion under Interagency Agreement AG-199, 40-193-69, with the AtomicEnergy Commission — Oak Ridge National Laboratory.

Figs. 2 & 3 Scanning electron micrographs of oribatid mites. 10 Ammarker indicated in lower left. Fig. 2 illustrates an Ere-maeidae isolated from mixed deciduous forest litter, whileFig. 3, a Galumnidae, was isolated from pine litter.(Specimens provided by C. Gist and D.A. Crossley, Jr.,University of Georgia.)

Figs. 4 & 5 Scanning electron micrographs of mycorrhizal colonization ofa white pine root. 10 m marker indicated in lower left.Fig. 4 illustrates the root segment with the attached charac-teristic fungal "club-like growth". Fig. 6 is a high magnifi-cation of the filamentous structure.

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Figs. 6-11 Scanning electron micrographs of decaying pine litter froma white pine forest floor. 10 gm marker indicated at lowerleft. Figs. 6-9 are an increasing magnification series ofan identical specimen taken from the "H" litter layer.Figs. 10 & 11 are micrographs from the "L" litter layerof the same pine plantation.

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Figs. 12-15 Scanning electron micrographs of decaying deciduous litterfrom a mixed hardwood forest floor. 10 A m marker indi-cated at lower left. Figs. 12 & 13 illustrate the topographyof a leaf surface taken from the "L" layer. Fig. 14 is amicrograph of a ground (60 mesh) sample. Note the inter-nal concentration of the fungal hyphae. Micrograph 15 isrepresentative of the "H" layer. The appearance of "pro-tuberances" (Gray, 1967) on some mycelia indicates thepresence of at least two distinct morphological forms.

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Figs. 16-17 Scanning electron micrographs of the soil horizon under-lying a mixed deciduous litter. 10 gm marker indicated inlower left. Fig. 16 represents a specimen for the A l lay-er, while Fig. 17 is a representative observation of theA2 horizon. Note the abundance of hyphae in the A l ascompared to the A2 layer.

Figs. 18-19 Scanning electron micrograph of a decaying smooth cordgrass, Spartina alterniflora, isolated from an estuarineenvironment. 10 Am marker is indicated in the lower left.Fig. 18 illustrates fungal colonization of the "air passages"and Fig. 19 is of bacterial cells associated with the decay-ing grass. (Specimens provided by M. May, BrunswickCollege, Brunswick, Georgia.)

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Figs. 20-23 Electron microprobe analyzer pictures of a rhizomorphsample isolated from decaying deciduous forest litter.10 urn marker is indicated in the lower left of each photo-graph. Fig. 20 is of an image made by secondary elec-trons, (r) indicates the rhizomorph and (b) the background.Fig. 21 is of the calcium X-ray image, Fig. 22, of thepotassium X-ray image and Fig. 23 of the magnesium X-ray image.

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References

Birks L.S. 1963. Electron probe microanalysis. Interscience Publishers, New York.Gray T.R.G. 1967. Stereoscan electron microscopy of soil microorganisms. Science 155: 1668-

1670.Hagen C.A., Jawrylewicz E.J., Anderson B.T., Tolkacz V.K. & Cephus M.L. 1968. Use of the

scanning electron microscope for viewing bacteria in soil. Appl. Microbiol. 16: 932-934.Humble G.D. & Raschke K. 1971. Stomata! opening quantitatively related to potassium

transport — evidence from electron probe analysis. Plant Physiol. 48: 447-453.Kaufman P.B., Soni S.L., La Croix I.D., Rosen J.J. & Bigelow W.C. 1972. Electron-probe micro-

analysis of silicon in the epidermis of rice (Oryza sativa L.) intemodes. Planta 104: 10-17.Kimoto S. & Russ J.C. 1969. The characteristics and applications of the scanning electron micro-

scope. Amer. Sci. 59: 112-133.Klainer A.S. & Betsch C.J. 1970. Scanning-beam electron microscopy of selected microorganisms.

J. Infec. Dis. 121: 339-343.Melhuish F.M. & Lang A.R.G. 1968. Quantitative studies of roots in soil. I. Length and diameters

of cotton roots in a clay-loam soil by analysis of surface ground blocks of resin-impregnatedsoil. Soil Sci. 106: 16-22.,

Olson, F.C.W. 1950. Quantitative estimates of filamentous algae. Trans. Amer. Microsc. Soc. 59:272-279.

Parkinson D. 1970. Methods for the quantitative study of heterotrophic soil microorganisms. In:Methods of study in soil ecology, ed. J. Phillipson, UNESCO, Paris.

Sawhney B.L. & Zelitch I. 1969. Direct determination of potassium ion accumulation in guardcells in relation to stomatal opening in light. Plant Physiol. 44: 1350-1354.

Todd R.L., Humphreys WJ. & Odum E.P. 1973. The application of scanning electron microscopy toestuarine microbial research. In: Methods of study in estuarine microbial ecology, eds. H.L.Stevenson & R. Colwell, University of South Carolina Press, (in press).

Todd R.L. & Kerr T.J. 1972. Scanning electron microscopy of microbial cells on membrane filters.Appl. Microbiol. 23: 1160-1162.

Discussion

E.L. Schmidt: Your SEM photomicrograph showed the mycorrhizia in a rather collapsed state,whereas the bacteria and fungi generally appeared plump and unwrinkled — what in your experienceis the best fixation procedure for soil microorganisms and SEM examination?R.L. Todd: All the electron micrographs in this presentation were made from dried metal-coatedspecimens. We have observed that a gradient fixation of glutaraldehyde-cacodylate enhancesspecimen stability. We are considering critical point drying technique as well.E.L. Schmidt: Have you been able to extend your electron probe analysis to bacterial cells in soils?R.L. Todd: Our bacterial observations with the electron microbe for chemical analysis have beenlimited to axenic cultures on membrane filters.T.R.G. Gray: Have you revised your opinion on the value of using this technique for the estimationof microbial biomass in view of the invisibility of cells embedded in particles and the relativelypoor contrast between cells and their background?R.L. Todd: Yes, it is extremely difficult to distinguish bacterial cells in a complex matrix such assoil. This fact, coupled with the heterogeneity of their distribution, make biomass determinationsmost difficult.T.R.G. Gray: Have you considered the use of X-ray microprobe analysis in conjunction with a label-led antibody technique to identify particular microorganisms?R.L. Todd: We are considering the use of cathodoluminescence to detect microbes in soil. If thistechnique proves satisfactory then the next step, labelled antibodies, would be a logical one. How-ever, to date we have not investigated this technique.J.S. Waid: One should draw attention to the difficulty of interpreting the true identity of particlesresembling bacteria that can be observed in mineral soils using the scanning electron microscope

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(SEM). Eswaran (1971) published some SEM photographs of fracture surfaces of soil peds whichshow structures he describes as globules of amorphous iron (Fig. 1 c) and halloysite crystals (Fig. 3).In Fig. lc the particles range in size from about 1.85 — 2.40 x 0.98 — 1.53 p , and in Fig. 3 fromabout 0.46 — 0.56 x 0.09 — 0.15 p. A soil bacteriologist might well describe these as large ovaland small rod-shaped cells respectively, although a pedologist has made a very different interpreta-tion. Therefore when examining mineral soils with the SEM we should take care to confirm thatstructures which resemble bacteria are indeed what they seem to be.Eswaran H. 1971. Electron scanning studies of the fabric of fracture surfaces. Soil Sci. Soc. Amer.Proc. 35: 787-790.R.L. Todd: Anyone who has used this system is certainly well aware of the difficulty in observingbacterial cells in a soil matrix. For this reason we generally have limited our observations to fungiand higher microscopic forms. However, it is premature to discount scanning electron microscopyfor bacterial observations. The application of refined techniques (such as cathodoluminescence) andfuture studies on the introduction of known cultures to sterile soils should provide a backgroundfor these assessments.J.S. Waid: Yes, I agree. Complementary observation techniques are essential.T.D. Brock: How can you tell that the structures you referred to are not bacterial?J.S. Waid: I do not see, with the evidence available, how one can identify these objects. Several dif-ferent methods need to be brought to bear upon this problem.J.A. Molina: Differentiation between bacterial cells and clay minerals observed by scanning electronmicroscopy could be obtained by use of X-ray diffraction.

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