unique advantages of using low temperature scanning ... · unique advantages of using low...

Protoplasma (1996) 195:133-143 PROTOPLASMA �9 Springer-Verlag 1996 Printed in Austria

Unique advantages of using low temperature scanning electron microscopy to observe bacteria

St6phane Roy 1' 2, Isabelle Babic 2, Alley E. Watada 2, and William P. Wergin i' *

Electron Microscopy Laboratory and 2 Horticultural Crops Quality Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland

Received November 10, 1995 Accepted January 2, 1996

Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology

Summary. Electron microscopy (EM) has greatly helped to eluci- date our understanding of bacterial structure and function. However, several recent studies have cautioned investigators about artifacts that result from the use of conventional EM preparation procedures. To avoid these problems, the use of low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leaves (Spinacia oleracea L. cv. New Jersey), which were naturally infected or inoculated with bacteria, were used as the experimental material. 1 cm segments of the infected leaves were plunge frozen in liquid nitrogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hydrated leaf segments were transferred onto agar medium to determine whether preparation for LTSEM was nondestructive to the bacteria. The other tissue segments were chemically fixed by freeze-substitution. The results indicated that after cryopreparation and observation in the LTSEM: (i) viable bacteria, which were recovered from the leaf sample, could be cultured on agar medium for subsequent study, and (ii) the frozen samples could be freeze substituted and embedded so that transmission electron microscopic (TEM) observations could be car- ried out on the same specimen. In conclusion, frozen, hydrated leaf tissue infected with bacteria can be observed using LTSEM and then can be either processed for TEM observation to obtain further structural details or recovered to culture the pathogenic bacteria for sup- plementary studies.

Keywords" Low temperature scanning electron microscopy; Freeze- substitution; Cell wall; Plant pathology.

Abbreviations: EPS extracellular polysaccharide; EM electron microscopy; LTSEM low temperature scanning electron microsco-

* Correspondence and reprints: Electron Microscopy Laboratory, ARS, U.S. Department of Agriculture, BARC-East, Bldg. 177B, Beltsville, MD 20705, U.S.A.

py; SEM scanning electron microscopy; TEM transmission electron microscopy; TSA tryptic soy agar; TSB tryptic soy broth.

Introduction

Our understanding of the structure and function of bacteria has been greatly expanded through the use of electron microscopy (EM) (Costerton 1979). For example, most bacteria can be easily distinguished using conventional scanning electron microscopy (SEM) (Davis et al. 1992). Colonization and the details of cellular adhesion have been illustrated in plant and animal tissues with conventional SEM and transmission electron microscopy (TEM) (Davis et al. 1992, Kim et al. 1994, Vasse et al. 1995). However, several recent studies document examples of artifacts that result from the preparation methods that are nor- mally used for conventional EM studies. For example, aqueous chemical fixation and dehydration frequently cause solubilization or aggregation of cellular components and result in altered morphology (Little et al. 1991). To avoid these problems, several alterna- tive procedures have been used to prepare bacteria for EM studies. To minimize the effects of aqueous chemical fixation and to preserve the extracellular polysaccharide (EPS) matrix in which the bacteria were embedded, Crang (1988) utilized osmium vapor for fixation. Graham and Beveridge (1990a, b) dem- onstrated that the ultrastructural features of most bac-

134

teria could be improved by using a nonchemical procedure, namely cryofixation followed by freeze-substitution. Chaphalkar et al. (1993) also avoided aqueous chemical fixation by using a synthetic polymer to create replicas that could be observed with the SEM, whereas Golecki and Heinrich (1991) embedded their samples directly in water soluble Nanoplast resin without any previous fixation. Collins et al. (1993) further minimized the use of preparation procedures by utilizing a variable pressure SEM to image hydrated microorganisms. Recent studies in our laboratory have allowed us to examine a variety of frozen, fully hydrated biological specimens with the low temperature SEM (LTSEM). The frozen specimens observed in this manner remain stable because they are placed on a cryostage that is maintained at -185 ~ At this temperature the vapor pressure of water is insignifi- cant; consequently neither sublimation nor recrystal- lization of water ice occurs at a detectable rate and the frozen, hydrated samples remain stable for several hours (Wergin and Erbe 1991a, b). The present study documents the advantages of imaging bacteria with this technique. The results not only emphasize the benefits generally associated with LTSEM, but also demonstrate several unique advantages that are appli- cable to studies in bacteriology.

Material and methods Plant samples and bacterial isolation

Spinach leaves (Spinacia oleracea L. cv. New Jersey), which contained natural infections of bacteria, were obtained from a local fresh-cut produce processor, placed in gas permeable bags and stored for 12 days at 10 ~ After the infected leaves were observed with the LTSEM, the native microorganisms were isolated from these samples by detaching the specimens from the SEM holder, which is described below, and placing them in sterile petri plates for about 1 h where they were allowed to attain room temperature. Next, a loop from the scalped leaf surface was streaked onto tryptic soy agar medium (Soybean-Casein Digest Agar medium; Fisher Scientific Co., Fair Lawn, NJ, U.S.A.) and incubated at 30 ~ for 2 h. Enumer- ation, differentiation, and identification of the microorganisms were performed as described by Babic et al. (1992). Isolation of single bacterial colonies was performed by randomly selecting the square root of the total number of colonies present on each plate. The isolated strains, which were purified and classified according to their mor-

S. Roy et al.: Low temperature scanning electron microscopy of bacteria

Fig. 1. Specimen holder that was used to observe infected leaves in the LTSEM and then to freeze-substitute the frozen, hydrated samples for TEM observation: a copper metal plate, b holder on which the copper metal plate can be inserted, c Oxford specimen carrier, and d assembled specimen holder. The holder was first mounted on the Oxford specimen carrier (vertical arrow), and then the copper metal plate was inserted in the holder (horizontal arrows). The copper metal plate can be transferred independently to the freeze-substitution chamber. The assembly was transferred to the cryostage in the cryochamber by attaching a cryotransfer arm to the threaded carrier. Bar: 3 cm

phological, biochemical, physiological and sexual characteristics, were maintained on TSA slants at 4 ~ (Babic et al. 1996). An inoculum was prepared by growing the bacteria, which had been previously observed with LTSEM and subsequently isolated from the spinach leaf, in tryptic soy broth at 30 ~ for 18 h. Next, each culture was centrifuged at 483 g; the media and residual cells were removed and the pellet was resuspended in a 0.85% NaC1 solution so that an inoculum with about 108 cells per ml was obtained. This inoculum was used to reinfect spinach leaves that had been fresh cut and not stored by abrading the leaf surface with a borer blade, placing 10 ~tl of inoculum on the wounded leaves and then incubating them in petri dishes at 10 ~ After 4 days these samples were also processed for observations with LTSEM, then removed from the instrument, chemically fixed by freeze-substitution, embedded and sectioned for TEM observation.

Low temperature SEM observations

Low temperature SEM observations were performed on an S-4100 field emission scanning electron microscope (Hitachi Scientific Instruments, Mountain View, CA, U.S.A.) equipped with a CT- 1500HF Cryotrans System (Oxford Instruments, Eynsham, Eng- land). Specimen preparation consisted of removing 1 cm segments

Figs. 2-4. LTSEM observations of frozen, hydrated palisade parenchyma cells beneath a ruptured cuticle (cu) of a leaf naturally infected with

bacteria

Fig. 2. Bacteria (b) form a meshwork that covers the cells (c). s Stomata. Bar: 60 ~tm

Fig. 3. The bacteria, which formed a meshwork that engulfed the cells, were barely distinguishable in the EPS matrix (arrows). Bar: 20 um

Fig. 4. Bacteria (b) embedded in EPS matrix (e) that forms a meshwork which interconnects the cells of the palisade parenchyma (c). Filaments appear to connect the bacteria to the cell wall (arrows). Bar: 0.5 gm

S. Roy et al.: Low temperature scanning electron microscopy of bacteria 135

136 S. Roy et al.: Low temperature scanning electron microscopy of bacteria

Figs. 5 and 6. Ambient temperature SEM observation of a naturally infected leaf

Fig. 5. Many bacteria (arrows) occur on the surface of the palisade parenchyma cells (c) beneath the raptured cuticle of the leaf. Bar: 20 gm

Fig. 6. The bacteria (b) are easily visible because of the absence of the EPS matrix. Bar: 2 ~tm

from the spinach leaves or 1 cm squares from the agar medium and placing them on flat specimen holders containing a layer of Tissue Tek (OCT Compound; Ted Pella, Inc., Redding, CA, U.S.A.). The holders were rapidly plunge frozen in liquid nitrogen slush and cryo- transferred under vacuum to a cold stage in the prechamber of the cryosystem. The frozen specimens were etched for 9 rain in the prechamber by raising the temperature of the stage to -90 ~ sputter coated with Pt and then transferred to the cryostage in the SEM for observation. An accelerating voltage of 10 kV was used to view the specimens; the images were recorded onto Polaroid type 55 P/N film.

Freeze-substitution, embedding, sectioning, and TEM observation

Samples that were intended for freeze-substitution were mounted on flat fabricated plates containing a layer of Tissue Tek that could be attached to modified Oxford specimen holders and easily withdrawn for further transfer (Fig. 1). After the samples were etched, sputter coated, observed and photographed in the SEM, they were moved back into the prechamber and then transferred under vacuum to liquid nitrogen. The fiat holders, on which the samples were mortared, were then transferred to 15 ml cryogenic vials that were filled with a

Figs. 7-9. LTSEM observation of a bacterial colony cultured on an agar medium after the ceils were isolated from the sample that had been observed with LTSEM

Fig. 7. Freeze-fracture and freeze-etching revealed the internal structure of the colony. The colony is characterized by two growth patterns: I an outer layer of tightly oppressed cells, and H an internal area of loosely associated cells. Bar: 5 gm

Fig. 8. Detail of the fractured surface of a bacterial colony. The surface of the colony is formed by bacteria ceils (b) that adhere closely to each other. Bar: 2 ~m

Fig. 9. Detail of the internal organization of a bacterial colony. The cells are loosely joined end-to-end. Bar: 3 ~tm

138

solution of 2% (w/v) osmium tetroxide in acetone precooled to -90 ~ The holders were supported vertically by a copper frame in the cryogenic vials that were placed in wells that had been drilled in an aluminum block. The aluminum block was put into a precooled

brass chamber that was then placed into an insulated encasement which had been precooled with liquid nitrogen. The insulated encasement was filled with dry ice to maintain a temperature of -80 ~ The temperature of the samples was monitored with a ther-

mocouple that was placed in a vial in the center well of the aluminum block. Substitution with the osmium solutions was allowed to pro- ceed for 3 days. Subsequently, the solution was slowly warmed (2 h at -60 ~ 2 h at -18 ~ 2 h at 4 ~ and 2 h at room temperature) and the substitution medium was replaced with fresh acetone. Next, the samples were detached from the holders, gradually infiltrated with Spurr's low viscosity resin and cured at 60 ~ The embedded samples were thin sectioned on an Ultracut ultramicrotome (AO Sci- entific Instruments, Buffalo, NY, U.S.A.) and mounted on copper grids. To improve contrast, some sections were post-stained in 2% aqueous uranyI acetate. TEM observations were performed on a Hitachi H500H operating at 75 kV.

Conventional fixation for TEM and ambient temperature SEM

observation

For conventional TEM fixation, the samples were placed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 2 h, then washed in cacodylate buffer. Next, the samples were postfixed in 1% osmium tetroxide for 1 h. After dehydration in an alcohol series, the samples were embedded in Spurr's resin. Thin sections were stained with 2% aqueous uranyl acetate for 20 min and lead citrate for 1 min. All thin sections were observed with a Hitachi H500H operating at

75 kV. For ambient temperature SEM observations, the samples were chemically fixed in glutaraldehyde as described above, dehydrated in an alcohol series and then critical point dried from liquid carbon dioxide. Next, the samples were mounted on aluminum stubs and sputter coated with Au/Pd. These samples were observed in a conventional SEM not equipped with a cryostage, namely a Hitachi $570 operating at 20 kV.

ResuLts

Infection of the spinach leaf by the bacteria

Observations with LTSEM revealed that the surface of the commercially purchased spinach leaves, which were stored for 12 days at 10 ~ exhibited areas with discrete ruptures of the cuticle. In these areas the

S. Roy e t al.: Low temperature scanning electron microscopy of bacteria

internal palisade parenchyma tissue could be observed (Fig. 2). These cells appeared to retain their hydrated condition; however, a layer consisting of numerous bacteria completely surrounded the palisade cells (Figs. 2 and 3). The material in which the bacteria were embedded was composed of a meshwork of filaments and sheets and appeared similar to the EPS matrix described by Van Doom et al. (1990). The bacteria appeared to be anchored to the cell wall by means of the filaments (Fig. 4). Similar infected tissues, which were conventionally prepared and observed with ambient temperature SEM, did not exhibit the layer of amorphous material surrounding the bacteria and covering the parenchyma cells (Fig. 5). Alternatively, the bacteria, which were clearly visible, appeared as discrete cells that aggregated in the intercellular spaces between palisade parenchyma cells (Fig. 6).

Organization of the bacterial colonies

Bacteria, which were isolated from the samples that were observed with LTSEM, grew on agar plates despite their previous exposure to freezing, etching and coating. These bacteria were identified as Pseu- domonas fluorescens bv. III (data not shown) (Babic et al. 1996). Further detailed information about the formation of the bacterial colonies was provided by in situ cryofixation. Figure 7 illustrates a portion of a colony that was cryofixed in liquid nitrogen slush, fractured, etched and coated. The fracture revealed that the bacteria in the colony occurred in two distinct growth patterns: (i) a continuous outer layer of tightly appressed cells that consisted mainly of small coc- ci (Fig. 8) and (ii) the internal portion of the colony where the cells were loosely organized, generally attached end-to-end, and separated by large intercellular spaces (Fig. 9). Observation of other colonies, which had not been etched as part of the preparation procedure, also

Figs. 10 and 11. Inner organization of a bacterial colony. LTSEM observation

Fig. 10. Freeze-fracture without etching before coating with Pt. The bacteria (b) are embedded in water-ice (i). Bar: 2 gm

Fig. 11. The sample observed in Fig. 10 was withdrawn to the cryochamber, cryofractured, etched and recoated. The organization of the loosely associated bacterial ceils (b) is apparent in the absence of the water-ice. Bar: 2 ~m

Figs. 12 and 13. TEM observations of a thin section illustrating a portion of a spinach leaf that had been inoculated with bacteria. 4 days after inoculation the infected areas were cryofixed, observed by LTSEM, removed from the instrument, freeze-substituted, and examined in the TEM

Fig. 12. The bacteria appeared as elongated cells with multilayered cell walls. The fine structure of the plant cell and the bacteria was well preserved, p Chloroplast, cw cell wall, er endoplasmic reticulum, mi mitochondria, pe peroxisome. Bar: 1 ~xm

Fig. 13. The bacteria were frequently elongated and appressed to the outer wall of the plant cell. Bar: 0.2 ~tm

140 S, Roy et al.: Low temperature scanning electron microscopy of bacteria

Fig. 14. Bacteria in inoculated leaf observed in situ with TEM after cryofixation and freeze-substitution. The bacteria, which have an angular outline, fill a narrow gap between adjacent plant cell walls. "Ribosome-free" zones (z) contain diffuse chromatin and some electron opaque inclusions. Note the connecting filaments between the bacterial cell and the plant cell wall (cw) (arrows). Bar: 0.2 ~m

Fig. 15. Bacteria in inoculated leaf observed in situ with TEM after conventional preparation. These bacteria, which are more spherical, also fill a gap between adjacent plant cells. The central area of the cytoplasm contains aggregated chromatin (arrows). Bar: 0.2 gm

exh ib i t ed the outer l aye r of cel ls s imi la r to that

desc r ibed above; however , in the in ternal por t ion o f

these co lonies , the l oose ly o rgan ized bac te r i a l cel ls

were found e m b e d d e d in a f rozen ma t r ix (Fig. 10).

W h e n these samples were w i thd rawn into the c ryo-

chamber , re f rac tured , e tched and coated , the l oose ly

o rgan ized bac te r i a l ce l ls cou ld be obse rved wi thout

the f rozen ma t r ix (Fig. 11). This obse rva t ion ind ica t -

ed that the mat r ix cons i s t ed o f wa te r - i ce that had been

subl imed.

Ultrastructural (TEM) aspects of the infected tissue

Af te r L T S E M observa t ion , the in fec ted leaves were

f reeze-subs t i tu ted for T E M observa t ion . Thin sec-

t ions o f the in fec ted leaves r evea l ed bac te r ia that


appeared to be colonizing the palisade parenchyma cells that lay beneath the epidermis (Fig. 12). The cytoplasm of the parenchyma cells contained well defined mitochondria, plastids, and endoplasmic reticulum. The bacteria, which were embedded in a layer of material similar to that observed with LTSEM, i.e., the EPS matrix, appeared as elongated cells having a multilayered cell wall (Figs. 12 and 13). They were frequently tightly appressed to the outer wall of the plant cell (Fig. 13) and tended to aggregate in the narrow intercellular spaces that formed between adjacent plant cells (Fig. 14). Bacte- rial chromatin formed a lightly-condensed, diffuse mass in the central "ribosome free" zone (Muller 1988) that was surrounded by clearly defined aggregates of ribosomes (Fig. 14). Electron opaque cyto- plasmic inclusions were frequently seen within the chromatin (Figs. 12-14). Bacterial colonies from the freeze-substituted samples occasionally contained electron translucent areas that may have been induced by ice crystal formation (Fig. 12). Samples that had been conventionally fixed did not appear to exhibit the presence of an EPS matrix or any fibrillar connections to the plant cell wall. The bacteria cells, which were more spherical, also tended to aggregate in the intercellular spaces bordered by plant cells (Fig. 15). The chromatin, which formed a fine fibrillar mass in the center of the cell, was surrounded by aggregates of ribosomes in the cytoplasm. The bacterial wail was covered by a thin outer membrane that was only rarely distinguished.

Discussion

The advantages of LTSEM, as opposed to ambient temperature SEM, have been extensively discussed (Echlin et al. 1970; Jeffree and Read 1991; Read and Jeffree 1991; Wergin and Erbe 1991a, b). The advantages associated with our samples included minimum specimen preparation time, rapid results, and no apparent mechanical distortion. In addition, our expe- rience with the bacterial samples indicated that this technique: (i) preserved the viability of at least some of the bacteria, which could be subsequently recovered and cultured on agar medium; (ii) permitted recovery of the infected leaf sample after LTSEM observation so that freeze-substitution could be used to prepare the sample for TEM observation; and (iii) maintained a layer that surrounded the bacterial colony and corresponded to the EPS matrix described by Van Doom et al. (1990). The extent to which the via-

bility of bacteria may have been affected by irradia- tion by the electron beam was not determined. How- ever, colonies could be easily obtained on all of the cultures that were inoculated with the bacteria obtained from the observed material. Previous studies have shown that ascospores of Sordaria macrospora also survived LTSEM preparation and observation (Read and Jeffree 1991). Because LTSEM observation did not appear to destroy the viability of our samples, bacteria as well as infected tissues could be recovered for further microscopic or analytical studies. Our study utilized the freeze-substitution procedure (Steinbrecht and Muller 1987) to dissolve the ice, chemically fix and dehydrate the cryoimmobilized specimens. This procedure has been previously used to prepare specimens for studies in mycology (Howard and O'Donnell 1987), bacteriology (Kellen- berger et al. 1992, Paul and Beveridge 1993), as well as phytopathology (Bonfante et al. 1994, Studer et al. 1992). However, to our knowledge ours is the first study that involved LTSEM observation prior to freeze-substitution and TEM observation. Our results indicated that the mode of cryofixation that we have chosen, i.e., plunge freezing in a liquid nitrogen slush, was adequate to prepare and observe bacteria. The ideal purpose of cryofixation is to freeze the water in the specimen in an amorphous or micro- crystalline (vitreous) state and thereby limit the dam- age caused by formation and growth of ice crystals (Gilkey and Staehelin 1986). The production of vitreous ice depends on the rapidity of thermal exchange between the specimen and the cryogen. Several freezing methods have been used to study bacterial ultra- structure including ultra-rapid plunge freezing (Gra- ham and Beveridge 1994), metal mirror freezing (Nicolas and Bassot 1993), and high-pressure freezing (Hohenberg et al. 1994). However, these methods of cryofixation used single cells of bacteria that were cultivated in suspension. The plunge method of cryofixation that we chose was relatively simple, versa- tile, and inexpensive. The ultrastructural features exhibited by the bacteria that were plunge frozen and freeze-substituted appeared to be similar to those that were obtained using other cryofixation methods. For example, we observed the "ribosome-free" zone described by Muller (1988) rather than a vacuole-like empty space containing a reticulate of DNA aggregates. However, in bulk samples of infected tissue the bacteria were more poorly preserved as one moved further from the freezing front. Using other cryofixa-

142 S. Roy et al.: Low temperature scanning electron microscopy of bacteria

tion methods would undoubtedly improve results of bulk specimens. Although most investigators agree that bacteria generally grow within the EPS matrix, relatively little is known about the function of this material. Cryofixa- tion nicely preserved the matrix in which the bacteria appear to grow. Previously, Crang (1988) had used vapor fixation from crystalline osmium tetroxide followed by freeze drying to preserve the EPS matrix of bacterial colonies. Van Doom et al. (1990) compared the morphology of a bacterial colony after chemical fixation, dehydration and critical point drying with colonies that were cryofixed and observed with a low temperature SEM. This study showed that alcohol dehydration and critical point drying removed the EPS matrix layer. Our experiments using conventional preparation for ambient temperature SEM observation confirmed the absence of the EPS matrix of the bacterial colony, which could provide misleading information about how bacteria colonize the leaf and invade the cell. Future LTSEM observations should enhance our knowledge of the function(s) of the EPS matrix. Manipulations of frozen specimens in cryochambers have allowed investigators to gain additional information about the structure and composition of a sample. In our study, after the initial observation of the external structure of the colony, fracturing the sample in the cryochamber was used to reveal its internal structure. This sequence of manipulations, namely the initial observations, transfer of the sample to the cryochamber for freeze-fracturing and coating followed by a second observation has also been used to gain information on other types of specimens including dauer larvae of Steinernema carpocapsae (Wergin et al. 1993) and the egg mass of the root knot nema- tode Meloidogyne incognita (Orion et al. 1994). The general organization of a stomatal complex (Jeffree and Read 1991) and a mite egg (Roy et al. 1994) was also revealed by similar manipulations of the frozen sample. By examining and comparing frozen, fractured samples of etched and nonetched material, Pearce and Ashworth (1992) were able to determine the presence and specific location of free water in leaves of overwintering wheat. We have applied this procedure to a bacterial study in an effort to provide information on the organization of colonies and their mode of growth. Amako and Umeda (1977) attempted to gain similar information about the formation of bacterial colonies using in situ fixation of bacteria on the agar

H I ~]Conventionall I InOculatiOn infectedSample ~ ' ~ ' 1 preparati~ I

T SEN [ TEN observation I observat on

' t liqCi~turediin m _LTSEM ! .=1 Freeze- I . / ~ 1 7 6 I "1 substituti~

/ Culture I [ maniMpulat]ons on Agar I I I

Fig. 16. Diagram that indicates several possibilities for processing and observing bacteria. After LTSEM observation, the bacteria can be directly processed for freeze-substitution and TEM observation or recovered and cultured for further studies

surface. However, after a short period of growth, the colony disintegrated in the fixative solution. Because this problem is eliminated by cryofixation, LTSEM appears to be an appropriate method for following the process of colony formation. We were able to observe colonies at all stages of growth. The colonies were always organized in the same manner: an external layer of tightly appressed cells that covered loosely associated cells within the colony. Observation of the fractured colonies prior to and after etching suggests that the underlying cells are bathed in an aqueous solution. The tightly appressed cells in the outer layer would appear to protect the colony from dehydration. In conclusion, LTSEM is a very effective tool for viewing bacteria that are not subjected to chemical preparation. Figure 16 summarizes the potential manipulations that can be pursued with this technique. An infected sample can be initially observed with LTSEM and subsequently either processed for TEM observation to obtain fine structural details of the bacterial ceils or colonies or they can be recovered and cultured to determine the different species that may be involved in the infected tissue.

Acknowledgements The authors thank Christopher Pooley for the realization of Fig. 1 and for converting the negatives to the digital images that were used to illustrate this study.


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