JOURNAL OF BACTERIOLOGY, Apr. 1990, p. 2150-2159 Vol. 172, No. 40021-9193/90/042150-10$02.00/0Copyright C) 1990, American Society for Microbiology

Effect of Chemical Fixatives on Accurate Preservation ofEscherichia coli and Bacillus subtilis Structure in Cells

Prepared by Freeze-SubstitutionLORI L. GRAHAM* AND T. J. BEVERIDGE

Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, Ontario, Canada NIG 2WJ

Received 25 August 1989/Accepted 5 January 1990

Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure duringfreeze-substitution of select Escherichia coli and BaciUus subdlis strains. Radioisotopes were specificallyincorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and intothe peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed byliquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator ofcell structural integrity and retention of cellular chemical composition. Subsequent visual examination byelectron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol)osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate;2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate.All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituentsdepended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2%gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In bothgram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids inwhich 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alonewere used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence ofdifferent chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone forroutine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalyticalprocessing necessitates the omission of osmium.

Freeze-substitution is rapidly becoming an important tech-nique for the electron microscopic examination of bacterialultrastructure. The envelope of Escherichia coli (2, 17; T. J.Beveridge, R. Harris, and R. Humphrey, Proc. Microsc.Soc. Can. 12:22-23, 1985; L. L. Graham and T. J. Bever-idge, Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, J4, p.205; L. L. Graham, Proc. Microsc. Soc. Can. 16:18-19,1989; L. L. Graham, R. Harris, and T. J. Beveridge, Proc.Microsc. Soc. Can. 16:58-59, 1989), the capsule of Vibriovulnificus (3), Lactobacillus casei (23), L. acidophilus (11),Klebsiella pneumoniae (1), E. coli (1; T. J. Beveridge, R.Harris, and R. Humphrey, Proc. Microsc. Soc. Can. 12:22-23, 1985), and Leptothrix discophora (5), and the walls ofStaphylococcus aureus (27) and Bacillus subtilis (4; T. J.Beveridge, R. Harris, and R. Humphrey, Proc. Microsc.Soc. Can. 12:22-23, 1985; L. L. Graham, Proc. Microsc.Soc. Can. 16:18-19, 1989; L. L. Graham, R. Harris, andT. J. Beveridge, Proc. Microsc. Soc. Can. 16:58-59, 1989)have been described for cells prepared by this technique. Asa preparatory method, freeze-substitution appears to besuperior to conventional embedding procedures since itcombines physical fixation (ultrarapid freezing to arrestphysiological processes) with slow chemical fixation (toincrease bonding and thereby stabilize structure) and dehy-dration (to allow plastic infiltration) (14). Since fixation anddehydration are performed at temperatures below the freez-ing and recrystallization points of cellular water, redistribu-tion and loss of cellular components as well as shrinkage ofsamples should be minimized, yet samples should still be

* Corresponding author.

miscible with standard resins so they may be handled easilyby ultramicrotomy.

In an examination of several conventionally fixed gram-negative species, Silva and Sousa (25) showed that theultrastructure of the bacterial envelope in thin section isexquisitely dependent on the conditions used for chemicalfixation. Best results were achieved by maintaining cells asclose as possible to in vivo conditions during fixation.Although numerous chemical fixative and solvent combina-tions have been applied to a variety of biological samplesprocessed by freeze-substitution, no systematic evaluationof their contribution to cellular preservation has been re-ported to date.

Relative to eucaryotic cells, bacteria are structurally sim-pler systems (6) whose ease of genetic manipulation hasenabled construction of specific mutant strains which lendthemselves to systematic study. Accordingly, E. coli K-12and B. subtilis strains, as representative gram-negative andgram-positive bacteria, respectively, were used to evaluate arange of chemical fixatives for freeze-substitution. Mutantstrains allowed the strategic radiolabeling of distinct struc-tural components. Liquid scintillation counting was thenused to detect the labeled components extracted during eachstep of the freeze-substitution process. Thin sections of cellsprocessed by each regimen were also examined for struc-tural integrity. This study allowed us to suggest a freeze-substitution protocol which yields optimal results for theroutine processing of eubacteria.(A portion of this work was presented previously [L. L.

Graham and T. J. Beveridge, Abstr. Annu. Meet. Am. Soc.Microbiol. 1988, J4, p. 205].)

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CHEMICAL FIXATIVES FOR FREEZE-SUBSTITUTION 2151

MATERIALS AND METHODS

Bacterial strains. All E. coli mutant strains were deriva-tives of K-12. Strain SFK11 was kindly supplied by S. F.Koval, University of Western Ontario. E. coli W7 waskindly provided by J.-V. Holtje, Max-Planck-Institut furVirusforschung, Tubingen, Federal Republic of Germany. B.subtilis W23 was kindly supplied by H. Pooley, Universitede Lausanne, Lausanne, Switzerland. All strains were main-tained on slants as described before (14).

Radiolabeling of bacteria. Radiolabeling of bacterial strainswas done as reported in Graham and Beveridge (14). Briefly,the peptidoglycan and RNA fractions of E. coli SFK11 werelabeled by the addition of 25 ,uCi of [3H]DPM (DL-meso-2,6-diamino[G-3H]pimelic acid dihydrochloride; specific ac-tivity, 1 Ci/mmol; Amersham Corp.) and 2.5 ,uCi of [2-'4C]uracil (specific activity, 54 mCi/mmol; Amersham). Labelingof lipopolysaccharide (LPS) and DNA fractions of this strainwas accomplished with 10 ,uCi of D-[1-_4C]galactose (specificactivity, 61 mCi/mmol; Amersham) and 25 pCi of [5-3H]thymidine (specific activity, 14.2 Ci/mmol; Amersham).The peptidoglycan and RNA fractions of E. coli strain W7

were labeled with 25 ,uCi of [3H]DPM and 2.5 ,uCi of['4C]uracil-Labeling of B. subtilis 168 and W23 was performed in

Spizizen minimal medium (26) by the method of Mobley etal. (21) by the addition of 25 ,uCi of N-acetyl-D-[1-3H]glucosamine (specific activity, 1.7 Ci/mmol; Amersham)and 2.5 ,uCi of [14C]uracil.

Freeze-substitution of radiolabeled bacteria. After beingharvested by centrifugation, radiolabeled cells were incu-bated in 18% (vol/vol) glycerol as a cryoprotectant in 0.05 MHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid) buffer, pH 6.8, for 20 min at room temperature. Cellswere pelleted in an Eppendorf centrifuge, a volume ofmolten 2% (wt/vol) Noble agar equal to the pellet was addedand mixed, and the suspension was immediately layeredonto a sterile cellulose-ester membrane filter (Gelman Sci-ences) (14). Wedge-shaped portions of this filter wereplunge-frozen in liquid propane at - 196°C with a simpleimmersion device designed and built at the University ofGuelph, Guelph, Ontario (L. L. Graham, E. Bullock, R.Harris, R. Humphrey, and T. J. Beveridge, unpublished).Frozen samples were then transferred to glass vials contain-ing frozen substitution media and a molecular sieve (sodiumaluminosilicate; pore diameter, 0.4 nm; Sigma Chemical Co.)maintained at -196°C. The following chemicals were dis-solved in anhydrous acetone for use as substitution media:2% (wt/vol) osmium tetroxide (OS04; Fisher Scientific) and2% (wt/vol) uranyl acetate (UA; Fisher Scientific); 2%(vol/vol) glutaraldehyde (Marivac Ltd.) and 2% (wt/vol) UA;2% (vol/vol) acrolein (Sigma) and 2% (wt/vol) UA; 2%(wt/vol) gallic acid (Fluka); and 2% (wt/vol) UA. Acetonealone was used as a control. Substitution medium wasprepared fresh prior to use and stored in sealed glass vials in2-ml portions. The time elapsed between sample layering onthe membrane and transfer of plunge-frozen cells into sub-stitution medium rarely exceeded 2 min. After transfer, vialswere sealed and the specimens were cryosubstituted at-80°C for 72 h (substitution step). During this period, themedium gradually melted and the cells were chemically fixedand dehydrated. After substitution, vials were removed andallowed to come to room temperature. Samples were washedsix times for 15 min each in anhydrous acetone to removeexcess fixative (acetone step) and then infiltrated overnightat room temperature in an acetone-Epon 812 mixture (1:1)

(infiltration step). Samples were embedded in fresh Epon 812and polymerized at 60°C for 36 h.

Radioassays of freeze-substituted cells. Triplicate samplewedges were collected immediately before and after plunge-freezing, after substitution but before washes in anhydrousacetone, and after overnight infiltration in the acetone-Epon812 mixture. All samples were mixed with 0.5 ml of Protosol(New England Nuclear Corp.) for solubilization prior to theaddition of scintillation cocktail. In addition, all substitutionfluids, all acetone washes, and all infiltration resins wereretained for analysis by liquid scintillation counting. Count-ing was performed in a toluene-based cocktail containingOmnifluor (New England Nuclear) with a Beckman LS-3150T scintillation counter. All data were corrected forquenching and are presented as the percent counts perminute (cpm) detected in samples or washes, representingthe mean of three independent experiments for each labelingcondition.

Electron microscopy. Thin sections of freeze-substitutedcells were cut on a Reichert-Jung Ultracut E ultramicrotomeand mounted on Formvar carbon-coated copper grids. Sec-tions were post-stained in aqueous 2% (wt/vol) UA and leadcitrate (22) prior to examination on a Philips EM300 electronmicroscope operating at 60 kV.

Unstained, thick sections (0.12 pLm) of B. subtilis 168 cellsfreeze-substituted in the different fixatives were mounted onFormvar-coated aluminum grids and analyzed on a PhilipsEM400T electron microscope operating at 100 kV equippedwith a goniometer stage and an EDAX energy-dispersiveX-ray detector coupled to a Tracor Northern TN5500 series1 microanalyzer. Both the EM400T and the EM300 wereoperated with a liquid nitrogen-cooled anticontaminationdevice in place at all times. Energy-dispersive X-ray spec-troscopy (EDS) was conducted with electron beam spotsizes of 200 nm or less, and counts were done for 100 s (livetime).

Optical densitometry. High-magnification, high-contrastnegatives were constructed on orthochromatic Graphic Artsfilm (Du Pont) from 35-mm negatives obtained by electronmicroscopy of cells processed in different substitution me-dia. These larger negatives, printed at the same magnifica-tion as the micrographs used in this text, were scanned by alinear tract 40 ,m in width perpendicular to a cross sectionof a bacterial cell envelope with a Joyce-Lobel 3CS opticaldensitometer (Joyce-Lobel Ltd., Gateshead, England) toproduce a tracing in which peak height was correlated withfilm grain density.

Statistical analyses. A one-way analysis of variance and theTukey test for multiple comparisons (30) were used toestablish significant differences in label retention under eachof the six fixative conditions examined.

RESULTS

Detection of cellular components during freeze-substitution.To establish an optimal protocol for freeze-substitution ofeubacteria, E. coli SFK11 and W7 and B. subtilis 168 andW23 were freeze-substituted in the presence of five differentfixatives as well as in the absence of any fixative. Detectionof radiolabel(s) in sample wedges and processing fluids byliquid scintillation counting was used as an indication of celldamage during processing. The radioisotope uptake proto-cols used in this study resulted in the efficient and specificincorporation of radiolabels into the major macromoleculesof the cell wall and nucleic acids of E. coli and B. subtiliscells. Table 1 presents the total percent 3H and "4C counts

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TABLE 1. Total percent 3H and 14C cpm detected in processing fluids during freeze-substitution of E. coli SFK11 and W7 andB. subtilis 168 and W23 in different fixatives

% of added cpma

E. coli B. subtilisFixative

SFK11 SFK11 W7 168 W23

[3HIDPM ['4C]Ura [3H]Thy [14C]Gal [3H]DPM [14C]Ura [3H]GlN ['4C]Ura [3H]GlN [14C]Ura

OS04-UA 12.05 54.16 21.45 30.88 18.34 37.69 43.46 52.68 36.64 44.98Glutar.-UA 86.19 96.43 58.36 71.75 41.28 69.05 80.70 82.44 88.68 92.29Acrolein-UA 99.00 99.00 43.73 56.26 69.08 73.95 82.93 65.10 80.05 46.13Gallic acid 25.23 23.49 26.63 12.64 22.75 20.78 22.59 7.53 32.31 19.01UA 85.31 91.81 NAb NA 81.47 89.73 73.89 74.14 78.51 63.83Acetone 33.39 28.98 NA NA NA NA 27.96 14.91 47.84 22.90

a DPM, Diaminopimelic acid; Ura, uracil; Thy, thymidine; Gal, galactose; GIN, N-acetyl-D-glucosamine; Glutar., glutaraldehyde.b NA, Not assayed.

detected in processing fluids during freeze-substitution in thefixatives. Similarities in the cpm detected for nucleic acidand wall labels implied that neither component was selec-tively extracted during the entire process. For both gram-positive and gram-negative cells, lower levels of radiolabelwere detected when cells were processed in the presence ofOs04-UA or in gallic acid. UA could not be used in thepresence of gallic acid because the uranyl ion is complexedand precipitated by the acid. As indicated by the high levelsof label detected in processing fluids, glutaraldehyde-UA,acrolein-UA, and UA alone were very ineffective fixatives.Surprisingly, acetone alone extracted low levels of thelabeled cell components from both gram-negative and gram-positive cells.The stage of processing at which radioisotopes were

leached from bacterial cells varied as a function of thechemical fixative used (Fig. 1). When substitution wasperformed in Os04-UA or gallic acid, most of the label wasextracted during the acetone washes, with the first washreleasing the most and succeeding washes releasing succes-sively decreasing amounts of the isotopes. This trend wastrue of E. coli and B. subtilis strains for both 3H and 14Clabels. However, when substitution was performed in glu-taraldehyde-UA, greater amounts of label were detected inthe substitution medium and the infiltration resin. Whenacrolein was used as a fixative, the peptidoglycan label ofboth gram-positive and gram-negative cells was detected atthe highest levels in the infiltration resin. This was incontrast to the LPS-labeled SFK11 strain, in which leachingwas greatest during the acetone washes; this difference inextraction is probably due to the greater solubility of thelipid than of peptidoglycan in acetone. With UA-acetone,extraction of the nucleic acid labels was prevalent duringsubstitution, while wall labels were leached during infiltra-tion. Relatively low levels of radiolabel were detected in theacetone washes of cells substituted in UA alone. Whensubstitution was carried out in acetone alone, cytoplasmicand wall isotopes were detected in the acetone washes, whileonly low levels were detected during the substitution andinfiltration stages of processing.Morphology and ultrastructure of cells. Bacteria freeze-

substituted in different fixatives showed variation in ultra-structural detail, variation substantiated by optical densi-tometry of cell envelope profiles. All fixatives yieldedrod-shaped cells with no evidence of shrinkage, with theexception of E. coli cells substituted in acetone alone; thesecells were irregularly shaped (Fig. 2F). Figures 2 and 3 are

representative E. coli SFK11 and B. subtilis 168 cells proc-essed by freeze-substitution in each of the fixatives exam-ined.

E. coli W7 and SFK11 cells (Fig. 2A) processed inOs04-UA exhibited a multilayered cell wall, including thedistinct "periplasmic gel" described by Hobot et al. (17).Although this layer was also visible in cells processed inglutaraldehyde, acrolein, and gallic acid, the gel appeared tobe compressed into a narrower electron-dense band in thesecells. This layer was difficult to observe in cells processed inUA or acetone alone. Deposition of osmium and UA atmembrane locations was indicated by the contrast impartedby these heavy metals in unstained sections. Cytoplasmicmembranes were most prominent with Os04-UA but werealso visible in cells processed in glutaraldehyde, acrolein,and gallic acid. In these cells, visibility was greatly enhancedby poststaining procedures, a step unnecessary in OS04-UA-substituted cells. Dense, granular cytoplasm was bestobserved in 0s04-UA- and acrolein-UA-treated cells. Al-though ribosomes were evident under all conditions, fibrousmaterial (DNA?) and cytoplasm of inconsistent density(ribosomes?) were characteristic of cells prepared in gallicacid, UA, acetone, and, most noticeably, glutaraldehyde.Optical densitometry tracings of the envelopes of E. coliillustrated diminished electron density and, in some cases,apparent loss of one or more envelope layers when cellswere treated with fixatives other than Os04-UA (data notshown). A distinct asymmetrically staining outer membrane(the outer face being more intensely stained) was character-istic of E. coli cells. Figures 4 and 5 show the envelopeprofile of strain SFK11 processed in Os04-UA or acrolein.Note the condensed periplasmic gel and poorly stainedcytoplasmic membrane of acrolein-treated cells.

Unlike E. coli, B. subtilis cells were always rod-shapedregardless of the fixative used. In all cases, a fibrous layerwas present on the outermost portion of the cell wallradiating outwards and extending 10 to 30 nm from the cellsurface, depending on the fixative used. The electron densityof this structure was graduated, appearing densest at itsinner face and decreasing in electron density as distancefrom the cell increased. Each fibril was well stained, and thedecreased stain density is presumed to indicate a decreasedmass in this area of the wall. This structure was especiallynoticeable in cells processed in Os04-UA or acrolein-UA.Visualization of the cytoplasmic membrane was, however,difficult in all cases, even when sections were poststainedprior to viewing. Ribosomes were clearly visible in all

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CHEMICAL FIXATIVES FOR FREEZE-SUBSTITUTION 2153

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SUBSTITUTION ACETONE INFILTRATION SUBSTITUTION ACETONE INFILTRATIONFIG. 1. Percent 3H and 14C cpm extracted from bacterial cells during substitution, acetone washing, and infiltration stages of

freeze-substitution carried out in the presence of different chemical fixatives. Isotopes used: DPM, diaminopimelic acid; URA, uracil; THY,thymidine; GAL, galactose; GLN, N-acetyl-D-glucosamine. Fixatives used: solid bars, Os04-UA; left-to-right hatched bars, glutaraldehyde-UA; cross-hatched bars, acrolein-UA; stippled bars, gallic acid; right-to-left hatched bars, UA; open bars, acetone.

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2154 GRAHAM AND BEVERIDGE

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EM,t ,f- ;'7A ,=:V"FIG. 2. Thin sections of E. coli SFKll prepared by freeze-substitution in different chemical fixatives. (A) Os04-UA; (B) glutaraldehyde-

UA; (C) acrolein-UA; (D) gallic acid; (E) UA; (F) acetone. Bars, 100 nm.

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CHEMICAL FIXATIVES FOR FREEZE-SUBSTITUTION 2155

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PL.S:4¼fWis? -r W 4~*P... -.iFIG. 3. Thin sections of B. subtilis 168 prepared by freeze-substitution in different chemical fixatives. See Fig. 2 legend for details. Bars,

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FIG. 4-6. Thin sections and optical densitometry.FIG. 4. Thin section of E. coli SFK11 prepared by freeze-substitution in Os04-UA. Bar, 100 nm. The corresponding optical densitometry

scan was taken perpendicular to the plane of the cell envelope (arrow in micrograph) with a negative printed at the same magnification as thismicrograph and a path width of 40 p.m. OM, Outer leaflet of the outer membrane; G, periplasmic gel; CM, cytoplasmic membrane; C,cytoplasm.

FIG. 5. Thin section of E. coli SFK11 prepared by freeze-substitution in acrolein-UA and a scan of its cell envelope. Bar, 100 nm. OM,Outer leaflet of outer membrane; PG, collapsed peptidoglycan; C, cytoplasm; CM, cytoplasmic membrane.

FIG. 6. Thin section of B. subtilis 168 prepared by freeze-substitution in Os04-UA and a scan of its cell wall. Bar, 100 nm. C, Cytoplasm;CM, cytoplasmic membrane; OW, cell wall. OW arrow in the densitometry scan indicates the extent of the cell wall on the bacterium on theleft side of the micrograph.

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CHEMICAL FIXATIVES FOR FREEZE-SUBSTITUTION 2157

preparations, although the appearance of the cytoplasmvaried from very fine granular material (Fig. 3A, Os04-UA-treated cells) to dense fibrillar material (Fig. 3B, E, and F).Optical densitometry of B. subtilis walls prepared in OS04-UA (Fig. 6) indicated a region, possibly an inner wall, withinthe fibrous matrix. Although all densitometry tracing pat-terns were similar, cells processed in substitution mediaother than Os04-UA showed walls in which peak height wasdiminished and in which scans were shortened along the xaxis (data not shown).

Semiquantitative analysis of unstained B. subtilis cellwalls by EDS revealed that phosphate (indicative of cell wallteichoic acid) levels were greater in cells processed in gallicacid and UA alone and lowest in cells processed in glutaral-dehyde, acrolein, and acetone. Corresponding measure-ments of cells processed in Os04-UA could not be obtainedbecause phosphorus and osmium have overlapping X-raylines (Os, Ma = 1.910 keV; P, Ka = 2.015 keV).

Together, the data obtained from biochemical leachingexperiments, cell envelope profiles determined by opticaldensitometry, EDS, and visual observation suggest thatfreeze-substitution in osmium tetroxide-UA yields superiorresults.

DISCUSSION

Over the last decade, freeze-substitution has been usedmost frequently for the examination of eucaryotic systems(10, 12, 13); it has rarely been used on procaryotic cells. Forthis reason, it has been difficult to ascertain the power andscope of freeze-substitution as a technique for the preserva-tion of microbial systems. Clearly, the smallness and sim-plicity of bacterial cells make them easier model systems toinvestigate, but this smallness creates difficulties in physicalmanipulation during processing. Previously, Weibull et al.(29) investigated the extraction of radiolabeled lipids duringfreeze-substitution of Acholeplasma laidlawii cells, a wall-less bacterium. Although mycoplasma cells may representthe simplest procaryotic system, they are not representativeeubacterial organisms. We believe that E. coli and B. sub-tilis, representative of gram-negative and gram-positive eu-bacteria, respectively, are more suitable organisms, andbecause their gross morphology and ultrastructure are wellknown (20), they make ideal systems for systematic study.In an accompanying manuscript (14), we used autoradiogra-phy to show that loss of radiolabel from E. coli and B.subtilis cells during freeze-substitution in Os04-UA wasinduced by ice crystal formation and consequent cell damageduring the initial freezing step. In this study we show that inaddition to rapid freezing, specific substitution medium isrequired for adequate label retention and cell preservation.

Since the percentage of leached nucleic acid label gener-ally reflected the percentage of wall label detected, thefreeze-substitution method used did not selectively extractwall or cytoplasmic components. Of the fixatives examined,the combination of Os04-UA or 2% gallic acid appeared tobe the best preservative agents, as evidenced by the lowestoverall levels of radiolabel detected during processing. Sincethe methods employed provided efficient radiolabeling ofspecific wall components, detection of low levels of walllabel in processing fluids of cells prepared in Os04-UA orgallic acid suggests that the cells remained intact throughoutprocessing. Similarly, low levels of nucleic acid labels indi-cated that cell walls were not breached during processingand that cytoplasmic integrity was retained in these cells.

Retention of label by cells fixed in Os04-UA was not

surprising, since OS04 is known to react with thiol andionizable amino groups as well as with unsaturated lipids toform stable diesters resistant to extraction in organic sol-vents (19). Superior fixation by osmium tetroxide in thisstudy was probably enhanced by the addition of UA, whichexhibits an affinity for the phosphate groups on phospholip-ids (8) and is able to stabilize membranes at low tempera-tures in the presence of acetone (7).

Extensive ultrastructural detail was revealed in bacteriaprocessed in Os04-UA. Each of the multilayered cell wallcomponents observed by Amako et al. (2), Graham andBeveridge (14; Abstr. Annu. Meet. Am. Soc. Microbiol.1988, J4, p. 205), and Graham (Proc. Microsc. Soc. Can.16:18-19, 1989) was present in E. coli and was readilydistinguishable in the optical density tracings. The mostdistinguishing characteristic of B. subtilis was the extensivecell wall, visualized as a dense fibrous matrix extending up to30 nm outwards from the cell membrane. Fibrous cell wallshave been reported for Lactobacillus casei (23) and L.acidophilus (with our system) (11), S. aureus (27), and B.subtilis W23 (4). Although Hobot et al. (18) observed ribo-some-free spaces and fibrillar material in the cytoplasm of E.coli B cells slam-frozen in liquid helium and freeze-substi-tuted in OS04 alone, a dense, granular cytoplasm wasconsistently observed for all cells processed in Os04-UA inthis study.

Biochemical leaching experiments suggested that amongthe other fixatives tested, only gallic acid provided sufficientlevels of chemical stabilization. Tannic acid was initiallyassessed as a potential fixative in this work, but the poorimages observed by electron microscopy suggested the useof an alternative agent. Gallic acid, a small subunit (Mr170.12) of the larger tannic acid molecule (Mr 1695.4), wasselected as the best candidate. Gallic acid is known to act as

a mordant (8), and its presence in the substitution fluidappears to have effectively blocked extraction of cellularcomponents during later stages of processing. Both OS04-UA- and gallic acid-treated cells lost most of their labeledmaterial during the first change of acetone following substi-tution, consistent with the concept that a defined populationof cells were freeze-damaged by our plunging system (14).Cells stressed by ice crystal damage during the initial freez-ing step would have their first opportunity to lose cellularcomponents at this time, while that material adequatelystabilized by Os04-UA or gallic acid would be retainedthroughout processing. However, electron microscopy re-

vealed that gallic acid-substituted cells lacked most of theultrastructural detail observed in Os04-UA-treated samples;a peptidoglycan layer was seen rather than the periplasmicgel of Os04-UA-substituted cells. Even though chemicalmordants facilitate binding of heavy metal stains to biologi-cal structures, thereby enhancing contrast (16), the cytoplas-mic membrane was difficult to observe despite post-staining.However, the asymmetrically staining outer membrane was

visible. Loss of ultrastructure, particularly in the cell wall,was also apparent in gallic acid-treated B. subtilis. Insuffi-cient detail and poor preservation are consistent with previ-ous findings for eucaryotic samples treated with this com-

pound (8). We suggest that gallic acid is the fixative of choicefor EDS microanalysis of freeze-substituted eubacteria, as

this organic compound has no overlapping spectral lines(osmium overlaps the phosphorus signal). Thus, when cellsmust be chemically stabilized prior to analysis, gallic acid isa suitable fixative.The remaining chemicals were poor fixatives, as deter-

mined by detection of high levels of radiolabel throughout

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2158 GRAHAM AND BEVERIDGE J. BACTERIOL.

the freeze-substitution process. Although glutaraldehyde, adialdehyde, is able to react with free amino groups ofproteins, phospholipids, and nucleic acids to form covalentcross-links, it is least effective at temperatures below 4°C (7).For this reason, little material would be chemically fixedduring the substitution step. Considerable rearrangementand loss of cellular components would probably occur assamples were warmed to room temperature and duringsubsequent processing. This is consistent with the detectionof high cpm in the substitution medium and during theremaining stages of processing.

Ineffective fixation in the presence of acrolein was anunexpected finding, since this monoaldehyde is known topenetrate and react faster than other aldehydes and is one ofthe few fixatives known to be reactive at the temperaturesused for freeze-substitution (7). When eucaryotic organismsare prepared, it is not uncommon to use 5 to 10% acrolein(22), and often these levels are used in combination withadditional fixatives (15). Our lower acrolein concentration isfrequently used in more conventional regimens to fix bacte-ria (9), and the total amount of acrolein within our systemshould be sufficient to saturate all available chemical siteswithin the limited number of cells undergoing rapid freezingand subsequent processing.Leaching of peptidoglycan and LPS from E. coli cells

occurred at distinctly different stages when cells were proc-essed in acrolein. Acetone is known to cause leaching ofcellular lipids during conventional embedding procedures(28), and it is possible that insufficient cross-linking of LPS atlow substitution temperatures was responsible for loss ofthis cell surface label during washing, whereas loss ofpeptidoglycan during infiltration may have been enhanced bythe viscosity of the resin and the recent permeability changeto the outer membrane (i.e., LPS removal).UA alone was a poor fixative, as evidenced by the high

levels of label detected during processing of both gram-positive and gram-negative cells. By its very nature, theuranyl ion and its various polymeric forms must bond to thestructural components of cells through charge interaction.These interactions were not sufficient to prevent loss ofcytoplasmic material and wall polymers.That little material was detected in the substitution me-

dium of acetone-treated cells was surprising. Indeed, itattests to the relative strength and impermeability of eubac-terial cell walls. The major differences between the two wallsin this study was that the B. subtilis wall resisted collapseduring processing and retained a semblance of cellularshape, whereas that of E. coli was deformed but notbreached (i.e., no radiolabel escaped). It has been suggestedthat the addition of fixatives to substitution fluids may in factincrease membrane permeability, thereby distorting andrearranging cellular constituents (15). If this is the case,leaching should be decreased in the absence of chemicalfixatives.

E. coli freeze-substituted in glutaraldehyde-UA, acrolein-UA, or UA or acetone alone revealed less ultrastructuraldetail than cells processed in Os04-UA. Despite their poten-tial to stabilize specific biological components, glutaralde-hyde and acrolein are organic in nature and impart noelectron density; structures are poorly visible before post-staining. Although the presence of UA imparted contrast tocell membranes, considerable loss of detail relative to OS04-UA treatment was apparent.

B. subtilis cells processed in acrolein and UA retained thefibrous cell wall despite losses of 82.9 and 73.9%, respec-tively, of the peptidoglycan label. Optical densitometry

scans revealed these walls to be of a much lower density(i.e., diminished peak height) and to be shortened along thex axis, indicating loss of substance and wall collapse. EDSalso showed diminished amounts of phosphorus (presumablyas teichoic acid) in the cell walls. This information suggeststhat wall polymers were released and partial collapse oc-curred, yet the basic fibrous format of the structure wasretained.

Unlike eucaryotic cells and their tissues, bacteria areextremely small life forms which have relatively simpledesign features (6). This smallness has made accurate struc-tural evaluation difficult, since light microscopy of livingbacteria can only provide information about basic shape,form, and internal organization. Our most powerful tool forstructural determination is the electron microscope with itsattendant pitfalls. Obviously, conventional embedding pro-tocols create artifacts, both recognized and undiscovered,and cryoelectron microscopy requires systems that few canafford or have the expertise to operate. An attractive alter-native, relatively new to microbiology, is freeze-substitu-tion. It offers the advantages of cryopreservation and chem-ical fixation and requires relatively simple protocols. Ourstudy represents the first attempt to correlate morphologicalappearance with chemical and structural integrity in bacte-rial cells which have been processed by freeze-substitutionwith different chemical fixatives. Although the processingprotocol must ultimately reflect the specimen's form and thedesired information, our data for both E. coli and B. subtilissuggest the use of Os04-UA in anhydrous acetone as amedium for routine freeze-substitution of eubacteria. How-ever, when microanalytical studies are required in whichinformation may be lost by fixation with osmium, gallic acidis the chemical of choice.

ACKNOWLEDGMENTS

We thank Bob Harris, Veterinary Microbiology and Immunology,University of Guelph, and Eric Bullock, Department of Chemistry,Memorial University, for their helpful input, suggestions, and stim-ulating discussion.

This research was entirely supported by a Medical ResearchCouncil of Canada operating grant to T.J.B. The microscopy wasperformed in the NSERC Guelph Regional STEM Facility, which ismaintained by funds from the Department of Microbiology, theCollege of Biological Sciences, and the Natural Sciences andEngineering Research Council of Canada.

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