JOURNAL OF BACTERIOLOGY, Feb. 1973, p. 953-962 Vol. 113, No. 2 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Ultrastructure of the Cell Wall and Cytoplasmic Membrane of Gram-Negative Bacteria with Different Fixation Techniques M. T. SILVA' AND J. C. F. SOUSA Electron Microscopy Center, I. A. C. Biochemistry Center and Department ofMicrobiology of the Faculty of Pharmacy, University of Porto, Portugal Received for publication 26 October 1972 The ultrastructure of the cytoplasmic membrane and cell wall of two strains of Escherichia coli, Proteus morganii, P. vulgaris, Acinetobacter anitratum, Moraxella lacunata, Erwinia amylovora, Acinetobacter sp., and of a plant pathogen, unclassified gram-negative, fixed by the Ryter-Kellenberger proce- dure, was found to be significantly affected by the use or omission of the uranyl postfixation included in that procedure, and by the presence or absence of calcium in the OsO fixative. The omission of the uranyl treatment results in a less clear profile of both the outer membrane of the cell wall and of the cytoplasmic membrane. The observation of these two membranes is further limited when both uranyl and calcium are omitted. The R-layer and the material covering the surface of the cell wall appear more distinct when the uranyl postfixation is not used. Evidence is given suggesting that the influence of uranyl and calcium ions on the appearance of the outer and cytoplasmic membranes would be primarily due to their action as fixatives, whereas the influence of uranyl on the appearance of the R-layer would be due to a direct action on the peptidoglycan component of this layer. When uranyl acetate is used as a section stain after the embedding in plastic, it improves the observation of the R-layer. In this case, a well contrasted R-layer is consistently observed in all strains studied, provided that the postfixation has been omitted. The frequent difficulty in clearly observing the R-layer in many published micrographs probably results from the common use of uranyl postfixation. Previous studies (3, 18, 20, and M. T. Silva, MATERIALS AND METHODS Proc. 6th Int. Cong. Electron Microscopy, Kyo- Microorganisms. The following strains were used: to, 1966, p. 275-276) using electron microscopy Escherichia coli (NCTC 8196); E. coli (enteropatho- of thin sections have shown that the ultrastruc- genic O,,,B4, clinical isolate); Proteus morganii ture of the membranous components of several (strain K from the collection of the Department of gram-positive bacteria is dependent on the Bacteriology and Parasitology, Faculty of Medicine, conditions of chemical fixation. In the present University of Porto, Portugal); P. vulgaris (CCM paper, additional observations are described 1956); Acinetobacter anitratum (NCTC 8102); Mo- regarding the influence of calcium and uranyl raxella lacunata (NCTC 7784); Erwinia amylovora (strain 595, National Collection of Plant Pathogenic ions, as used in the Ryter-Kellenberger (R-K) bacteria, Harpeden, Herts, England); Acinetobacter technique (15), on the ultrastructure of the cell sp. isolated and kindly supplied by Dinah Abram. wall and cytoplasmic membrane of several University of Pittsburgh, and previously considered gram-negative microorganisms. Part of this as Herellea vaginicola (J. M. Klemencic and D. work was previously presented in abstract (J. Abram, Bacterial Proc., p. 43, 1971); and an unclassi- C. F. Sousa and M. T. Silva, Proc. 6th Annu. fied gram-negative isolated from the roots of a Meet. Portug. Soc. Electron Microscopy, Triticale, (Triticum vulgare Linnaeus), currently Abstr. 13, 1971). under study and hereafter referred to as strain 2. Culturing conditions. E. amylovora was grown in 'Permanent address: Department of Bacteriology and nutrient-yeast extract-glucose broth (23). Acineto- Parasitology, Faculty of Medicine, University of Porto, bacter sp. was grown in Trypticase (BBL, 0.2%)- Porto, Portugal. yeast extract (BBL, 0.04%), plus Ca(NO)2 (0.013%); 953 954 SILVA AND SOUSA J. BACTERIOL. pH 6.8. All other strains were grown in tryptone broth prefixation step of the R-K technique is omitted. All (1% tryptone [Difco], 0.5% NaCl; pH 7.2). All cul- the micrographs presented in this work, regardless of tures were carried out at optimal temperatures with the use or omission of the uranyl acetate postfixation aeration by shaking, until the middle of the exponen- or of the presence or absence of calcium in the OSO4 tial phase was reached. fixative, are of bacteria prepared by the R-K proce- Production of ethylenediaminetetraacetic dure without prefixation. acid-lysozyme spheroplasts. E. coli NCTC 8196, To determine the thickness of the envelope layers, grown as described above, was treated with lysozyme a Bausch & Lomb Measuring Magnifier was used on (muramidase, EC 3.2. 1.17; Sigma Chemical Co., egg prints enlarged to 200,000 x. The electron micro- white, 3x crystallized, specific activity 18,000 units/ scopes were periodically calibrated with a grating mg) by a modification of the method of Repaske (13): replica grid (Ladd Research Industries, Inc.). a pellet of centrifuged cells was washed once with The terminology which has been used in the 0.01 M tris(hydroxymethyl)aminomethane (Tris)- literature concerning the layers of the cell wall of hydrochloride buffer (pH 8.0) and suspended in the gram-negative bacteria is not uniform. For the sake same buffer (0.3 M) supplemented with sucrose (0.5 of clarity, in the present paper the following terms M), ethylenediaminetetraacetatic acid (EDTA) (1.6 will be used: (i) outer membrane (indicated in the mg/ml), and lysozyme (1 mg/ml). After 20 to 30 min figures by an 0), for the double-track component; (ii) at room temperature (24 C), the resulting sphero- R-layer (indicated in the figures by an R) for the plasts were fixed as described below. Cells of the single dense component located between the outer same culture treated as above, except that EDTA or membrane of the cell wall and the cytoplasmic lysozyme were omitted from the buffer, served as membrane. control. Electron microscopy. Intact bacteria were fixed RESULTS at room temperature (24 C) by the R-K procedure The profile of the cytoplasmic membrane of (15) usually without the prefixation step, as de- all strains studied is clearest when both cal- scribed elsewhere (18). In some experiments with E. cium and uranyl ions are used in the fixation coli 8196 and A. anitratum, calcium was omitted procedure (Fig. la and b, 6, 9a and b, 11). from the R-K OSO4 fixative. After brief washing in When the postfixation with uranyl is omitted R-K buffer, the fixed specimens were either directly dehydrated or postfixed with uranyl acetate (E. or substituted by the treatment with acidic Merck AG, Darmstadt, Germany) (15, 20) using a buffer, bacteria fixed by the R-K OsO with 0.5% solution either in R-K buffer (final pH 5.1) or in calcium usually exhibit a less defined profile water (final pH 3.9) for 30 to 120 min at room of the cytoplasmic membrane (Fig. 2, 7, 10, temperature (24 C). In experiments with E. coli 8196, 12). A poor definition of the membrane profile the uranyl postfixation was also carried out for 24 hr. is also observed in cells fixed by the R-K OsO0 Samples of the same specimens were treated under without calcium followed by the uranyl post- the same conditions with 0.2 M aceto-acetate buffer fixation (Fig. 3). When the uranyl postfixation (pH 3.9) to serve as a control for the standard uranyl is omitted in preparations fixed under condi- treatment. To test the action of uranyl acetate after the dehydration step, the standard uranyl treatment tions of calcium deficiency, the cytoplasmic was applied to samples of E. coli 8196 and strain 2, membrane is usually unobservable in the dehydrated to 100% ethanol, and then rehydrated strains studied (E. coli 8196 and A. anitratum, back to water, as previously described (20). Fig. 4 and 8). For the fixation of the spheroplasts, glutaralde- An outer cell wall membrane (Fig. la and b, hyde (TAAB Laboratories, Reading, England; stock 6, 9a and b, 11) with a very distinct profile is 25% solution) was added to the suspension of E. coli visible in all bacteria studied when the R-K 8196 in sucrose-EDTA-Tris-lysozyme to a final con- OsO fixative contains calcium and the postfix- centration of 1.0%. After 1 hr at room temperature ation with acetate is used. In all the (24 C), the cells were collected by centrifugation uranyl (2,000 x g, 10 min) and the pellets were fixed in R-K strains studied, mainly in E. coli 8196, a OSO4 fixative without any intermediate washing. The slightly asymmetric profile (the outer layer postfixation in uranyl acetate, when used, was car- being denser than the inner layer) is sometimes ried out as described above. apparent (Fig. la). This is less frequent in The fixed specimens were processed for electron samples postfixed with uranyl acetate for 24 hr. microscopy as described previously (20). The sections In the space between the outer membrane and were contrasted with uranyl acetate (see legends to the cytoplasmic membrane, a third layer is Fig.) and lead citrate (22) and, in some experiments, occasionally visible. This layer, due to its with thallium sulfate (10). For observation, an AEI EM 6 G electron microscope and a Siemens Elmiskop location in the cell wall (6) as well as its I A electron microscope were used at 80 kv, with sensitivity to lysozyme in the presence of ED- double condenser, 50 um objective apertures, and TA (10) (see below), corresponds to the R-lay- anticontamination devices. er. Frequently it has a loose structure and Preliminary observations with E.