APPLIED MICROBIOLOGY, Sept. 1972, p. 477-487Copyright 0 1972 American Society for Microbiology

Vol. 24, No. 3Printed in U.S.A.

Improved Staining of Extracellular Polymer forElectron Microscopy: Examination ofAzotobacter, Zoogloea, Leuconostoc,

and BacillusG. D. CAGLE,1 R. M. PFISTER, AND G. R. VELA

Department of Microbiology, Ohio State University, Columbus, Ohio 43210, and Department of BiologicalSciences: Microbiology, North Texas State University, Denton, Texas 76203

Received for publication 3 April 1972

Phase contrast, ultraviolet microscopy, and freeze-etching were used to de-termine the amount of exocellular polymer surrounding unfixed cells of fourgenera of bacteria: Azotobacter vinelandii, Zoogloea ramigera, Leuconostoc mes-

enteroides, and an acid-tolerant, floc-forming Bacillus species. Thin-sectionalelectron microscopy was employed to measure the effectiveness of a modifiedruthenium red staining method. The results obtained with this modification ofruthenium red staining technique were compared to results obtained when pre-

viously proposed ruthenium red methods of fixation were employed. The re-

sults of these relations were then compared to the amounts of exocellular mate-rial as determined with phase-contrast microscopy, ultraviolet microscopy, andfreeze-etching. The data obtained indicate that improved fixation of exocellularpolymer is achieved when cells are pretreated with ruthenium red as describedherein. In addition, the modified methods also reveal cytological detail notapparent when other methods of ruthenium fixation are employed.

Early investigations of extemal layers ofmicroorganisms revealed that certain compo-nents (e.g., slime, capsules, walls) could bedemonstrated with the light microscope. Elec-tron microscopy visualization of external bac-terial polysaccharide has been achieved withthe employment of negative staining (5) orshadow-casting. However, capsular materialhas been observed to crack, whereas slimelayers sometimes absorb stains, decreasing theefficiency of negative staining. Althoughshadow-casting allows visualization of nonso-luble extracellular polymer, internal cellulardetail is not apparent. Attempts to adequatelyfix this material for ultrastructural study havenot been very successful. Pate and Ordal (8)have achieved improved staining of Chondro-coccus columnaris when ruthenium red wasemployed during prefixation with glutaralde-hyde. Other workers have applied theirmethods to better visualize extracellular pol-ymer in animal (11), plant (10), and microbial(1) thin sections. More recently, freeze-etching

1 Present address: Tarrant County Junior College Dis-trict, Northeast Campus, Hurot, Texas 76053.

47'

techniques have been employed to study thestructure and distribution of extracellular pol-ysaccharide, as well as internal cellular detail(7).This report describes a modification of the

Luft ruthenium red staining technique, as re-ported by Pate and Ordal (8). The results ofthis study demonstrate improved stainingqualities of extracellular polysaccharide, aswell as improved internal cytological detail.The results obtained when these methods areused are compared to those obtained whenLuft's methods are employed, as well as acomparison of these methods to light micros-copy and frozen-etched preparations.

MATERIALS AND METHODSThe four organisms used in this study are Azoto-

bacter vinelandii ATCC 12837, Zoogloea ramigera1-115 (The Ohio State University, Culture Collection),Leuconostoc mesenteroides (OSU 553, The OhioState University, Culture Collection), and an acid-tolerant, slime-forming Bacillus sp., previously char-acterized (4).

A. vinelandii ATCC 12837 was grown at 26 C inBurk's nitrogen-free salts (13) supplemented with 1%

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478 CAGLE, PFISTER, AND VELA

glucose. Vegetative forms were induced to encyst bytransferring them to the same basal medium supple-mented with 0.3% n-butanol and 2% agar. Z. rami-gera 1-115 was cultured in the following modificationof the arginine medium of Crabtree et al. (2): argi-nine-hydrochloride, 0.5 g; alanine, 1.0 g; MgSO4.7H20, 0.2 g; K2HPO4, 2.0 g; KH2PO4, 1.0 g; glucose,5.0 g; vitamin B12, 1.5 x 10-6 g; and distilled waterto 1 liter. Cultures of Z. ramigera were grown for 18to 24 hr at 26 C. L. mesenteroides was cultured inthe medium of deMan et al. (3), with sucrose re-placing glucose as the carbohydrate source, and wasincubated for 18 hr at 25 C. The previously described(4) acid-tolerant, slime-producing Bacillus sp. wasgrown in the following modification of the mediumof Thome et al. (12): glycerol, 80 g; citric acid, 12 g;glutamic acid, 20 g; NH4Cl, 7 g; MgSO4-7H20, 0.5g; FeCl 36H2O, 0.04 g; K2HPO4, 0.5 g; CaCl3, 0.15g; MnSO4 H2O, 0.42 g; to 1 liter with distilled wa-ter, pH adjusted to 3.0. Cultures of this organismwere grown for 24 hr at 24 C, at which time largeflocs of the bacillus had formed.Phase-contrast and ultraviolet microscopy.

Aqueous solutions (0.1%) of Violet R monastral, Pon-tamine, and Paper White BP (diamine stilbene di-sulfonic acid dyes possessing high affinities for cellu-lose; E. I. DuPont de Nemours & Co., Inc., Chicago,Ill.) were used to stain cultures. Wet mounts of thecells, stained with fluorescent dye, were examinedunder phase-contrast and ultraviolet illuminationwith a Zeiss Universal microscope equipped forphase-fluorescence epi-illumination with ultravioletlight. Photomicrographs were taken on Kodak Tri-Xfilm using a Nikon AFM microflex sensor.

Freeze-etching. Cells of each of the four orga-nisms were prepared for freeze-etching by centri-fuging into a pellet. The bacteria were placed on acopper disc (3 mm) that had been scratched to aid inadherence of the cells, quickly frozen in Freon 22and rapidly transferred to liquid nitrogen. Thecopper disc was removed from the liquid nitrogen,placed on a precooled stage (-100 C) in a "Balzers"

APPL. MICROBIOL.

apparatus (model 360 M), and frozen-etched (9). Fol-lowing etching, the copper disc was removed anddipped in distilled water until the replica floatedfree. The replica was then transferred to 70% H2SO4and treated for a period of 1 hr, rinsed in distilledwater, treated for another hour with Eau de Javell(9), and finally rinsed several times with distilledwater. The replicas were then collected on an un-coated 400-mesh copper grid, allowed to dry, andexamined with the electron microscope.Thin sections. The methods of pretreatment, fix-

ation, and staining are described below. All cellswere dehydrated by passage through a graded al-cohol series, followed by two rinses in propyleneoxide. The bacteria were then embedded in Epon812, and the resin was polymerized for 24 to 48 hr at60 C. Thin sections were obtained on a Reichert Om-12 ultramicrotome, stained with uranyl acetate andthen with lead citrate for 7 to 10 min. Thin sectionswere collected on 300-mesh copper grids and exam-ined with a Phillips EM-300 electron microscope atan 80-kv accelerating voltage:-Method 1. The method employed when cells were

prepared for electron microscopy was similar to thatof Pate and Ordal (8). Cells were removed from thegrowth medium by centrifugation and were sus-pended in a solution containing 3.6% glutaralde-hyde-0.15% ruthenium red (w/v) in 0.1 M cacodylatebuffer for 1 hr at ambient temperature. The cellswere washed twice with buffer and resuspended foran additional hour in a solution of 1% Os04-0.15%ruthenium red in 0.1 M cacodylate at 4 C. Followingfixation, the bacteria were washed twice with buffer,dehydrated, and embedded as described above.Method 2. Cells were collected from the growth

medium by centrifugation and were suspended in a0.15% solution (w/v) of ruthenium red for periodsvarying from 2 min to 1 hr prior to treatment withglutaraldehyde. The bacteria were centrifuged andtreated as described in Method 1, except that allwashings were performed with 0.15% ruthenium redin 0.1 M cacodylate buffer.

FIG. 1. Phase-contrast photomicrograph of A. vinelandii during the vegetative phase of growth.Marker=10gm. x2,200.

FIG. 2. Same field as shown in Fig. 1, except that ultraviolet illumination has been employed. The fluores-cent dye (Violet R monastral) employed acts as a negative stain, illustrating the extent of the exocellular pol-ymer. Marker= 10 ,um. x2,200.

FIG. 3. Frozen-etched preparation of A. vinelandii showing polymer (P) distribution around the cells (C).One cell has been cleaved through the center to reveal several granules of poly-,8-hydroxybutyric acid (PHB).Marker= 1 um. x25,000.

FIG. 4. Thin sections of A. vinelandii fixed according to the methods of Pate and Ordal. Polymer (P) isdistributed around the cells (C). Marker= 1gm. x17,000.

FIG. 5. Thin section of A. vinelandii as shown in Fig. 4 except that these cells have been pretreated withruthenium red. Polymer (P) surrounding the cells (C) is much more evident than that in Fig. 4. Marker= Igim. x13,400.

FIG. 6. Higher magnification of cells treated as in Fig. 5. The appearance of the cell wall-cell membrane(CW-CM) is much clearer and more defined than in Fig. 7. Polymer distribution (P) is also more distinct.Several membrane-bound vesicles (V) and membranous invaginations (MI) can be seen at the periphery ofone cell. Marker= 1 Am. x28,000.

FIG. 7. A portion of the cell wall-cell membrane (CW-CM) from the cells shown in Fig. 4. Polymer (P)surrounding the cells is also apparent. Marker= I gm. x34,000.

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CAGLE, PFISTER, AND VELA

RESULTSIndividual cells and flocs of each of the four

organisms were examined with phase-contrastand ultraviolet microscopy, and with the elec-tron microscope, utilizing frozen-etched prepa-rations and thin-sections prepared by the twopreviously described procedures. Initially, twogram-negative organisms, A. vinelandii and Z.ramigera, both observed to produce largeamounts of extracellular polymer, were chosenfor this study.When A. vinelandii was examined with

phase-contrast microscopy, after staining withViolet R monastral, a fluorescent dye, onlyfaintly defined areas of exocellular materialwere observed surrounding the individual cells(Fig. 1). When these same cells were observedwith ultraviolet illumination, the extent of theexocellular material became more evident, asthe entire background fluoresced, whereas ex-tracellular polymer or cells did not (Fig. 2).Aggregates of A. vinelandii also fluorescedwhen stained with other fluorescent dyes usedin this study. The examination of frozen-etched replicas of A. vinelandii (Fig. 3) indi-cated that exocellular polymer (P) is present inlarge quantities around cells of the organism.We had previously employed ruthenium red,after Pate and Ordal (8), to stain polysaccha-ride surrounding the cells of Azotobacter (1)although the results (Fig. 4) obtained did notindicate the extent of polymer distribution asshown with fluorescent microscopy (Fig. 2) orwith frozen-etched replicas (Fig. 3). The ad-vantage of preincubation in ruthenium redprior to treatment with glutaraldehyde andruthenium red is apparent in Fig. 5, which il-lustrates the relation and distribution of the

cells of A. vinelandii to the extracellular pol-ymer. Cells of A. vinelandii fixed after Pateand Ordal (8) (Fig. 4) show the fibrous capsulepolysaccharide (P), which extends beyond thecells, gradually decreasing in amount. On theother hand, when the same cells are preparedwith pretreatment with ruthenium red, thebacteria appear embedded in a polysaccharidematrix (Fig. 5). Preincubations of as little as 3min were observed to improve staining quali-ties in the bacteria studied. Periods longerthan 30 min did not appear to improvestaining qualities of exocellular polymer, orcytological detail. The material which sur-rounds the cells appears of the same fibrousconsistency as when fixed with the previouslydescribed methods (8), although the quantityof polymer is evidently greater. The extracel-lular polymer appears to surround all of thecells, and, without exception, cells of A. vine-lkndii were not observed outside this matrix.Improved cytological staining was also ob-served in the cells preincubated in rutheniumred prior to prefixation with glutaraldehyde-ruthenium red, particularly in the region of theouter envelope of the organism (Fig. 6). In Fig.6, the cell wall-cell membrane of bacteria pre-treated with ruthenium red appears muchmore defined than those prepared by Luft'smethod as reported by Pate and Ordal (8) (Fig.7). In addition to the increased resolution af-forded by ruthenium red pretreatment, theamount of intracellular membrane is also ob-served to increase. Numerous membrane-bound vesicles, membranous invaginationsaround the periphery of the cell, are apparent(Fig. 6).

Z. ramigera, a pseudomonad, was also exam-

FIG. 8. Phase-contrast photomicrograph of Z. ramigera stained with Pontamine and illuminated with vis-ible light. Area of polymer is evident around cells. Marker= 10 sm. x2,100.

FIG. 9. Phase-contrast photomicrograph of the same cells shown in Fig. 8, except illumination is by ultra-violet light. Capsular areas are much more evident and extend much further around cells. Marker= 10 Mm.x2,100.

FIG. 10. Low-magnification electron micrograph of Z. ramigera fixed with the glutaraldehyde-ruthenium(Method 1). A nearly homogeneous slime layer (S) appears between cells (C). Faintly visible (arrows) is acapsular area, while inside the cells deposits of poly-,3-hydroxybutyric acid (PHB) and polyphosphate (PP)are visible. Marker= 1 gm. x 12,600.

FIG. 11. Cells are from the same culture as those in Fig. 10, except fixed with ruthenium red pretreatment(Method 2). Capsular areas (Ca) surround individual cells or cell groups, with slime (S) interconnecting cellgroups. Intracellular deposits of PHB are also apparent. Marker= 1 /m. x 12,500.

FIG. 12. Electron micrograph of a single cell of Z. ramigera fixed according to Pate and Ordal (Method 1).Polysaccharide (P) surrounding the cell (C) appears indistinct, whereas the cell wall-cell membrane complex(CW-CM) appears very electron dense. Within the cell, PHB and a structure which appears to be a polyphos-phate (PP) deposit are clear. Marker=1 gm. x29,000.

FIG. 13. Cell similar to that observed in Fig. 12, except treated, prior to treatment with glutaraldehyde,with ruthenium red (Method 2). The fine structure of the exocellular polymer (P), the cell wall-cell mem-brane (CW-CM), and a structure analogous to the polyphosphate deposit in Fig. 12, now apparent as a meso-some (M), are shown. Marker= 1 jAm. x27,500.

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CAGLE, PFISTER, AND VELA

ined by using the procedures described above.When the organism was observed with phase-contrast microscopy, a distinct exocellularpolymer was visible surrounding the cells (Fig.8). Although this capsular matrix is evidentwith phase-contrast microscopy alone, the ex-tent of this material beyond the cell is shownmuch more clearly in Fig. 9, which illustratesthe same cells stained with the fluorescentdye, Pontamine, and photographed with ultra-violet illumination. When Z. ramigera wasfixed for electron microscopy, as described byPate and Ordal (8), large quantities of exocel-lular polymer were observed (Fig. 10). Faintlyvisible around the bacterium, a poorly definedcapsular layer (arrows) was evident, with slimedispersed between the cell groups. Quantitiesof poly-f,-hydroxybutyric acid and structureswhich appear to be polyphosphate deposits arealso observed in Fig. 10. When cells of thesame culture are prepared for electron micros-copy by the methods described in this study,notable improvement in general fixationquality is achieved. Improved capsular fixationis illustrated in Fig. 11; a discrete capsule sur-rounds each of the cell groups, with slime in-terdispersed only between cell group capsularareas. Improved fixation and staining qualitiesof cytoplasmic structures in Z. ramigera werealso noted, when pretreatment with rutheniumred was employed. Structures previously de-scribed as polyphosphate bodies (Fig. 10), andenlarged in Fig. 12, demonstrate no distinctinternal structure. In contrast, cells pretreatedwith ruthenium red (Fig. 13) appear to containdistinct mesosomes at sites corresponding topolyphosphate deposits in Fig. 12. During ourobservations when ruthenium red was used inconjunction with glutaraldehyde (8), inclusionsin Z. ramigera appeared only as polyphosphatedeposits (Fig. 12), but, when the bacteria weretreated with ruthenium red prior to rutheniumred-glutaraldehyde fixation, the sites corres-ponding to a polyphosphate deposit appeared

as a distinct mesosome (Fig. 13). The resultsobtained with bacterial thin sections preincu-bated with ruthenium red correlate well withdata obtained for polymer distribution as re-vealed by frozen-etched preparations (5).Two gram-positive bacteria, L. mesenter-

oides and B. cereus were also used in thesestudies to test the applicability of thesemethods to other bacterial types. L. mesenter-oides grown in media containing sucrose pro-duces copious quantities of polysaccharide.Phase-contrast microscopy of cellular flocs il-lustrates the nature and distribution of thismaterial (Fig. 14). These same cells (flocs)were observed to fluoresce when stained withthe fluorescent dye, Paper White BP (Fig. 15),and observed with ultraviolet illumination. L.mesenteroides pretreated with ruthenium red,as described in this study, demonstrates amore clearly defined capsular area than inthose bacterial cells fixed with ruthenium red-glutaraldehyde alone (Fig. 16). Although dis-persed capsular polysaccharide can be ob-served in portions of Fig. 16, the amount anddistribution of this material are clearly greaterin Fig. 17, in which the cells were pretreatedwith ruthenium red. In the case of L. mesen-teroides, mesosomes were observed with bothmethods of preparation of cells for electronmicroscopy. General cytological detail is, how-ever, improved. Areas such as the cell wall-cellmembrane, fine structure of mesosomes, andcell cytoplasm are more evident in the cellsdepicted in Fig. 17. When the results obtainedwith ruthenium red pretreatment are com-pared to those obtained with frozen-etchedpreparations, the amount of exocellular pol-ymer demonstrable with each method appearsquantitatively similar (Fig. 18).The acid-tolerant, slime-forming Bacillus

fluoresced differently when stained with thefluorescent dye Pontamine than did the otherbacteria used in this study. In Fig. 19, a phase-contrast photomicrograph of the Bacillus sp.

FIG. 14. Phase contrast of L. mesenteroides stained with Paper White BP and observed with visible light.Large quantities of extracellular polysaccharide are visible in Fig. 15 around chains of cells. Marker= 10 Mm.x2,100.

FIG. 15. Phase contrast of the same field shown in Fig. 14, except illuminated with ultraviolet light. Theamount of polysaccharide surrounding the cells is evident. Marker= 10 Jm. x2,100.

FIG. 16. Electron micrograph of L. mesenteroides fixed with glutaraldehyde-ruthenium red, according toPate and Ordal. Surrounding the chain of cells, a polysaccharide sheath (arrows) is evident, with dispersedpolymer (P) also visible. A mesosome (M) is also visible in these cells. Marker= 1 gm. x25,000.

FIG. 17. Electron micrograph of cells from the same culture as in Fig. 17, treated with ruthenium red priorto fixation. The polymeric sheath (arrows) surrounding the cells is apparent, although much more dispersedpolysaccharide (P) is apparent. A mesosome (M) can be seen inside one of the cells comprising the chain.Marker= 1 lm. x27,000.

FIG. 18. Frozen-etched preparation of L. mesenteroides showing the polysaccharide which encases thecells, as well as dispersed polymer (P). Marker= 1 um. x20,000.

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CAGLE, PFISTER, AND VELA

(4), no exocellular polymer is evident about thecells. However, when examined with ultravi-olet illumination, individual cells fluoresce,indicating a distinct slime coat surrounds eachof the cells (Fig. 20). Frozen-etched prepara-tions (Fig. 21) confirm the presence of thisslime layer around each of the cells, as well asindicating the presence of exocellular fibrils.Thin-sectional studies of the bacterium (Fig.22) support the presence of both exocellularfibrils and a slime coat. Bacillus sp. fixed ac--cording to the methods of Pate and Ordal (8)(Fig. 22) is observed to possess a thick, ruth-enium-red-positive coat surrounding the cells.Several strands are apparent around each ofthe cells in Fig. 22 which appear to have be-come detached from the organism during theprocesses of preparation for electron micros-copy. The employment of ruthenium red as apretreatment prior to suspension of the cells inglutaraldehyde-ruthenium red improves thestaining properties of the bacterial cells. Theexocellular slime coat (Fig. 23) appears similarto that seen in Fig. 22, whereas exocellular fi-brils (Fig. 24) appear much more defined whencells are pretreated with ruthenium red. Thecomposition of the slime coat in this organismappears qualitatively different from those inAzotobacter, Zoogloea, and Leuconostoc. Thesheath surrounding this acid-tolerant Bacillusmay be composed of mucopeptide, rather thana polysaccharide, as in the other three bac-teria. If this is the case, this would partiallyexplain the presence of exocellular fibrils andthe unique type of fluorescence demonstratedby this organism. Internal structure of thisorganism appeared to vary little regardless ofthe manner of fixation involved. Therefore, nostatement can be made concerning the effectof these fixation methods on improved internalstructure.

DISCUSSIONAlthough ruthenium was developed and

implemented for electron microscopy stainingof polysaccharide by Luft (8) approximately 8years ago, no modifications of this stainingmethod have been proposed. To demonstratethat the methods employed in this study do fixextracellular polymer better than those pre-viously proposed, we had to demonstrate theamount of exocellular material present aroundcells used in this study. For this reason, weemployed phase-contrast and ultraviolet mi-croscopy to inspect the amount of materialpresent surrounding the cells at the light mi-croscopy level. In addition, we used freeze-etching procedures, which do not incorporatechemical fixatives, to compare the extent ofpolymer demonstrable at the electron micros-copy level to chemically fixed thin sections.Initially, we observed that, when rutheniumred was preincubated with cells for periods oftime as short as 2 to 3 min, capsular fixationwas improved. Using A. vinelandii as a prelim-inary system, we observed a faint ruthenium-red-positive area around cell aggregates of thebacterium. By increasing the contact time ofruthenium red with the cells prior to treat-ment with glutaraldehyde-ruthenium red, wefound that capsular fixation could be im-proved. Optimal preincubation time in ruth-enium red was determined to be 30 min, withlonger periods of pretreatment not notablyimproving fixation and staining qualities ofexocellular polymer, or internal cytologicaldetail.

In addition to A. vinelandii, we chose to testthe applicability of these methods with an-other gram-negative bacterium, Z. ramigera,and two gram-positive bacteria, L. mesenter-oides and an acid-tolerant variety of Bacillus.In each case, comparative studies of the orga-nisms from the same cultures fixed by themethods of Pate and Ordal (8) and by ourmethods indicated that improved fixation andstaining qualities were achieved with our mod-ification. The most obvious improvement in

FIG. 19. Phase-contrast photomicrograph of B. cereus stained with Pontamine and examined with visiblelight. No exocellular polymer is visible around cells of this organism. Marker= 10 gm. x2,500.

FIG. 20. Phase-contrast photomicrograph with ultraviolet illumination of the same cells shown in Fig. 20.Only the cells in Fig. 20 are observed to fluoresce, indicating a distinct layer surrounds each cell. Marker= 10,gn. x2,500.

FIG. 21. Frozen-etched preparation of B. cereus showing the slime layer (SL) which surrounds the bacillus,as well as several fibrils (F) which radiate from it. The slime layer (L) has been cleaved away to reveal thecell wall (CW) of the organism. 24,000. Marker= 1 gim. x21,000.

FIG. 22. Thin-sectional electron micrograph of B. cereus fixed as described by Pate and Ordal. Detachedfibrils (F) are apparent around a cell which is encased in a ruthenium-red-positive slime layer (SL).Marker=l gm. x21,500.

FIG. 23. Electron micrograph from the same culture as that seen in Fig. 23, except fixed according to themethods described here. A more defined slime layer (SL) surrounds these cells. Marker= 1 gim. x22,500.

FIG. 24. Exocellular fibrils (F) surround the cell. Marker= 1 gim. x25,000.

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CAGLE, PFISTER, AND VELA

capsular fixation and staining was apparent inthe gram-negative organisms. In addition toimproved capsular and cytological detail no-

table in A. vinelandii, improved extracellularpolymer appearance was observed with Z. ra-migera (Fig. 10-13). When the two methods ofpreparation were compared (Fig. 10 versus 11and 12 versus 13), differences were apparent.Improved cytological staining of internal or-

ganelles was most apparent in Z. ramigera, as

evidenced by mesosomal structures (Fig. 12,13). We propose that the presence of polyphos-phate observed in normal thin sections as an

electron-dense granule be reexamined in thoseorganisms in which it has been found. Fromthe data presented it is evident that, at leastin Z. ramigera, adequate cellular fixation dras-tically improved cytological detail. The densegranules which have been referred to as poly-phosphate (5) and which appear similar togranules in other bacteria also labeled as poly-phosphate appear to be a series of concentricmembranous layers that could just as easily becalled a mesosome. We observed many sec-

tions during this study, and we did not findany dense nondefined granules in the Method2 treatment. On the other hand, we found no

well-defined membranous bodies as seen inMethod 2 in the Method 1 samples. Further-more, the location and distribution of thegranules (Method 1) and mesosome-like struc-tures (Method 2) appeared to be the same. Weconcluded that we were looking at the same

structure in either case, but under greater reso-

lution or more suitably stained in our modifi-cation (Method 2). In A. vinelandii, as well, we

observed that the degree of detail discernablealong the cell envelope indicates more mem-

branous activity (Fig. 6 and 7).With the gram-positive organisms, L. mes-

enteroides and the acid-tolerant Bacillus, thecontrasted results of the two staining methodsstudied here are not as apparent as in thegram-negative organisms. Exocellular polymerin both organisms appears in greater quantitieswhen our methods are employed, and this isparticularly noticeable in L. mesenteroides(Fig. 17). Although mesosomes were observedwhen both methods of fixation were employed,we observed a generally improved appearanceof cellular detail (Fig. 17). The Bacillus yieldedresults which revealed the fewest differencesbetween the techniques employed. We pro-posed that this was due primarily to differ-ences in the exocellular material surroundingthe organism, which do not appear to be of a

true polysaccharide nature.The use of ruthenium red, as described by

Pate and Ordal (8), in cell preparation for elec-tron microscopy has provided the bestmethods by which extracellular polysaccharidecould be studied in thin sections. It has beenshown that ruthenium red deposits itself ran-domly in polysaccharides containing two nega-tively charged sites, which possess an inter-charge distance of approximately 0.183 nm.The deposition of ruthenium red was first ob-served in pectins, which contain numerouscharged sites at which ruthenium red is oxi-dized. We suggest that improved fixation ofexocellular material, as well as improved cyto-logical fine structure, is a result afforded byruthenium red in that the materials which dooxodize ruthenium red become fixed prior tointroduction of OsO, into the system. The neteffect of ruthenium red pretreatment is to in-crease the concentration of ruthenium redwhich can react. When ruthenium red is em-ployed with glutaraldehyde, the action of thealdehyde is to partially mask the fixation ofpolysaccharides obtained when ruthenium redis incubated alone with bacterial cells. In addi-tion, polysaccharide fixed with ruthenium redallows a more rapid and complete oxidationwhen OSO4 is added. Apparently these meth-ods are less detrimental to cellular fine struc-ture.

In light of the wide range of applicabilitywhich ruthenium red has had in the visualiza-tion of exocellular polymer, we feel that themethods proposed here will have a like effect,since it appears that these methods not onlyimprove fixation and staining qualities of ex-tracellular polymer, but also improve cytolog-ical detail as well.

LITERATURE CITED

1. Cagle, G. D., and G. R. Vela. 1971. Giant cysts andcysts with multiple central bodies in Azotobactervinekandii. J. Bacteriol. 107:315-319.

2. Crabtree, K., E. McCoy, W. C. Boyle, and G. A. Rob-lich. 1965. Isolation, identification and metabolic roleof the sudanophilic granules of Zoogloea ramigera.Appl. Microbiol. 13:218-226.

3. deMan, J. C., M. Rogosa, and M. E. Sharp. 1960. Asemi-defined medium for the growth of Leuconostocmesenteroides. J. Appl. Microbiol. 23:130-135.

4. Dugan, P. R., C. B. MacMillan, and R. M. Pfister. 1970.Aerobic heterotrophic bacteria indigenous to pH 2.8acid mine water: predominate slime-producing bac-teria in acid streamers. J. Bacteriol. 101:982-988.

5. Friedman, B. A., P. R. Dugan, R. M. Pfister, and C. C.Remsen. 1968. Fine structure and composition of thezoogloeal matrix surrounding Zoogloea ramigera. J.Bacteriol. 96:2144-2153.

6. Hall, C. E. 1955. Electron densitometry of stained virusparticles. J. Biophys. Biochem. Cytol. 1:1-12.

7. Moor, H., and K. Miihlethaler. 1963. Fine structure offrozen-etched yeast cells. J. Cell Biol. 17:609-628.

8. Pate, J. L., and E. J. Ordal. 1967. The fine structure ofChondrococcus columnaris. J. Cell Biol. 35:37-51.

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