Cells and Materials Cells and Materials

Volume 2 Number 1 Article 5

1992

Evaluation of Bacterial Glycocalyx Preservation and Staining by Evaluation of Bacterial Glycocalyx Preservation and Staining by

Ruthenium Red, Ruthenium Red-Lysine and Alcian Blue for Several Ruthenium Red, Ruthenium Red-Lysine and Alcian Blue for Several

Methanotroph and Staphylococcal Species Methanotroph and Staphylococcal Species

Theresa A. Fassel University of Wisconsin-Madison

James E. Van Over Medical College of Wisconsin, Milwaukee

Christopher C. Hauser Medical College of Wisconsin, Milwaukee

Lorie E. Buchholz University of Wisconsin-Milwaukee

Charles E. Edmiston Medical College of Wisconsin, Milwaukee

See next page for additional authors

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Recommended Citation Recommended Citation Fassel, Theresa A.; Van Over, James E.; Hauser, Christopher C.; Buchholz, Lorie E.; Edmiston, Charles E.; Sanger, James R.; and Remsen, Charles C. (1992) "Evaluation of Bacterial Glycocalyx Preservation and Staining by Ruthenium Red, Ruthenium Red-Lysine and Alcian Blue for Several Methanotroph and Staphylococcal Species," Cells and Materials: Vol. 2 : No. 1 , Article 5. Available at: https://digitalcommons.usu.edu/cellsandmaterials/vol2/iss1/5

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Evaluation of Bacterial Glycocalyx Preservation and Staining by Ruthenium Red, Evaluation of Bacterial Glycocalyx Preservation and Staining by Ruthenium Red, Ruthenium Red-Lysine and Alcian Blue for Several Methanotroph and Ruthenium Red-Lysine and Alcian Blue for Several Methanotroph and Staphylococcal Species Staphylococcal Species

Authors Authors Theresa A. Fassel, James E. Van Over, Christopher C. Hauser, Lorie E. Buchholz, Charles E. Edmiston, James R. Sanger, and Charles C. Remsen

This article is available in Cells and Materials: https://digitalcommons.usu.edu/cellsandmaterials/vol2/iss1/5

CELLS & MATERIALS, Vol. 2, No. 1, 1992 (Pages 37-48) 1051-6794/92$3. 00 +. 00 Scanning Microscopy International , Chicago (AMF O'Hare) , IL 60666 USA

EVALUATION OF BACTERIAL GLYCOCALYX PRESERVATION AND STAINING BY RUTHENIUM RED, RUTHENIUM RED-LYSINE AND ALCIAN BLUE

FOR SEVERAL METHANOTROPH AND STAPHYLOCOCCAL SPECIES _./ 9 8b1 ,/ v 3

Theresa A. Fassel 1•2 , James E. Van Over2 , Christopher C. Hauser2 , Lorie E. Buchholz , Charles E. Edmist9114 , James R. Sange1} · and Charles C. Remsen 3/

1 Present Address: Muscle Biology Laboratory , Univ. Wisconsin-Madison , Madison, Wisconsin 53706 2Dept. Plastic and Reconstructive Surgery, Medical College of Wisconsin, Milwaukee, WI 53226

3Center for Great Lakes Studies, Univ. Wisconsin-Milwaukee, Milwaukee, Wisconsin 53204 4Surgical Microbiology Res. Lab. , Dept. Surgery , Medical College of Wisconsin, Milwaukee, WI 53226

(Received for publication September 24 , 1991, and in revised form March 2, 1992)

Abstract

The cationic reagen ts , ruthenium red (RR), alcian blue (AB) and ruthenium red -lysine were compared for ultrastructural preservation and staining of the bacterial glycocalyx. En bloc RR , AB and RR-lysine procedures, and a glutaraldehyde/Os04 fixation wi thou t cationic re­agent , were evaluated by transmission electron micros­copy (TEM) for several methanotroph and staphylococ­cal species. The glutaraldehyde/Os04 fixation was total ­ly ineffective in preserving or staining the glycocalyx material in all species studied . The RR procedure was more effective than the glutaraldehyde/Os04 fixation but was generally less so than the RR-lysine procedure. By the RR-lysine procedure , extensive layers that were ofte n filamentous or fibrou s were observed in the meth ­anotrophs, and extended fibrous elaborations were seen in the staphylococci. By the AB procedure, staphylococ­ci glycocalyx material consisted of condensed curved structures or net-like features not directly comparable to the RR-lysine images. In the methanotrophs, filamen­tous or fibrous layers were often comparable between AB and RR-lysine . Thus , ultrastructural observation of the bacterial glycocalyx in all species studied was improved by use of RR-lysine , whereas the use of AB showed species variation.

Key Words: alcian blue, ruthenium red, ruthenium red­lysine, bacterial glycocalyx, polysaccharide , staphy­lococci , methanotroph , en bloc reagent , staining , transmission electron microscopy.

*Address for correspondence: C.C. Remsen, Center for Great Lakes Studies, University of Wisconsin-Milwaukee , 600 E. Greenfield Avenue , Milwaukee , WI 53204

Telephone No. : (414)-649-3000

37

Introduction

Extracellular polysaccharides are a major compo­nent of structures external to the outer surface of micro­bial cells (Sutherland, 1990). If these structures are of bacterial origin and lie outside the integral elements of the outer membrane of gram-negative cells and outside the peptidoglycan of gram-positive cells, then by defi ni ­tion we are dealing with the bacterial glycocalyx (Costerton et al., 1981). The term glycocalyx was first used in this context by Bennett (1963). In general , there are two types of bacterial glycocalyx (Costerton er al., 1981) . One is composed of globular glycoproteins, of­ten in regular arrays. These are also known as S layers (Sleytr, 1972). The other consists of fibrous matri ces of polysaccharide with variation in thickness and other characteristics such as flexibility and strength of associa­tion with the bacterial cell body (Costerton et al., 1981; van Herson, 1984). The polysaccharide is highly poly ­meric in nature and consists of numerous anionic moie­ties of variable composition (Costerton et al ., 1981; van Herson, 1984). The bacterial glycocalyx is also highly hydrated , as much as 99 % water (Sutherland , 1972).

These properties make effective observation of the bacterial glycocalyx by TEM difficult and often subject to uncertainty of interpretation . The aldehydes and os­mium tetroxide generally are considered to have a low affinity for polysaccharide (Hayat , 1981). Insufficient stabilization by these chemical fixatives may result in observation of glycocalyx material not preserved. An alternative interpretation is that due to a lack of innate electron density and an inability to stain with conven­tional reagents (Roth, 1977), any preserved glycocalyx material is not visible in the TEM. During dehydration, the step-wise removal of water from the highly hydrated bacterial glycocalyx (Sutherland, 1972) can cause defor­mation or condensation (Roth, 1977; Springer and Roth, 1973). This adverse modification may be decreased or minimized by reagents with multiple attachment sites or by cross-linking agents (Costerton et al. , 1981). Lectins (Molinari et al., 1988; Orefici et al ., 1986) and specific antibodies (Molinari et al., 1987; Progulske and Holt , 1980) are two possible cross-linking approaches.

Another approach is the use of cationic reagents

T.A. Passel, et al.

traditional for specific or semi-specific staining of poly­anions by light microscopy, such as ruthenium red (RR) and alcian blue (AB). Ruthenium red was first used to semi-specifically stain pectin (Jensen, 1962). This was extended to include acidic mucopolysaccharides, includ­ing acidic protein polysaccharides or glycoaminoglycans (Luft 1971a, 197lb). Many other substrates react with RR. The generalities concluded by Luft (197la, 1971b, 1976) were that reaction of RR is favorable with polyan­ions of high charge density. Neither high charge density alone, nor a high polymeric character alone, makes a fa­vorable reaction. A relationship to the number of ioniz­able carboxylic acid groups of a polymer is possible. Neutral polysaccharides, polypeptides or proteins are generally unreactive (Luft, 197 la). Ruthenium red alone has been applied to study bacterial outer layers (Pate and Ordal 1967; Springer and Roth, 1973; Titus et al., 1982; Passel et al., 1990, 1992). For some bacterial species , considerable additional outer material of various descrip­tions are observed. In other cases , condensation or col­lapse, perhaps due to dehydration artifacts of the glycoc­alyx, is still reported (Costerton et al., 1981; Molinari et al., 1988). In general, where a high proteinaceous component (Buckmire and Murray, 1973; Mc Cowan et al., 1979; Costerton et al., 1974) or a glycocalyx well anchored to many attachment sites (Costerton et al., 1981) occurs, an extended structure is observed . Also, cross-linking antibodies applied in conjunction with RR have improved observation of extended fibrillar struc­tures (Mackie et al., 1979; Patterson et al., 1975).

Ruthenium red was coupled with the diamine, ly­sine, and glutaraldehyde in another approach that makes use of the cross-linking concept. Jacques and Graham (1989), by inclusion of lysine in a prefixation with glutaraldehyde and RR, stabilized and visualized exten­sive glycocalyces for several bacterial species. This procedure has since been applied to ruminal bacteria (Akin and Rigsby, 1990), Clostridium difficile (Davies and Borriello, 1990) and Streptococcus suis (Jacques et al., 1990). It is based on the development of a glutaral­dehyde-lysine fixation by Boyles (Boyles, 1982, 1984; Boyles et al., 1985). Lysine, which is positively charged at physiological pH, forms large polymers with glutaraldehyde. Cross-linkages of various lengths and directions with anionic sites of the glycocalyx could form stable configurations resistent to later stages of processing (Boyles, 1982, 1984; Boyles et al., 1985). Where the RR molecule exactly fits in this scheme, i.e., acting to stabilize or stain, is not yet established.

Another cationic reagent, alcian blue, was charac­terized by Scott (1972a, 1972b, 1972c, 1973, 1980). Alcian blue is thought to bind negatively charged moie­ties primarily through electrostatic or ionic linkages (Behnke and Zelander, 1970; Luft, 1971a; Scott et al., 1964). It has been used to stain acidic mucopolysaccha­ride (Luft, 1971a; Kiernan, 1990). For light micros­copy, it reacts specifically only with sulphate-ester groups at pH 1.0 and with both carboxyl and sulfate groups at pH 2.5 (Kiernan, 1990; Jones and Reid, 1973).

38

Figure 1. Transmission electron micrographs for Methylomonas albus BG8 (A, C, D, E) and for Methylo­sinus trichosporium Ob3b (B, C, D, F) are shown by the glutaraldehyde/Os04 fixation (A, B), the ruthenium red procedure (C), the alcian blue procedure (D) and the ruthenium red-lysine procedure (E,F). (A) Methylomonas albus BG8 by the glutaraldehyde/ Os04 fixation, shows cup-like features (arrows) in LR White resin. (B) Methylsinus trichosporium Ob3b by the glutaralde­hyde/Os04 fixation, shows limited (arrow) and spike­like structures (curved arrow). (C) By the ruthenium red procedure, Me1hylomonas albus BG8 (cell indicated by large black arrow) cup features (arrow) with good definition are seen in a complete surrounding layer. For Methylosinus trichosp­orium Ob3b (cell indicated by large white arrow) round bead features (white arrowhead), thick filaments (curved arrow) and filamentous or fibrous material (arrow) are observed. (D) By the alcian blue procedure for Methylomonas albus BG8 (cell indicated by large black arrow), both the cup layer (arrow) and material within the cup (curved arrow) are retained. For Methylosinus trichosporium Ob3b (cell indicated by large white arrow), an inner electron dense layer (long arrow) is surrounded by an less dense outer fibrous layer (arrow). (E) By the ruthenium red-lysine procedure for Methylo­monas albus BG8, a complete cup layer (arrow) with fibrous material and elaborate external material (curved arrow) between cells are observed. (F) By the ruthenium red-lysine procedure for Methylo­sinus trichosporium Ob3b, a dense thick innermost layer (long arrow), a gradation of material of varying electron density and a less dense fibrous outermost material (arrow) suggest complex layers.

All magnification bars are 0.5 µm.

In electron microscopy, it has been used in bacterial studies to demonstrate outer material in Eikenella strains (Progulske and Holt, 1980) , Pseudomonasfragi (Herald and Zottola, 1988) and several methanotrophs (Passel et al., 1992).

In this study, the use of RR and AB in previously reported procedures (Passel et al., 1990; 1991) are com­pared to the RR-lysine approach based on Jacques and Graham (1989). The comparison is carried out for sev­eral species of methanotrophs Methylomonas albus BG8, Methylosinus trichosporium Ob3b, Methylocystis parts OBBP, and Methylocystis species strain Lake Washing­ton. They have been shown to play an important role in cycling of carbon in natural environments (Whittenbury and Dalton, 1981). Work with a totally unrelated group of organisms was desired to evaluate the utility of these approaches with other bacterial species. Species of the gram positive coagulase negative staphylococci were se­lected. Staphylococcal species studied are Staphylococ­cus aureus ATCC 25923, Staphylococcus epidermidis RP62 and Staphylococcus hominis SP2. These species

Cation reagent effect on the bacterial glycocalyx

E

39

T .A. Passel, et al.

c

Figure 2. Transmission electron micrographs for Methylocystts species strain Lake Washington and Methylocystts parts OBBP are shown for the ruthenium red procedure (A), the alcian blue procedure (B) and the ruthenium red-lysine procedure (C, D). (A) By the ruthenium red procedure, an electron dense fibrous layer (arrow) surrounds cells of Methylocystts parts OBBP. (B) By the alcian blue procedure, material in a fibrous layer (arrow) surrounds cells of Methylocystts species strain Lake Washington. (C, D) By the ruthenium red-lysine procedure, an ex­tensive fibrous layer (arrow) is seen for (C) Methyloc­ystts species strain Lake Washington and (D) Methyloc­ystts parts OBBP. Some fibrous and/or filamentous material is observed near cells (curved arrow) of Methy­locystts parts OBBP.

All magnification bars are 0.5 µm.

40

are widespread potential opportunistic pathogens and in­fectious agents (Edmiston et al., 1989). Through im­proved visualization of the glycocalyces of both groups of bacterial species, our environmental and medical stud­ies will be facilitated. We hope other laboratories will also find this useful in their applications.

Material and Methods

Cell Culture

Methanotrophic species studied are Methylomonas albus BG8, Methylostnus trtchosportum Ob3b, Methylo­cystts parts OBBP, and Methylocystts species strain Lake Washington. They were grown as suspension cultures at 30 °C in nitrate mineral salts medium under 50:50 air: methane atmospheres until mid-to late log phase, 36 hours (Whittenbury and Dalton, 1981). Staphylococcal species are Staphylococcus aureus ATCC 25923, Staphy-

Cation reagent effect on the bacterial glycocalyx

3A

lococcus epidermidis RP62 and Staphylococcus hominis SP2 . These organisms were recovered from frozen stor­age (-70 °C) and plated on blood agar plates for test of viability. After 24 hrs the organisms were inoculated to trypticase soy broth and incubated for 18 hrs at 35 °C.

Cells were handled as pellets until all fixation/ wash procedures were completed. Then, they were en­robed in 4 % agar and handled as 1 mm 3 blocks.

Glutaraldehyde/Os04 fixation

Fixation was in 2.5% glutaraldehyde in buffer for 2 hours, followed by washes of 3X 10 minutes each. Postfixation was in 1 % (w/v) Os04 in buffer for 2.5 hours, followed by washes of 3X 10 minutes duration each. The buffer for these solutions was 0.1 M cacodyl­ate pH 7. 0-7. 3. Dehydration was in a graded ethanol series of 10%, 25% , 50%, 70%, 95%, 100% anhydrous, and 100 % anhydrous. Infiltration and embedment was in Spurr resin, Standard Medium Mixture (Polysciences

41

Figure 3. Transmission electron micro graphs for Staphylococcus aureus ATCC 25933 is shown by the glutaraldehyde/Os04 fixation (A), the ruthenium red procedure (B), the alcian blue procedure (C) and the ruthenium red-lysine procedure (D). For Staphylococcus aureus ATCC 25933: (A) no glycocalyx material is seen by a glutaralde­hyde/Os04 fixation. This is also observed for Staphylo­coccus hominis SP2 and Staphylococcus epidermidis RP62 (electron micrographs not shown). (B) By the ruthenium red procedure, sparse fibrous material (arrow) appears irregularly around cells. (C) By the alcian blue procedure, curved irregular electron dense structures (arrow) occur between and are frequently continous with cells. A net-like structure of fine strands (curved arrow) also is observed. (D) By the ruthenium red-lysine procedure, fibrous material (arrow) is extensively elaborated and surrounds cells.

All magnification bars are 0.5 µm.

T.A. Fassel , et al .

42

Cation reagent effect on the bacterial glycocalyx

Figure 4. Transmission electron micrographs for Staphylococcus hominis SP2 (A, C, E) and Staphylococ­cus epidermidis RP62(B, D, F) are shown by the ruthe­nium red procedure (A, B), the alcian blue procedure (C, D) and the ruthenium red-lysine procedure (E, F). (A, B) By the ruthenium red procedure , Staphylococcus hominis SP2 shows some fibrous material (arrow). Staphylococcus epidermidis RP62 shows considerably more glycocalyx material (arrow) around and between cells. (C, D) By the alcian blue procedure for both species, curved electron dense (arrow) structures occur that are frequently continuous with cells (shown for Staphylococ­cus hominis SP2 in C). A fine netlike structure (curved arrow) is continuous with cells of Staphylococcus hominis SP2 (C). It is shown completely surrounding a cell of Staphylococcus epidermidis RP62 (D). (E, F) By the ruthenium red-lysine procedure, the fibrous material completely surrounds each cell of Staphylococcus hominis SP2 (E). The fibrous material (arrow) is more extended and elaborated for Staphylo­coccus epidermidis RP62 (F). Additional extended material is seen between cells for both species (E and F).

All magnification bars are 0.5 µm .

Data Sheet #127), except for Methylomonas albus BG8 , where an LR White embedment proves more effective. This processing followed London Resin Company Limit­ed, London, England; Data Sheet 305A. Thin sections were cut on a Porter-Blum MT-II using a diamond knife, and post-stained in 25 % ethanolic 2 % uranyl acetate and Reynolds lead citrate. Sections were studied on a Hitachi H-600 at 75 kV.

Ruthenium red en bloc procedure

Prefixation was in 0. 2 % glutaraldehyde and O. I5 % RR in buffer for 30 minutes. Fixation immedi­ately followed in I% glutaraldehyde and 0.05 % RR in buffer for 2 hours. The washes were 3X 10 minutes each with 0. 05 % RR included in the wash buffer. Post­fixation was in 2 % Os04 and 0.05 % RR in buffer for 2.5 hours. The washes were 3X 10 minutes each with 0.05 % RR included in the wash buffer. The buffer for these solutions was O. IM cacodylate pH 7.0-7.3. Dehy­dration in the graded ethanol series included 0.05 % RR through the 70 % stage. All remaining steps were car­ried out as above.

Alcian blue en bloc procedure

Prefixation was in 0.2 % glutaraldehyde and 0.15 % AB in buffer for 30 minutes. Immediately fol­lowing fixation was in I% glutaraldehyde and 0.05 % AB in buffer for 2 hours. The washes were 3X 10 minutes each with 0.05 % AB included in the wash buffer. Post­fixation was in 2 % Os04 and 0.05 % AB in buffer for 2.5 hours. The washes were 3X 10 minutes each with 0.05 % AB included in the wash buffer. The buffer for these solutions was 0.1 M cacodylate pH 7.0-7.3. Dehy­dration in the graded ethanol series included 0.05 % AB

43

through the 70% stage. All remaining steps were car­ried out as above.

Ruthenium red-lysine en bloc procedure

Prefixation was in 0.075 % RR, 2.5 % glutaralde­hyde and 75 mM lysine in buffer for 20 minutes. Fixa­tion was in 0.075 % RR and 2.5 % glutaraldehyde in buf­fer for 2 hours (Jacques and Graham, I989). This was followed by washing in buffer 3X 10 minutes each. Postfixation was in I% Os04 for 2 hours followed by 3X 10 minutes each washes. The buffer for these solutions was O.I M cacodylate pH 7.0-7.3. Further processing was identical to the glutaraldehyde/Os04 fixation procedure above.

Results

By the glutaraldehyde/Os04 fixation, in the ab­sence of en bloc reagent, glycocalyx material is absent or limited for all species examined. In general, im­provement is seen with cationic reagents.

In Figure I Methylomonas albus BG8 and Meth­ylosinus trichosporium OB3b are shown. For Methylo­monas alb us BG8 (Figure IA), accessory cup features (arrow) are observed external to the outer membrane of the gram-negative cell wall for a glutaraldehyde/osmium tetroxide fixation in LR White resin. These cup features are not preserved in Spurr resin without cationic re­agents ruthenium red or alcian blue as previously shown (Passel et al., I990, 1992). For Methylosinus trichospo­rium OB3b (Figure lB), a few spike-like structures (curved arrow) and suggestion of some limited material (arrow) are observed external to the outer membrane irrespective of embedding resin. This figure and all following electron micrographs are from Spurr resin embed men ts.

Figure lC shows the ruthenium red procedure. The Methylomonas albus BG8 cell is indicated by a large black arrow. Cup features (arrows) are retained with good definition . As previously reported (Passel et al., I990), cups are seen in a gap-less layer surrounding the outer membrane. The Methylosinus trichosporium OB3b cell in Figure IC is indicated by large white arrow. Features seen with ruthenium red include round bead­like structures (white arrowhead), thickened electron dense filaments (curved arrow), and some fibrous mate­rial (arrow). To aid description of glycocalyx, the term filamentous is used where indivjdual strands or filaments can be easily identified. The term fibrous is used for outer material where separate and distinct strands or individual filaments are not easily discerned.

By the alcian blue procedure (Figure ID) for Methylomonas a!bus BG8 (cell indicated by large black arrow), fibrous material (curved arrow) appears in cups (arrow). For Methylosinus trichosporium OB3b (cell in­dicated by large white arrow), two layers of different electron density are observed. A dense inner layer (long arrow) is completely surrounded by a more amorphous or fibrous outer layer (arrow), as detailed previously (Passel et al., I 992).

T.A. Passel , et al.

By the ruthenium red-lysine procedure , several observations similar to the above are seen for Methylom­onas albus BG8 (Figure lE) . A well defined and com­plete cup layer is preserved (arrow). Fibrous material appears within cups. Additionally, more elaborate mate­rial frequently extends between cells (curved arrow). For Methylosinus trichosporium OB3b (Figure IF) by the ruthenium red-lysine procedure , an electron dense inner material (long arrow) is frequently seen. A gradation of electron density in the other glycocalyx material occurs with frequent suggestion of an outermost fibrous layer (arrow) around many cells.

For Methylocystis species strain Lake Washington and Methylocystis paris OBBP by a glutaraldehyde/Os04 tetroxide fixation, observation of glycocalyx is limited to remnants of spikes , or strands , or is completely ab­sent (electron micrographs not shown), as was seen for Methylosinus trichosporium OB3b. By the ruthenium red procedure (Figure 2A) , a matted fibrous layer (ar­row) is found for some cells , as shown here for Methylo­cystis paris OBBP. For other cells , abundant filamen­tous material is often seen . Additionally , for some cells of Methylocystis species strain Lake Washington , dis­crete bead-like structures were observed (Passel et al. , 1992). By the alcian blue procedure , a fibrous layer (arrow) completely surrounds cells of Methylocystis species strain Lake Washington (Figure 2B) as is simi­larly found in cells of Methylocystts parts OBBP (Passel et al. , 1992).

By the RR-lysine procedure , cells are also sur­rounded by an extensive fibrous layer (arrow) for Meth­ylocystts species strain Lake Washington (Figure 2C) and Methylocystis parts OBBP (Figure 2D) . For the lat­ter , filamentous or fibrous material (curved arrow) is also seen frequently in a mass between cells. An elec­tron translucent region, possibly artifact, between the fibrous layer and the cell wall is noted for some cells.

For Staphylococcus aureus ATCC 25923 (Figure 3A) and the other staphylococci species studied, no sug­gestion of glycocalyx material is seen by a glutaralde­hyde/Os04 fixation. In S. aureus (Figure 3B-3D) , by the ruthenium red procedure (Figure 3B) sparse fibrous material (arrow) occurs irregularly around cells. Where there is a grouping of cells , more material is frequently seen on the outermost cells. By the alcian blue proce­dure (Figure 3C), curved irregular electron dense struc­tures (arrow) appear continuous with cells and extend between cells. A netlike structure that appears to consist of fine strands (curved arrow) is occasionally seen . In contrast, by ruthenium red-lysine (Figure 3D) , an elabo­rate fibrous material (arrow) surrounds cells and extends between cells.

By the ruthenium red procedure for Staphylococ­cus hominis SP2 (Figure 4A), some fibrous material (arrow) is seen. For Staphylococcus epidermidis RP62 (Figure 4B), more fibrous material (arrow) is observed. By the alcian blue procedure, for Staphylococcus hom­inis SP2 (Figure 4C) and Staphylococcus epidermidis RP62 (Figure 4D), curved electron dense structures

44

(arrow) are seen. A netlike structure (curved ran"Jw) is shown continuous with some Staphylococcus hJminis SP2 cells (Figure 4C) and completely surroundiin~ a cell of Staphylococcus epidermidis RP62 (Figure t4D). By the ruthenium red-lysine procedure for Staphy1lowccus hominis SP2 (Figure 4E) , fibrous material (arrrov) sur­rounds the cell body as a more complete layer. I addi­tion, more elaborate material extends between c1el ls . For Staphylococcus epidermidis RP62 (Figure 4F), <elaborate and extensive fibrous material surrounds cellls md is maintained between cells (arrow).

Discussion

The bacterial glycocalyx is defined as s;tnctures containing polysaccharide of bacterial origin arnc lying outside the integral elements of the outer merrnbrane of gram-negative cells and outside the peptidog ly:an of gram-positive cells (Costerton et al. , 1981) . It is rich in highly varied and diverse polymeric polysac ct.arides (Costerton et al. , 1981 ; van Herson , 1984) an d! is also a highly hydrated structure containing 99 % water (Sutherland , 1972) . These characteristics make:s ptimi­zing their observation for transmission electron micros­copy challenging. One approach is the use of cationic reagents , RR and AB , in en bloc procedures wi tth chemi­cal fixatives . A previous successful method util izing RR or AB improved observation of glycocalyx mate rial over a glutaraldehyde/Os04 fixation for several sp ecies of methanotrophs. This material bore a resemblance to freeze etch images without cationic reagent or chemical fixative (Passel et al ., 1990; 1992). The use of a cross­linking reagent , the diamine lysine , with glutar aldehyde was developed by Boyles (Boyles , 1982, 1984; Boyles et al. , 1985) and applied to fi xation of actin filaments, extracellular matrix and the mammalian gly cocalyx. This approach was combined with the use of RR by Jacques and Graham (1989) and applied to the bacterial glycocalyx by themselves and several authors (Akin and Rigsby, 1990; Davies and Borriello, 1990 ; Jacques et al. , 1990).

Two general types of bacterial glycocalyx have been described. One is composed of Slayers of globular glycoproteins often in regular arrays (Sleytr, 1972). The other is a complex fibrous matrix (Costerton et al., 1981). Both are represented in the species of the present study.

Many gram-negative species have regular arrays of wine glass- , globet- or cup-shaped subunits that are considered by Sleytr (1972) as special types of S layers. Examples include Flexibacter polymorphus (Ridgway, 1977), several species of Chromattum (Remsen et al., 1970; Hageageand Gherna, 1970, 1971) andMethylomo­nas alb us. The cup layer of Methylomonas albus strain BG8 , fits the Slayer definition of Sletyr (1972), and was identified by negative staining and freeze etch electron microscopy by Haubold (1978) and in thin section by Wilkinson (1971).

By TEM , the cup layer was not observed in Spurr

Cation reagent effect on the bacterial glycocalyx

embedments without RR (Passel et al., 1990), AB (Passel et al., 1992) or RR-lysine. It was seen without these reagents in LR White. However, the cups lacked strong defi nition as individual structures and were often not in a complete layer around the outer membrane (Passel et al., 1990). Further, the improvement in the cup layer by RR is not increased by either AB or the use of lysine. The appearance of the cup layer by RR com­pares directly with the images obtained by freeze etch, where cationic reagents or chemical fixatives were not used (Passel et al., 1990). Based on the Spurr observa­tions alone , one cannot differentiate between the im­proved preservation of the cups or the alternative possi­bility of improved staining of otherwise invisible cup features. However , the inferior appearance of cups in LR White (Passel et al. , 1990), their improvement with RR in either resin (Passel et al., 1990) and the agree­ment with the freeze etch cup layer (Haubold , 1978; Passel et al., 1990) suggest an improvement in preserva­tion of the cup layer by RR processing. This improve­ment also agrees with the suggestions of a cup layer composition that includes acidic mucopolysaccharides, acidic protein polysaccharides or glycoprotein.

Additionally, fibrous material appears within cups by the AB or RR-lysine procedure. Elaborate material that frequently extends between cells is seen by RR­lysine processing. For Methylomonas a/bus BG8, RR alone is inadequate for observation of this glycocalyx material. Perhaps this composition is sufficiently differ­ent from the cup layer that RR alone is insufficient to effect its preservation or staining.

Glycocalyces of the remaining three methanotr­ophs appear to fall under the fibrous matrix type. In previous work, freeze etch was compared to a glutaral­dehyde/Os04 fixation without cationic reagent. For Methylosinus trichosporium Ob3b and the two Methyloc­ystis species, large filamentous and complex layers were revealed outside the outer membrane of these gram-nega­tive cells by freeze etch after ultra-rapid freezing (Passel et al, 1990; 1992). When fixed by a glutaraldehyde/ Os04 procedure, spike-like structures or a complete lack of any glycocalyx material was observed. This can be interpreted as suggestive of collapse and/or condensation perhaps due to an ethanol dehydration artifact (Bayer and Thurow, 1977) that is minimized or avoided by ul­tra-rapid freezing and by freeze etch. An alternative explanation is this material lacks innate electron density and fails to stain by conventional procedures (Roth, 1977). Perhaps some combination of both dehydration artifact and inability to stain may occur.

When cells of Methylosinus trichosporium Ob3b and the two Methylocystis species were processed in a procedure with AB extensive layers that were filamen­tous or fibrous were observed. For Methylosinus tricho­sporium Ob3b, this glycocalyx material appears as two layers of distinct electron density. This was interpreted as suggesting a segregation of different polyanions (Passel et al., 1992). By the RR procedure for these three species, although some fibrous layers were ob-

45

served, a variety of other features have consistently been seen for some cells. This includes round bead-like structures, thick filaments, and filamentous layers (Passel et al., 1990, 1992; Titus et al., 1982).

The use of lysine with RR, resulted in images that were markedly similar to the AB fibrous layer surround­ing the two Methylocystis species. However, there was a clear zone between the cell outer membrane and the fi­brous layer. This may be suggestive of an artifactual cell shrinkage, an area of washout, lack of stain or other artifact. Where cells were observed with a fibrous layer by RR processing, or for all cells by AB processing, this clear zone was not seen. However, a few cells on occa­sion had evidence of shrinkage that was internal to the outer membrane . Also, for Methylocystis paris by RR­lysine a more massive congregation of filamentous or fi ­brous material was noted between cells . For Methylos­inus trichosporium Ob3b by RR-lysine, the gradation of electron density in glycocalyx material may again sug­gest a separation of polyanions. The clear zone was not seen for this species. This may suggest a greater vulner­ability to this type of artifact by the Methylocystis species. On the basis of the observation of bead-like structures and thick filaments by RR and their absence by RR-lysine or AB , one could conclude these features are perhaps due to collapse of more filamentous or fi­brous material, and their insufficient stabilization or stain reaction with RR. However , the bead-like struc­tures have been observed by scanning electron microsco­py (SEM) in the absence of RR or other cationic reagent (Passel et al., 1990; Titus et al. , 1982). There is some suggestion of this structure by freeze etch (Passel et al., 1990). Alternatively, their lack of observation by RR­lysine or AB might mean an insufficient stabilization or other damage that results in loss of their structural integ­rity. Perhaps, the composing polymers of the beads are split apart and the structures broken to merge visually with other fibrous or filamentous material.

It was desirable to study the effects of these procedures on glycocalyx preservation or staining on a different group of bacteria. For the staphylococcal species studied, there was no evidence of a glycocalyx by the glutaraldehyde/Os04 fixation even in species known for production of external polysaccharide as a slime layer. By RR some fibrous material was observed for all species studied. A considerable increase in the amount of fibrous material in extended configuration around and between cells was seen in RR-lysine process­ed cells for all species. For SP2, an incomplete layer close to the cell body by RR was replaced by a more complete layer surrounding the cell by RR-lysine. This suggests an improvement in preservation and/or staining of this glycocalyx, possibly by a more efficient cross­linkage and stabilization. For RP62, the difference between RR and RR-lysine processing is less obvious with considerable material observed for both.

A very different image was seen by AB process­ing for all species of staphylococci studied . Curved electron dense structures and net-like structures may be

T.A . Passel , et al.

the result of condensations or distortions of glycocalyx material for these cells. This is very likely due to insuf­ficient stabilization and subsequent collapse. The lack of agreement with the extended configurations seen with RR-lysine also suggests the inability of AB to interact as effectively with the components of the staphylococci glycocalyx .

In summary , for the methanotrophs , extended fila­mentous or fibrous configurations were generally similar between AB and RR-lysine procedures. The additional cross-linking ability of the lysine procedure does not ap­pear superior to the interaction of alcian blue with these glycocalyces. For the staphylococci , the most abundant and extended glycocalyces were observed for cells by RR-lysine processing. Thus perhaps , the glycocalyx for staphylococci are of a biochemical and/or charge distri ­bution that are preferentially preserved and/or stained by processing that cross-links glutaraldehyde , lysine and polysaccharide polymers and interacts with RR . The difference in effectiveness of the methods using RR , AB or RR-lysine appears somewhat genera/species depen­dent. Di fferences in complex polyanionic polysaccha­ride composition of the glycocalyx for the different bacteria could account for these observations .

Acknowledgements

Th e authors wish to acknowledge Dr. Ken Seigesmund , Medical College of Wisconsin , for access to electron mi croscopy instrumentation and Marilyn J . Schaller for her continued and valuable insight and positive support. This work was supported in part by grant 2022-IRP-79 from NASA and in part by grant MCB-9118653 from NSF . This is contribution No. 360 from the Center for Great Lakes Studies.

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Discussion with Reviewers

T. Zelander: Having in mind the highly hydrated con­dition of the glycocalyx material, it occurred to me, that the spinning of unfixed bacteria to a pellet might influ­ence the distribution and appearance of the glucocalyx material as seen in some of the pictures. Did you con­sider this possible influence or have I totally misun­derstood your paragraph on methods?

T.A. Passel, et al.

Authors: We cannot conclusively rule out any effect of the centrifugation on the glycocalyces. However, of the two types of glycocalyces we feel the S layers to be less likely effected. The fibrous/filamentous matrices may be the more susceptible in the extended nature of their layers. Previous freeze etch comparisons for the metha­notrophs with fibrous/filamentous matrices suggested a good correspondence between freeze etch and images ob­tained by the alcian blue procedure (Passel et al., 1992). Therefore, we feel confident with our methods.

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