Ultramicroscopic localization of cationized antigen in the glomerular basement membrane in the course of active, in situ immune complex glomerulonephritis

The sequence of antigen localization and the interaction of immune deposits with the anionic sites of the glomerular basement membrane (GBM) were investigated in an active model of in situ immune complex glomerulonephritis using a cationized ferritin. Three weeks after immunization with native horse spleen ferritin, the left kidneys of rats were perfused with 500 micrograms of cationized ferritin through the left renal artery. One h after renal perfusion, most of ferritin particles localized subendothelially, corresponding to the anionic sites of the lamina rara interna. In the glomerular capillary loops, infiltrating polymorphonuclear leukocytes and monocytes were seen. Some of these monocytes were in direct contact with immune complexes containing ferritin aggregates associated with anionic sites of the lamina rara interna. At 24 h, numerous ferritin aggregates were present subepithelially, preferentially beneath the slit membrane. The subepithelial location of ferritin did not always correspond to the anionic sites of the lamina rara externa. From days 3 to 7, there was remarkable endocapillary cell proliferation in some loops and pronounced effacement of epithelial foot processes. Focal detachment of epithelium from the GBM was observed occasionally. From days 14 to 28, most of ferritin aggregates were located intramembranously and subepithelially. Membranous transformation has already begun around the subepithelial deposits. This morphological study provides insight into the fate of immune deposits and injury to the GBM in the glomerulonephritis.     

Freeze-fracture cytochemistry of rat glomerular capillary tuft. Determination of wheat germ agglutinin binding sites and localization of anionic charges

We propose here the use of freeze-fracture to gain access and to label in vitro glomerular components and locate WGA receptors and anionic sites. Tissues are frozen, fractured under liquid nitrogen, and thawed. Freeze-fracture rendered all glomerular structures directly accessible to the reagents. This made possible study of the nature and topology of cationized ferritin and WGA binding sites. WGA-gold complexes were observed over plasma membranes of podocytes and of endothelial and mesangial cells. Labeling of podocytes and endothelial cells was similar in the mesangial area and in the peripheral part of the capillary loop. Cross-fractures of extracellular matrices showed that WGA bound uniformly to the glomerular basement membrane (GBM) as well as to mesangial matrix. In fractured specimens treated with neuraminidase, WGA was no longer observed over podocytes but it consistently labeled the surface of endothelial and mesangial cells. Whereas in GBM cross-sections WGA binding was greatly reduced or even abolished, it remained unmodified in the mesangium. This shows that only NeuNAc (sialic acid) might account for the binding of WGA to podocytes, whereas GlcNAcs appear to be the main WGA binding sites on endothelial and mesangial cells and in the mesangial matrix. Both NeuNAc and GLcNAc residues are probably associated in GBM. With cationized ferritin (pI 8.3) at pH 7.4, intense, continuous labeling was seen all over the different plasma membranes, denser in podocytes than in endothelial cells. CF was also observed in cross-fractured profiles of extracellular matrices and never appeared agglutinated in discrete sites.     

Assembly of the glomerular filtration surface. Differentiation of anionic sites in glomerular capillaries of newborn rat kidney

Glomerular development was studied in the newborn rat kidney by electron microscopy and cytochemistry. Glomerular structure at different developmental stages was related to the permeability properties of its components and to the differentiation of anionic sites in the glomerular basement membrane (GBM) and on endothelial and epithelia cell surfaces. Cationic probes (cationized ferritin, ruthenium red, colloidal iron) were used to determine the time of appearance and distribution of anionic sites, and digestion with specific enzymes (neuraminidase, heparinase, chondroitinases, hyaluronidases) was used to determine their nature. Native (anionic) ferritin was used to investigate glomerular permeability. The main findings were: (a) The first endothelial fenestrae (which appear before the GBM is fully assembled) possess transient, negatively charged diaphragms that bind cationized ferritin and are impermeable to native ferritin. (b). Two types of glycosaminoglycan particles can be identified by staining with ruthenium red. Large (30-nm) granules are seen only in the cleft of the S-shaped body at the time of mesenchymal migration into the renal vesicle. They consist of hyaluronic acid and possibly also chondroitin sulfate. Smaller (10-15-nm) particles are seen in the earliest endothelial and epithelial basement membranes (S-shaped body stage), become concentrated in the laminae rarae after fusion of these two membranes to form the GBM, and contain heparan sulfate. They are assumed to be precursors of the heparan sulfate-rich granules present in the mature GBM. (c) Distinctive sialic acid-rich, and sialic acid-poor plasmalemmal domains have been delineated on both the epithelial and endothelial cell surfaces. (d) The appearance of sialoglycoproteins on the epithelial cell surface concides with the development of foot processes and filtration slits. (e) Initially the GBM is loosely organized and quite permeable to native ferritin ;it becomes increasinly impermeable to ferritin as the lamina densa becomes more compact. (f) The number of endothelial fenestrae and open epithelial slits increases as the GBM matures and becomes organized into an effective barrier to the passage of native ferritin.     

Role of molecular charge in glomerular permeability. Tracer studies with cationized ferritins

Mouse kidneys were perfused with Krebs-Ringer bicarbonate buffer (KRB) containing native, anionic horse spleen ferritin or various cationized derivatives, and the glomerular localization of the probe molecules determined by electron microscopy. Ferritins cationic with respect to the medium (KRB, pH 7.45) accumulated in the subendothelial layers of the glomerular basement membrane (GBM) in amounts far exceeding those observed with anionic ferritins, the degree being greater for the more cationized derivatives. Strongly cationized ferritins, in addition permeated the full thickness of the GBM in considerable amounts, but appeared to be retarded from entry into the urinary spaces at the level of the filtration slits. Very strongly cationized derivatives adhered to glomerular endothelium and GBM and formed aggregates in the outer layers of the latter. The results suggest that intrinsic negative charges are present in the GBM and endothelium, and that the barrier function of the glomerular capillary wall may be ascribed in part to its electrophysical properties.     

Preservation of fixed anionic sites in the GBM in the acute proteinuric phase of cationic antigen mediated in-situ immune complex glomerulonephritis in the rat

Cationic antigens have been observed to bind with the negatively charged glomerular basement membrane (GBM). Using the cationic reagent polyethyleneimine (PEI), the distribution of glomerular anionic sites was evaluated ultrastructurally in the early stage (2 h-day 7) of cationic antigen mediated in-situ immune complex formation type glomerulonephritis (GN) in the rat. Renal perfusion via the renal artery with 100 micrograms of cationized human IgG(pI greater than 9.5), followed by the i.v. injection of specific antibodies, led to an initial increase in urinary albumin excretion, subsequent massive globulinuria and the formation of numerous subepithelial deposits on day 7. The most striking alteration in glomerular anionic sites was observed on the epithelial cell surface coat; the PEI deposition on the epithelial cell surface was almost identical to that in control glomeruli at 2 and 4 h after the induction of GN; thereafter, on day 7, a broad loss of anionic sites was observed on flattened epithelial foot processes. In contrast, fixed anionic sites of the laminae rarae of the GBM showed no apparent alterations in the distribution and number from 2 h to day 7 and did not disappear even in the lamina rara externa adjacent to subepithelial deposits. These findings not only show that fixed anionic sites of the GBM, in contrast to the rapid decrease in those of the epithelial cell surface, are not completely neutralized or destroyed even in GN, in which cationic antigen participates in the in-situ formation of GBM-deposits.(ABSTRACT TRUNCATED AT 250 WORDS)     

The anionic barrier system in the mesonephric renal glomerulus of the arctic lamprey,Entosphenus japonicus (Martens) (Cyclostomata)

Summary To examine the selective permeability of the nephrons of lower vertebrates, the permeability of the glomerulus in the kidney of an arctic lamprey, Entosphenus japonicus (Martens), to native anionic ferritin or cationized ferritin was studied by observing the distribution of ionized anionic groups in renal tissues. The cationized ferritin molecules injected into the dorsal aorta penetrated rapidly into the glomerular basement membrane layer through fenestrae present in the capillary endothelium and were subsequently excreted into the urinary spaces via the interstices between foot processes of the visceral epithelial cells. Native anionic ferritin, on the other hand, passed only minimally through the capillary wall. Cytochemical staining of fixed tissue or perfusion of the kidney in situ with cationic cacodylate-iron colloid revealed that the ionized anionic groups of acid mucopolysaccharides were distributed on both the luminal and abluminal surfaces of endothelial cells, and in the thick fibrous lamina rara interna of the glomerular basement membrane; they were especially dense on the surfaces of visceral epithelial cells and their foot processes. These results suggest that the mesonephric glomerulus of the arctic lamprey possesses a functionally well developed anionic barrier system comparable to that of the mammalian metanephric glomerulus.     

Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rabbit Peyer's patch

Summary M cells in Peyer's patch epithelium conduct transepithelial transport of luminal antigens to cells of the mucosal immune system. To determine the distribution of specific lectin-binding sites on luminal membranes of living M cells and to follow the transport route of membranebound molecules, lectin-ferritin conjugates and cationized ferritin were applied to rabbit Peyer's patch mucosa in vivo and in vitro. The degree to which binding enhances transport was estimated by comparing quantitatively the transport of an adherent probe, wheat germ agglutinin-ferritin, to that of a nonadherent BSA-colloidal gold probe. When applied to fixed tissue, the lectins tested bound equally well to M cells and columnar absorptive cells. On living mucosa, however, ferritin conjugates of wheat germ agglutinin and Ricinus communis agglutinins I and II bound more avidly to M cells. Absorptive cells conducted little uptake and no detectable transepithelial transport. Lectins on M cell membranes were endocytosed from coated pits, rapidly transported in a complex system of tubulocisternae and vesicles, and remained adherent to M cell basolateral membranes. Cationized ferritin adhered to anionic sites and was similarly transported, but was released as free clusters at M cell basolateral surfaces. When applied simultaneously to Peyer's patch mucosa, wheat germ agglutinin-ferritin was transported about 50 times more efficiently than was bovine serum albumin-colloidal gold.     

A simple two-step labeling procedure for ultrastructural localization of cell surface anionic sites

We propose a new method for ultrastructural localization of cell surface anionic sites. The method consists of sequential interaction of aldehyde-fixed cells with a polycationic reagent, poly-L-lysine (PL), followed by secondary interaction with a negatively charged marker, ferritin. By use of PL of low molecular weight (4000) on aldehyde-pre-fixed red blood cells and macrophages, the reaction resulted in binding of ferritin particles to cell surface anionic sites with a density distribution resembling that of cationized ferritin (CF). The density of the attached ferritin molecules increased in direct correlation with the MW of PL used. The primary PL interaction can be carried out at low pH (less than 2), thus restricting the labeling mainly to membrane-bound sialyl residues.     

The appearance of photosynthetic proteins in developing maize leaves

Soybean (Glycine max (L.) Merr.) protoplasts have been surface-labelled with cationized ferritin, and the fate of the label has been followed ultrastructurally. Endocytosis of the label occurs via the coated-membrane system. The pathway followed by the label, once it has been taken into the interior of the protoplast, appears to be similar to that found during receptor-mediated endocytosis in animal cells. Cationized ferritin is first seen in coated vesicles but rapidly appears in smooth vesicles. Labelled, partially coated vesicles are occasionally observed, indicating that the smooth vesicles may have arisen by the uncoating of coated vesicles. Structures which eventually become labelled with cationized ferritin include multivesicular bodies, dictyosomes, large smooth vesicles, and a system of partially coated reticula.Abbreviation CF cationized ferritin     

Localization and distribution of anionic charges in the glomerular mesangium of normal and nephrotic rats

Sulfated glycosaminoglycans and sialoglycoproteins are thought to play a pivotal role in the glomerular capillary wall barrier to filtration since these anionic charged elements are important in the maintenance of capillary wall integrity and constitute a charge-selective filter. The development of proteinuria in puromycin aminonucleoside (PAN) nephrosis is associated with polyanion loss from the glomerular capillary wall structures. Since in PAN nephrosis the permeability of the mesangial area to plasma proteins and tracer substances has also been shown to be increased, the purpose of this study was to analyse the localization and distribution of anionic charges in the glomerular mesangium in this experimental model. Glycosaminoglycans were labeled by perfusion of the kidneys with ruthenium red solution (RR). Electron microscopic examination revealed the presence of distinct small RR granules ("anionic sites") in the mesangial intercellular matrix substance and in the laminae rarae of the glomerular basement membrane (GBM). The center-to-center spacing of the granules was measured and a frequency distribution of intervals in different interval classes was constructed. In normal glomeruli the anionic sites in the mesangial matrix showed a distribution pattern identical to the GBM with a maximal interval incidence at the 31-40 nm class. In nephrotic rats anionic site distributions in matrix and GBM did not change significantly. Sialoglycoproteins were labeled with colloidal iron (CI). In PAN nephrosis a decrease of CI binding was observed at the epithelial-basement membrane junction of the glomerular capillary wall. However, CI labeling of the mesangial matrix and mesangial cell membranes did not differ from that of normal glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes

Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (approximately 60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (approximately 3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells. When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites. CF remained firmly bound after prolonged perfusion with 0.1-0.2 M KCl or NaCl. It was displaced by perfusion with buffers of high ionic strength (0.4-0.5 M KCl) or pH (less than 3.0 or greater than 10.0). CF also bound (clustered at approximately 60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix. The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations.     

Cortical granule exocytosis is coupled with membrane retrieval in the egg of Brachydanio

Activation of the teleost (Brachydanio) fish egg includes the exocytosis of cortical granules, the construction of a mosaic surface consisting of the unfertilized egg plasma membrane and the limiting membranes of the cortical granules, and the appearance of coated and smooth vesicles in the cytoplasm (Donovan and Hart, '82). Unfertilized and activated eggs were incubated in selected extracellular tracers to (1) determine experimentally if cortical granule exocytosis was coupled with the endocytosis of membrane during the cortical reaction, and (2) establish the intracellular pathway(s) by which internalized vesicles were processed. Unfertilized eggs incubated in dechlorinated tap water or Fish Ringer's solution containing either horseradish peroxidase (HRP; 10 mg/ml), native ferritin (12.5 mg/ml), or cationized ferritin (12.5 mg/ml) were activated as judged by cortical granule breakdown and elevation of the chorion. Cells treated with HRP and native ferritin exhibited a delay in cortical granule exocytosis when compared with water-activated eggs lacking the tracer. Each tracer was internalized through the formation of a coated vesicle from a coated pit. Since coated pits appeared to be topographically restricted to the perigranular membrane domain of the mosaic egg surface, their labeling, particularly with cationized ferritin, strongly suggested that the retrieved membrane was of cortical granule origin. Cationized ferritin and concanavalin A (Con A) coupled with either hemocyanin or ferritin labeled the surface of the unactivated egg and both domains of the mosaic egg surface. Transformation of the deep evacuated cortical granule crypt into later profiles of exocytosis was accompanied by increased Con A binding. Within activated egg cortices, HRP reaction product, native ferritin, and cationized ferritin were routinely localized in smooth vesicles, multivesicular bodies, and autophagic vacuoles. Occasionally, each tracer was found in small coated vesicles adjacent to the Golgi and within Golgi cisternae. The intracellular distribution of HRP, native ferritin, and cationized ferritin suggests that internalized membrane is primarily processed by organelles of the lysosomal compartment. A second and less significant pathway is the Golgi complex.     

Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages

The participation of cell surface anionic sites on the interaction between tachyzoites of Toxoplasma gondii and macrophages and the process of phagosome-lysosome fusion were analyzed using cationized ferritin as a marker of cell surface anionic sites and albumin-colloidal gold as a marker for secondary lysosomes. Incubation of either the macrophages or the parasites with cationized ferritin before the interaction increased the ingestion of parasites by macrophages. Anionic sites of the macrophage's surface, labeled with cationized ferritin before the interaction, were internalized together with untreated parasites. However, after interaction with glutaraldehyde-fixed or specific antibody-coated parasites, the cationized ferritin particles were observed in endocytic vacuoles which did not contain parasites. Macrophages previously labeled with albumin-gold at 37 degrees C, were incubated in the presence of cationized ferritin at 4 degrees C and then incubated with untreated or specific antibody-coated parasites. After interaction with opsonized parasites, the colloidal gold particles were observed in the parasitophorous vacuoles while the cationized ferritin particles were observed in cytoplasmic vesicles. However, when the interaction was carried out with untreated parasites, the parasitophorous vacuoles exhibited ferritin particles while the colloidal gold particles were observed in cytoplasmic vesicles. These observations, in association with studies previously reported, suggest that the state of the parasite surface determines the mechanism of parasite entry into the macrophage, the composition of the membrane lining the parasitophorous vacuole and the ability of lysosomes to fuse with the vacuoles.     

Redistribution of surface anionic sites on the luminal front of blood vessel endothelium after interaction with polycationic ligand

The ability of anionic groups on the luminal surface of blood vessels to redistribute by lateral migration under the influence of multivalent ligands was analyzed by electron microscopy, using cationized ferritin (CF). In vitro interaction of blood vessel segments with CF results in rapid aggregation of most anionic sites on the luminal fromt of the endothelium, followed by internalization or detachment of the CF patches, leaving most of the luminal surface devoid of anionic sites. Further incubation of such endothelial cells without CF results in regeneration of binding capacity for the polycationic label. Transport of CF, but not of native ferritin, across the endothelium by vesicle transport, followed by exocytosis of the interiorized CF clusters on the tissue front of the endothelium, was also observed. The possibility that such activities in the blood vessels in vivo may be associated with local changes in the normal distribution of the surface anionic sites as well as in accumulation of debris in the subendothelial layers of the vessels is suggested.     

Glomerular extracellular matrices and anionic sites in aging ddY mice: a morphometric study

A morphometric study was undertaken to examine age-related changes in glomerular ultrastructure and anionic sites in ddY male mice at various ages. A progressive increase in glomerular extracellular matrices, including thickening of the glomerular basement membrane (GBM), formation of GBM nodules, and mesangial matrix increase, was found to be the primary age-related ultrastructural change in aging mice; there were also electron-dense deposits in mesangial and subepithelial regions. The extent of GBM thickening in mice was less than was reported in rats. Rather, the GBM nodules, which had the same electron density as the lamina densa (LD) and protruded on the subepithelial side of the GBM, were more striking. Quantitative evaluation showed that GBM thickness, number and size of GBM nodules, and the area of the mesangial matrix were significantly correlated with the age of the mice. The distribution of anionic sites in the glomeruli of aging animals was described for the first time. No statistically significant differences were noted between the number of glomerular anionic sites in the different age groups. These results indicate that the increase in glomerular extracellular matrices reported in aged rats was also present in aged mice, although the extent of various changes was different. The results also indicate that this increase in glomerular extracellular matrices with age was not accompanied by significant alteration in glomerular anionic sites.     

Endocytosis of native and cationized ferritin by intralobular duct cells of the rat parotid gland

Summary The ability of the intralobular ducts of the rat parotid gland to take up protein from the lumen was examined after retrograde infusion of native and cationized ferritin. At high concentrations (3–10 mg/ml), cells of both intercalated- and striated ducts avidly internalized the tracers. No differences were noted in the mode of uptake or fate of native or cationized ferritin. Large, apical ferritin-containing vacuoles up to 5 m in size were present in cells of the intercalated ducts after infusion for 15 min. Small, smooth-surfaced spherical or flattened vesicles and tubules containing ferritin were also observed, often in association with the large vacuoles. Ferritin uptake increased with increasing infusion time, up to 1 h. Uptake by the striated ducts was less consistent than by the intercalated ducts, and occurred mainly in small vesicles and tubules. Secondary lysosomes became labeled with ferritin in both cell types. Ferritin was not observed in the Golgi saccules, nor was it discharged from the cells at the basolateral surfaces. At low concentrations (0.3–1 mg/ml), uptake was reduced, especially by cells of intercalated ducts, and differences were noted in the behavior of the two tracers. Cationized ferritin was internalized mainly into vesicles and tubules of cells of striated ducts; little uptake of native ferritin occurred at low concentrations. These results demonstrate that the ductal cells of the salivary glands are capable of luminal endocytosis of foreign proteins. They also suggest that in addition to modifying the primary saliva by electrolyte reabsorption and secretion, and secretion of various glycoproteins, the ductal cells are able to reabsorb proteins secreted by the acinar cells.     

Preparation of ferritin-avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry.

An improved technique was developed for the unidirectional covalent binding of avidin to ferritin by reductive alkylation. The method is based on the oxidation of sugar moieties on avidin and subsequent coupling to amino groups of ferritin via Schiff's bases followed by reduction with sodium borohydride. The resultant conjugate was used as an ultrastructural marker for the localization of surface receptor sites on biotin-derivatized whole cells. Erythrocytes were treated chemically with sodium meta-periodate and biotin hydrazide in succession. The ferritin-avidin conjugates were used to label the biotin sites either before or after fixation of the cells. The density and distribution of ferritin avidin conjugates on cell surfaces were anlyzed on thin sections and compared with those of cationized ferritin, which were shown to bind anionic sites of the erythrocyte membrane. The extensions of this method for the visualization of other systems is discussed.     

Surface charges associated with fenestrated brain capillaries. I. In vitro labeling of anionic sites

Ferritin derivatives with different pI values and the basic dye ruthenium red have been used as cationic probes to localize anionic sites associated with fenestrated brain capillaries. Cationic ferritin was found in the endothelial basement membrane and the basement membrane of the perivascular cellular linings in amounts far exceeding those observed with anionic derivatives, the degree being greater for the more cationized ferritin molecules. Labeling of the luminal endothelial front with cationic ferritin was only achieved when a serum- or albumin-free medium was applied. Furthermore, the striated collagen fibers were coated with cationic ferritin molecules in a highly ordered fashion. Ruthenium red localized to the same sites. The findings suggest the existence of a perivascular charge filter around fenestrated capillaries of the brain. Some physiological roles of this filter are discussed, as related to its possible function in regulating homeostasis of cerebrospinal fluid.     

Electron microscopic examination of wastewater biofilm formation and structural components.

This research documents in situ wastewater biofilm formation, structure, and physiochemical properties as revealed by scanning and transmission electron microscopy. Cationized ferritin was used to label anionic sites of the biofilm glycocalyx for viewing in thin section. Wastewater biofilm formation paralleled the processes involved in marine biofilm formation. Scanning electron microscopy revealed a dramatic increase in cell colonization and growth over a 144-h period. Constituents included a variety of actively dividing morphological types. Many of the colonizing bacteria were flagellated. Filaments were seen after primary colonization of the surface. Transmission electron microscopy revealed a dominant gram-negative cell wall structure in the biofilm constituents. At least three types of glycocalyces were observed. The predominant glycocalyx possessed interstices and was densely labeled with cationized ferritin. Two of the glycocalyces appeared to mediate biofilm adhesion to the substratum. The results suggest that the predominant glycocalyx of this thin wastewater biofilm serves, in part, to: (i) enclose the bacteria in a matrix and anchor the biofilm to the substratum and (ii) provide an extensive surface area with polyanionic properties.     

Electron microscopic examination of wastewater biofilm formation and structural components

This research documents in situ wastewater biofilm formation, structure, and physiochemical properties as revealed by scanning and transmission electron microscopy. Cationized ferritin was used to label anionic sites of the biofilm glycocalyx for viewing in thin section. Wastewater biofilm formation paralleled the processes involved in marine biofilm formation. Scanning electron microscopy revealed a dramatic increase in cell colonization and growth over a 144-h period. Constituents included a variety of actively dividing morphological types. Many of the colonizing bacteria were flagellated. Filaments were seen after primary colonization of the surface. Transmission electron microscopy revealed a dominant gram-negative cell wall structure in the biofilm constituents. At least three types of glycocalyces were observed. The predominant glycocalyx possessed interstices and was densely labeled with cationized ferritin. Two of the glycocalyces appeared to mediate biofilm adhesion to the substratum. The results suggest that the predominant glycocalyx of this thin wastewater biofilm serves, in part, to: (i) enclose the bacteria in a matrix and anchor the biofilm to the substratum and (ii) provide an extensive surface area with polyanionic properties.