Effect of Penicillin on the Succession, Attachment, and Morphology of Segmented, Filamentous Microbes in the

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1 INFECTION AND IMMUNITY, Jan. 1976, p Copyright C) 1976 American Society for Microbiology Vol. 13, No. 1 Printed in U.S.A. Effect of Penicillin on the Succession, Attachment, and Morphology of Segmented, Filamentous Microbes in the Murine Small Bowel CHARLES P. DAVIS' AND DWAYNE C. SAVAGE* Department of Microbiology and School of Basic Medical Sciences, University of Illinois, Urbana, Illinois Received for publication 13 August 1975 Indigenous segmented filamentous microbes attach to murine ileal epithelial cells. These microbes can be seen on the epithelial surface with a scanning electron microscope. They colonize preferentially the distal ileum in mice. Penicillin, placed in the animal's drinking water, eliminates the microbes from the mouse ileum, but recolonization of the ileum is observed 4 to 5 weeks after the penicillin treatment is stopped. Within 3 to 5 h after rats are given penicillin, the morphology of the microbes is changed. Their external surfaces are wrinkled or broken. Vacated and partially vacated attachment sites are observed. Almost all of the organisms disappear from murine ilea after the animals are exposed to penicillin for 10 h. These observations are discussed in relation to the microbe itself and in its interaction with ileal epithelial cells. Suckling, weanling, and adult murine intestines contain various microbial types localized in specific regions of the tract (4, 6, 11, 13, 14). The habitat and succession of lactic acid bacteria, coliforms, enterococci, bacteroides, fusiform- and spiral-shaped bacteria in mice have been described (3, 6, 13). We recently reported the habitat, succession, attachment, and morphology of another microbial type found in the ilea of mice and rats (5). This microorganism is a segmented, filamentous prokaryote that interacts with ileal epithelial cells to form an attachment site. The microbes can contain intrasegmental bodies that may be components in the life cycle of the microbe. In this paper, we present the attachment and morphology of this microbe in the normal murine ilea as observed with scanning electron microscopy. In addition, we show the effects of penicillin on the colonization, morphology, and attachment of these microbes in murine ilea as observed with scanning, transmission, and light microscopy. (This paper is part of a dissertation submitted by Charles P. Davis to the Graduate School of the University of Texas at Austin in partial fulfillment of the requirements for the Ph.D. degree.) MATERIALS AND METHODS Animals. ARS Ha (ICR)f mice and ARS/Sprague Dawley rats were purchased from ARS/Sprague I Present address: Department of Medical Microbiology and Surgery, University of Wisconsin, Madison, Wis Dawley, Madison, Wis. COBS CD-1(ICR) BR and C57BL/6St Crl BR mice were purchased from Charles River Breeding Laboratories, Inc., Wilmington, Mass. The animals were housed and maintained as described previously (5). When penicillin was given to mice and rats, it was added to sterile tap water (0.6 g/liter) and given to the animals ad libitum. The solution was replaced by a fresh preparation every 24 h. Histological methods. Animals were killed with alcohol-chloroform-ether vapors (5). Pieces of ileum (1 to 2 cm) were removed from mice and rats about 1 to 5 cm from the ileocecal junction. Other pieces of mouse ileum (1 to 2 cm) were removed from a region about 8 cm distal to the pyloric sphincter. Tissues processed for light microscopy were frozen with contents intact in 2% methylcellulose in 0.85% saline and sectioned while frozen. The sections were tissue Gram stained (13). If the tissue was processed for transmission electron microscopy (TEM), it was placed in Millonig phosphate-buffered 2% glutaraldehyde at 4 C after the contents were brushed gently aside. The tissue then was cut into strips about 1 mm wide and postfixed in phosphatebuffered osmium tetroxide (4). Bowel preparations for scanning electron microscopy (SEM) were processed by first clamping the ileal region to be removed at both ends with hemostats. Then either Ringers salt solution or phosphate-buffered 2% glutaraldehyde was injected until the bowel just began to distend. The clamped region was taken from the animal, and the hemostats were removed. If Ringers solution was injected, the excised section was drained, reinjected with phosphate-buffered 2% glutaraldehyde, and immersed in cold (4 C), phosphatebuffered 2% glutaraldehyde. An ileal homogenate, prepared by pooling the homogenates of 2-cm sections of the distal ilea of

2 VOL. 13, 1976 MICROBES IN THE MURINE SMALL BOWEL 181 five mice, was examined microscopically to determine if it contained segmented filamentous microbes. About 1 ml of the homogenate was poured over the food pellets of mice for one experiment. TEM. Strips of fixed mouse and rat ilea were dehydrated and embedded by the method of Spurr (15). Sections were cut on a Reichert ultramicrotome with either glass or diamond knives. The sections were stained with 2% uranyl acetate followed by a lead citrate stain (10). A Hitachi HU-11C electron microscope (Hitachi Ltd., Tokyo, Japan) was used to examine the sections. SEM. Tissues were dehydrated for SEM in the same manner as those prepared for TEM. Then tissues were infiltrated with amyl acetate-ethanol solutions (10, 40, 70, and 100% amyl acetate) at 5- to 15- min intervals and dried in a critical point dryer, made for the Center for Electron Microscopy by E. Murphy. Another method was used to examine resin-embedded tissue by SEM. An apparatus was built after the design of Erlandsen et al. that allowed aged 2% sodium hydroxide in absolute methanol to flow constantly over resin-embedded tissue samples (8). The tissue was processed as above for SEM, starting with the infiltration of 10% amyl acetate. Samples were coated with carbon for 30 s and then coated with gold-palladium in a Denton 502 or 503 evaporator (Denton Vacuum, Cherry Hill, N. J.). SEM examinations were performed with either a Cambridge Mark 2A or a JEM HU-3 scanning electron microscope at 20 kv. Light microscopy. Sections stained by a Gram method for tissues were examined with either a Leitz Ortholux microscope or a Zeiss Universal microscope. Unstained 1- to 3-cm sections of resinembedded tissue cut with glass knives on a ultramicrotome were viewed with phase optics with a Zeiss Universal microscope. RESULTS SEM examination of normal mouse small bowel. Mice of two strains, C57BL/6St Crl BR and COBS CD-1 (ICR) BR, were used in this experiment. Five animals from each strain were killed and processed for SEM. All of the animals except one C57BL/6St Crl BR mouse showed many organisms attached to the epithelial cells on the villi located close to the ileocecal valve. The microbes were attached apparently to epithelial cells by only one end and were usually present along one-half to threefourths of the length of the villus from its tip to its base (Fig. 1). None of the animals showed any segmented filamentous microbes on jejunal pieces taken within about 8 cm from the pyloric sphincter (Fig. 2). The length of the microbes varied from about 1 to 100 cm in length. Segments were observable on many of the microbes. They varied in both length and width (Fig. 3, 5). Vacant attachment sites were viewed on some villus epithelial cells. Often they were difficult to distinguish from small indentations formed at epithelial cell junctions, but some were seen with ease (Fig. 3, 4). These sites were usually observed only near or at the tips of the villi. Rarely, an exfoliated epithelial cell was observed with a microbe attached (Fig. 6). 'One of the mice, a C57BL/6St Crl BR, was infected with a tapeworm, Hymenolepsis nana (Fig. 7). The tapeworm was identified by the characteristic appearance of its eggs. It was attached by its scolex to the base of the villi. Interestingly, the segmented filamentous microbes were still found on villi adjacent to the tapeworm. Tapeworms were not observed in any of the CD-1 mice. Modification of segmented filamentous microbial populations in mouse ilea by penicillin. Adult ARS Ha(ICR)f mice were given penicillin-water for 7 days and then acidified water (0.001 HCl) for the remainder of the experiment. Animals of one group were fed an ileal homogenate 18 days after penicillin was removed from their drinking water. The ileal homogenate was used to determine whether or not the microbes in it expedite recolonization of the gastrointestinal tract. Segmented filamentous microbes were not found in any of 20 mice given the penicillinwater for 7 days. Moreover, these microbes were not found in any of 50 animals treated with penicillin-water and then given acid-water for 17 days. Many of the animals exposed to the ileal homogenate were colonized about 28 to 32 days after penicillin-water was removed (Table 1). Animals not exposed to the homogenate were colonized about 32 to 35 days after penicillin-water was removed. Thus, penicillin removed the microbes from the gastrointestinal tract of adult mice. The mice, then, are not recolonized for about 4 to 5 weeks. Furthermore, the animals exposed to the ileal homogenate were colonized only a few days earlier than those animals not given the homogenate. It was not known how quickly the segmented filamentous microbes were removed from the ileum, so adult COBS mice were given penicillin-water for 10.5 to 86 h. Five mice were killed at 10.5-, 18-, 24-, 48-, 72-, and 86-h intervals after penicillin was added to their drinking water. Pieces of their ilea were processed for light microscopy and TEM. The microbes were detected only in the mice exposed to penicillin-water for up to 10.5 h (Table 2). A microbe attached to an ileal epithelial cell was found in the sample from one mouse taken 10.5 h after treatment. The microbe's cytoplasm, cell wall, and attachment site were substantially modified (Fig. 8). Outer layers of

3 182 DAVIS AND SAVAGE INFECT. IMMUN.

4 VOL. 13, 1976 the cell wall were absent, and part of the remaining wall possessed an unusually sinuous profile. Its attachment site had only a small amount of the electron-dense layer adjacent to the organism. Likewise, the apical cytoplasm was not as markedly altered as that in epithelial cells with microbes not exposed to penicillin. Exfoliated epithelial cells with microbes attached were not observed. The foregoing findings indicated that segmented filamentous microbes disappear from the ilea of mice during a short period of treatment with penicillin. In addition, they suggested that the attachment site on the epithelial cell may undergo modifications. Modification of segmented filamentous microbial populations in rat ilea by penicillin. The above experiment was repeated, but ARS rats were used and examined at earlier time intervals. The tissues were taken from approximately the same area of the distal ileum. Pieces of ileum from each of the rats were processed for light microscopy and TEM. Ileal tissues embedded in resin for TEM examination were also used for phase microscopy and SEM. SEM samples were prepared by removing the resin from the TEM blocks. The microbes and their attachment sites did not change discernibly when compared to controls after a 1-h exposure to penicillin (Fig. 9-11); the organisms remained intact and attached to epithelial cells along the upper twothirds of the villi. Segmented filamentous microbes were located in all of the animals given penicillin for 1 to 3 h (Table 3). Rarely an exfoliated epithelial cell was observed with an organism and an attachment site (Fig. 9). Apparently empty attachment sites were observed with phase optics only in animals that had been drinking penicillin-water for 3 to 5 h (Fig. 12). Some of the microbes, viewed by SEM, and their attachment sites were visibly altered during the same time period. The organism's external surface appeared wrinkled or broken (Fig. 13, 14). Vacant attachment sites were found occasionally, and partially vacated sites were viewed frequently (Fig. 13, 14). Partially vacated sites consist of the attachment site associ- MICROBES IN THE MURINE SMALL BOWEL 183 ated with an altered microbe (Fig. 14). Because the segmented filamentous organisms were either removed or modified by penicillin, the attachment sites were observed as dark holes. These dark holes were usually not observed in animals that drank penicillin-water for 7 or more h. Furthermore, microbes were not seen usually in the rats drinking penicillinwater for 7 or more h (Fig. 15; Table 4). TEM studies of the attachment site showed that the dense band adjacent to the epithelial cell membrane was either absent or less apparent (Fig. 16). The underlying apical cytoplasm, although less electron dense than that found in animals not treated with penicillin, still did not contain cellular organelles. Occasionally, however, golgi and membrane-bound vesicles were seen next to this attachment site structure (Fig. 16). TEM micrographs showed that the previously described intrasegmental bodies were modified extensively. The bodies appeared to have lost cell walls and were irregularly shaped (Fig. 17). Surprisingly, segmented filamentous microbes were found to contain structures resembling bacterial endospores. They were observed in one rat that was exposed to penicillin-water for 1 h. Several filaments possessed the spores. All of the spores were at about the same stage of development, i.e., coat formation (Fig. 18, 19). The spores may be unusual because several sections revealed ultrastructure, suggesting that the spore was subdivided into two internal bodies (Fig. 19). DISCUSSION The findings presented in this paper reaffirm and extend the previous work done in this and other laboratories on segmented filamentous microbes (5, 7, 8, 11). The SEM micrographs support previous findings with TEM, indicating that the microbes have segments with various lengths and widths. Furthermore, they support data gained by light microscopy suggesting that the microbes could be attached to exfoliating epithelial cells (5). In addition, the data support previous work showing that the segmented filamentous bacteria populate the FIG. 1. Segmented filamentous microbes attached to distal ileal villi in a COBS CD-i ICR)BR mouse. x270. FIG. 2. Proximal jejunal villi without attached segmented filamentous microbes. x270. FIG. 3. Tip of villus with segmented filamentous organisms attached. The organism in the center of the box is about 1O0 p.m long. The small black spot to the right ofthe long organism is a vacant attachment site. x790. FIG. 4. Enlargement of boxed area in Fig. 3 showing the vacant attachment site in the right corner of the micrograph. x8,150. FIG. 5. Segmented filamentous microbes attached to mouse ileal epithelial cells. Note the difference in the length and width of individual segments. x3,640. FIG. 6. Exfoliated epithelial cell with a microbe attached. x7,400.

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6 VOL. 13, 1976 MICROBES IN THE MURINE SMALL BOWEL TABLE 1. Recolonization of mice with or without exposure to an ileal homogenatea Source of bacteriab No. of animals possessing microbes/no. examined 22' Ileal homogenate 1/10 2/10 6/10 9/10 5/10 10/10 No ileal homogenate 1/10 0/20 0/10 2/10 7/10 9/10 a Penicillin was placed in the drinking water at a concentration of 0.6 g/liter for 7 days. The animals were then given acid-water. Animals were killed, and frozen sections were obtained from pieces of distal ilea. These sections were stained with a tissue Gram stain and examined with a light microscope. b Control animals, which were known to have the segmented filamentous microbes in their ilea, were killed and their distal ilea were homogenized. The homogenate was poured over the food of mice that had not had access to penicillin-water for 18 days. I Days off penicillin. TABLE 2. Sequence of removal of segmented filamentous microbes from COBS mice given penicillina Penicillin-water treatment No. of animals with mi- (h)b crobes/no. examined /5 18 0/5 24 0/5 48 0/5 72 0/5 86 0/5 a Organisms were located by examination of Gram-stained sections of distal ilea. bpenicillin was placed in drinking water (0.6 g/liter). TABLE 3. Presence of segmented filamentous microbes in rat small bowel after administration of penicillin Detection method 185 No. of animals possessing microbes/no. examined 0ha lh 3h 5h 7h 10h Light microscopy- NDb 5/5 5/5 1/5 1/5 1/5 Gram stain Light microscopy ND 5/5 5/5 5/5 0/5 0/5 phase SEM 2/2 2/2 2/2 2/2 1/2 0/2 a Hours on penicillin. Animals were given 0.6 g of penicillin per liter in their drinking water. b ND, Not done. distal ileum in large numbers, whereas they do not populate the proximal ileum to any great extent. Our SEM observations indicate that about two-thirds of the length of the distal villi of normal mice and rats was populated by the microbes. Erlandsen and Chase reported that the microbes were most abundant on the apical and lateral third of rat intestinal villi; however, they also stated that some attach to epithelial cells in the intervillous space. Furthermore, they report a complete disruption of the microvillus border in densely colonized rats (7). Although we have observed disruption of the microvillus border in the immediate area of attachment, the great majority of microvillus borders observed was intact in both mice and rats. Several factors, such as the differences in rat strains, microbial strain, housing conditions, or others, may have contributed to the differences in our observations. In previous experiments in this laboratory, C57BL/6 St Crl BR mice did not possess segmented filamentous microbes in their small intestines. We suggested previously that either the mice were genetically incapable of interact- FIG. 7. A tapeworm (Hymenolepsis nana) attached by its scolex to the base of the villi in a C57BL/6St Crl BR mouse. Segmented filamentous microbes are present on adjacent villi. x295. FIG. 8. The remains ofthe microbe attached to a mouse ileal villus. Penicillin-water was given ad libitum for 105 h to the mouse. Note the decreased electron density ofthe attachment site when compared to untreated animals (see reference 5) and the absence of some of the organism's cell wall (arrow). x34,1(90. FIG. 9. Tissue Gram stain ofa segmented filamentous organism attached to an exfoliating epithelial cell in a rat given penicillin-water for 1 h. x5,150. FIG. 10. Segmented filamentous microbe, viewed with phase optics, attached to an ARS rat ileal epithelial cell. The rat was exposed to penicillin-water for 1 h. x5,150. FIG. 11. Deresinated distal rat villus with microbes attached. The rat was given penicillin-water for 1 h. X1,520. FIG. 12. Light micrograph (phase optics) showing a microbe and its attachment site in an epithelial cell (upper left corner) and an attachment site with no apparent associating microbe (lower right corner). The rat was exposed to penicillin-water for 3 h. x3,760.

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8 VOL. 13, 1976 TABLE 4. Attachment sites on epithelial cells in rats drinking penicillin-watera Hours on penicillin Empty attachment sitese 1 0/5c 3 5/5 5 3/5 7 0/5 10 0/5 a Rats were given penicillin-water (0.6 g/liter) adlibitum. b Empty attachment sites were observed with phase optics in sections of resin-embedded pieces of distal rat ileum. c Number of animals with empty sites/number of animals examined. ing with the microbes to form attachment sites or the animals simply were not exposed to the microbes (5). Apparently, the mice were not exposed previously because most of the C57BL/6St Crl BR mice possessed the bacteria in this study. Empty attachment sites, observed in normal mice as dark holes, suggested to us that a variation may exist in the proposed life cycle (5) of this microbe. We suggest that, once the microbe matures and releases its intrasegmental bodies, the organism no longer participates in maintaining the attachment site, and either the microbe itself or the epithelial cell releases the attached segment. Self-release suggests to us that the microbe either stops synthesizing compounds that allow it to adhere to the epithelial surface or starts synthesizing material that causes its release from the attachment site. Release of the segment by the epithelial cell seems unlikely, because the epithelial cell is almost at the end of its life-span on the apical villus when the empty attachment sites are observable. The ileal epithelial cell would have little or nothing to gain by the release of the bacterium after being colonized by it for more than one-half of its life span. The microbe would have an advantage, however, if it pos- MICROBES IN THE MURINE SMALL BOWEL sessed the ability to synthesize a substance that would stimulate an epithelial cell to form an attachment site; it could be anchored to the cell and thus not removed from a favorable environment to another that could be more inhospitable. A continual supply of nutrients, flowing through the intestine, could be another advantage for the attached microbe. Once the organism completed its release of the intrasegmental bodies, it would have no need to be attached. If the microbe had not completed its release of intrasegmented bodies, it would remain attached to the epithelial cell until it did so. If a few segmented filamentous microbes began their life cycle on epithelial cells located near the villus tip, then they may not have completed their cycle before the epithelial cells were exfoliated. Our observations of microbes attached to exfoliating epithelial cells suggest that such a situation may occur. Thus, we suggest that the microbe is detached from the epithelial cell usually before the cell exfoliates. Previous investigations have shown that penicillin changes the microbial flora of mice substantially. After penicillin is no longer given to the animals, the microbial populations eventually return to normal levels. The recovery period depends on the length of exposure to penicillin (12). Our findings indicated that mice were not recolonized for 4 to 5 weeks after the 1- week exposure to penicillin was stopped. This time period is longer than that found for the recolonization of coliforms, enterococci, and anaerobes. Qualitatively, however, lactobacilli, enterococci, and coliforms do not reach normal levels until 3 or 4 weeks after penicillin administration is ceased. In suckling mice, the segmented filamentous microbes begin to colonize about 1 week (day 21) after the anaerobic coliform and enterococci are established in the large bowel (3, 5). Thus recolonization of segmented filamentous microbes in the adult mouse given penicillin mimics closely the succession of bacteria observed in normal suckling mice. This suggests to us that the normal FIG. 13. Segmented filamentous microbes in a rat given penicillin-water for3 h. Filaments appear ragged and wrinkled. Arrow indicates an empty attachment site. x2,800. FIG. 14. Wrinkled microbe partially displaced from the attachment site (dark hole) x15,500. FIG. 15. Distal ileal villi of a rat given penicillin-water for 10 h. No segmented filamentous microbes or attachment sites are present. x410. FIG. 16. Micrograph of the microbe in a rat exposed to penicillin for 3 h. Apical cytoplasm below the microbe is slightly more electron dense than the other cytoplasm and it has no cellular organelles within it. Arrow (top) shows golgi adjacent to the site. Membrane-bound vesicles are seen next to the electron-dense cytoplasm (arrows, bottom). x27,800. FIG. 17. Intrasegmental bodies show irregularly shaped cell walls (cf. reference 5). Rat was given penicillin-water for 3 h. x40,700. FIG. 18. Spore present in a segmented filamentous microbe. Rat was exposed to penicillin-water for 1 h. x49,600. FIG. 19. Cross section ofa spore-containing segment. There may be two separate structures (arrows) within this spore. x44,

9 188 DAVIS AND SAVAGE succession and climax populations of bacteria in the mouse intestine must occur before these microbes establish their normal populations. Examination of light, TEM, and SEM micrographs revealed more information on the microbes themselves and their interaction with epithelial cells. The microbes were very sensitive to penicillin. They were almost completely removed from the small intestines of mice and rats after the animals were exposed to penicillin-water for 10 h. Damage to the microbe's filament was easily observed after a 3-h exposure to penicillin. The whole filament, including the intracellular bodies, was disrupted. This indicates that probably the entire filament and the intrasegmented bodies were either rapidly synthesizing cell wall material or were affected by the cessation of cell wall synthesis. In either case, the data strongly suggest that the entire filament and intrasegmental bodies are actively metabolizing. Such rapid growth is also suggested by the rapid rate of epithelial cell renewal in the small intestine (1); thus the microbes need to complete their life cycle every 2 days. Attachment sites were observed to be either vacated or partially vacated only after the rats were exposed to penicillin-water for 3 to 5 h. A few of these sites may be found in mice drinking penicillin-water for 7 to 10 h, but are probably so infrequent that observation is difficult or impossible. Removal of the attachment site and resynthesis of lumenal membrane and microvilli by the epithelial cells are suggested by the presence of golgi and membrane-bound vesicles adjacent to the attachment site. Furthermore, there were no large numbers of epithelial cells, with microbes and attachment site, observed exfoliating into the lumen. Since the attachment sites were not present on the vast majority of epithelial cells after a 7- to 10-h exposure to penicillin, it seems most likely that the epithelial cells replaced the attachment site with either lumenal membrane or microvilli, or both. In one rat that had access to penicillin-water for 1 h, spores were found in segmented filamentous microbes. Erlandsen et al. (Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, G187, p. 57) also reported viewing spores infrequently in these microbes. Segmented filamentous microbes, to our knowledge, have not been cultured in recognizable form. Previously, we suggested that the organism shares characteristics of the family Arthromitis, but this family and genera are not described in detail inbergey's Manual (2). They INFECT. IMMUN. are considered to be in an uncertain taxonomic position (2). These microbes seem to be ubiquitous in mice and rats. A similar microbe is found also in chickens (9). Because they are found commonly in mammals and birds, interact intimately with epithelial cells, and may produce compounds that induce bacterial cellepithelial cell adherence, segmented filamentous microbes are promising subjects for future investigation. ACKNOWLEDGMENTS We wish to thank the Center for Electron Microscopy, University of Illinois, for the use of its facilities. This investigation was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Abrams, G. D., H. Bauer, and H. Sprinz Influence of the normal flora on mucosal morphology and cellular renewal in the ileum. Lab. Invest. 12: Buchanan, R. E., and N. E. Gibbons (ed.) Bergey's manual of determinative bacteriology, 8th ed., p The Williams & Wilkins Co., Baltimore, Md. 3. Davis, C. P., J. S. McAllister, and D. C. Savage Microbial colonization of the intestinal epithelium in suckling mice. Infect. Immun. 7: Davis, C. P., D. Mulcahy, A. Takeuchi, and D. C. Savage Location and description of spiralshaped microorganisms in the normal rat cecum. Infect. Immun. 6: Davis, C. P., and D. C. Savage Habitat, succession, attachment, and morphology of segmented, filamentous microbes indigenous to the murine gastrointestinal tract. Infect. Immun. 10: Dubos, R., R. Schaedler, R. Costello, and P. Hoet Indigenous, normal and autochthonous flora of the gastrointestinal tract. J. Exp. Med. 122: Erlandsen, S. L., and D. G. Chase Morphological alterations in the microvillous border of villous epithelial cells produced by intestinal micro-organisms. Am. J. Clin. Nutr. 27: Erlandsen, S. L., A. Thomas, and G. Wendelschafer A simple technique for correlating SEM with TEM on biological tissue originally embedded in epoxy resin for TEM. p In 0. Johari and I. Covin (ed.), Scanning electron microscopy. IIT Research Institute, Chicago. 9. Fuller, R Ecological studies on the Lactobacillus flora associated with the crop epithelium of the fowl. J. Appl. Bacteriol. 36: Reynolds, E. S The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: Savage, D. C., and R. Blumershine Surface-surface associations in microbial communities populating epithelial habitats in the murine gastrointestinal ecosystem: scanning electron microscopy. Infect. Immun. 10: Savage, D. C., and R. Dubos Alterations in the mouse cecum and its flora produced by antibacterial drugs. J. Exp. Med. 128: Savage, D. C., R. Dubos, and R. S. Schaedler The gastrointestinal epithelium and its autochthonous bacterial flora. J. Exp. Med. 127: Savage, D. C., J. S. McAllister, and C. P. Davis Anaerobic bacteria on the mucosal epithelium of the murine large bowel. Infect. Immun. 4: Spurr, A. R A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:31-43.