APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1977, p. 470-476 Copyright ©) 1977 American Society for Microbiology
Vol. 33, No. 2 Printed in U.S.A.
NOTES Scanning Electron Microscopy of Vaginal Colonization BRYAN LARSEN, * A. J. MARKOVETZ, AND R. P. GALASK Department of Microbiology and Department of Obstetrics and Gynecology, University of Iowa College of Medicine, Iowa City, Iowa 52242 Received for publication 21 September 1976
Changes
in the appearance of the vaginal epithelium of rats during the were seen by scanning electron microscopy. Bacterial colonization
estrous cycle
of this tissue appeared to be influenced by these changes. A valuable contribution to understanding host-microbial interaction has been made by using scanning electron microscopy to examine the microbial communities of the gastrointestinal tracts of various species (1-3, 7, 9). We have been studying the microecology of the vaginal epithelium of rats, and, in addition to identifying the bacterial species associated with the cervix and vagina (6), we have found that this represents an interesting tissue, in that the epithelium undergoes cyclic changes during the estrous cycle as does bacterial colonization. During the estrus phase of the cycle, bacterial counts measured by vaginal lavage and plate count (5) are several orders of magnitude greater than during other phases of the cycle. Because of these previous findings, scanning electron microscopy was used to examine the vaginal epithelium from rats during estrus and at other times during the cycle. The estrous cycle stage was determined by vaginal cytologic evaluation (8). Eight rats were sacrificed during various cycle stages. Tissue was collected by exposing the viscera with a midline incision, pouring ice-cold sodium cacodylate-buffered (0.1 M, pH 7.4) glutaraldehyde (2.5%) into the abdominal cavity and then resecting the internal genitalia in toto. The tissue was immersed in glutaraldehyde, and specimens of suitable size were cut from the upper one-third of the vagina. After glutaraldehyde fixation, samples were postfixed with osmium tetroxide, treated with thiocarbohydrazide (4), and treated again with osmium tetroxide. Specimens were dehydrated in increasing concentrations of ethanol, and were finally dehydrated by the criticalPresent address: Department of Obstetrics and Gynecology, General Hospital, University of Iowa, Iowa City, IA 52242.
point method, using the Sorvall (Norwalk, Conn.) critical-point-drying system, which employed CO, as the solvent. Critical-point-dried specimens were coated with gold-palladium (10 nm thickness) and examined in a Kent-Cambridge Stereoscan S-4 electron microscope. Tissue obtained during the diestrus phase of the cycle is characterized by the presence of epithelial cells organized in a single intact layer (Fig. 1). The surface of these cells gives the appearance of microvilli or a similar surface irregularity. Bacteria are notably absent. Bacteria may be seen on the surface of diestrus epithelium, but this is a rare observation. When present on the diestrus epithelium, bacterial forms are widely dispersed (usually seen singly) and distribution appears random (Fig. 2). Although bacteriological evidence presented elsewhere (5) shows a substantial diminution in colonization during diestrus, the extreme paucity of bacteria on the diestrus epithelium was nevertheless surprising, since a large number of bacteria are recoverable by vaginal lavage even during times of minimal colonization. A possible explanation, which may reconcile these two findings, is that bacterial colonization during diestrus is predominantly associated with the cervix rather than the vagina. Some bacteria of cervical origin would be expected to contribute to the counts determined by vaginal lavage and plate count. Actually, the cervix at diestrus does contain a substantial collection of cellular debris and microcolonies of characteristic bacterial forms (Fig. 3). This finding is consistent with the concept that diestrus counts primarily originate from the cervix. Vaginal epithelium obtained during estrus was densely colonized in contrast to the diestrus tissue (Fig. 4). Many bacterial forms ap470
VOL. 33, 1977
NOTES
471
FIG. 1. Scanning electron micrograph of vaginal epithelium. Tissue was taken from the upper one-third of the vagina during the diestrus phase of the cycle. Note the characteristic surface irregularity of the epithelium and the absence of bacterial forms. Bar represents 5 ,um.
472
NOTES
APPL. ENVIRON. MICROBIOL.
FIG. 2. Vaginal epithelium collected during diestrus. Bacteria are rarely seen on the diestrus epithelium but, where present, occur singly as illustrated. Bar represents 5 ,um.
NOTES
VOL. 33, 1977
_
473
.
FIG. 3. Scanning electron micrograph of the uterine cervix during diestrus. (A) Entire cervix; approximate magnification, x30. Note the collection of cellular debris in the cervical os, magnified in (B) (ca. x160). The cellular debris contains may microcolonies of bacterial forms (C). Bar represents 1 pum.
474
APPL. ENVIRON. MICROBIOL.
NOTES
4~~~~4
.
L;:f .I
FIG. 3C.
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FIG. 4. Appearance of the vaginal epithelium during estrus. Epithelial cells are flattened and detaching, and bacterial forms are more abundant than during the diestrus phase of the cycle. Bar represents 5 ,um.
476
APPL. ENVIRON. MICROBIOL.
NOTES
peared in each field examined. Bacteria frequently were distributed along the borders between cells or formed microcolonies. These findings have substantiated bacteriological studies presented previously (5), which indicate that the bacterial content of the rat genital tract is determined by events in the estrous cycle. The association of tissue changes with enhancement of colonization suggests the former may have some controlling influence on the genital flora. If the microflora of the vagina are a protective mechanism against genital tract infections, as is axiomatically believed by many, then it will be interesting to examine the relationship of fluctuations in the colonization density of the vaginal epithelium to the susceptibility of this tissue to infection. LITERATURE CITED 1. Akin, D. E. 1976. Ultrastructure of rumen bacterial attachment to forage cell walls. Appl. Environ. Microbiol. 31:562-568. 2. Erlandsen, S. E., and D. G. Chase. 1974. Morphological
3. 4.
5.
6. 7.
8. 9.
alterations in the microvillous border of villous epithelial cells produced by intestinal microorganisms. Am. J. Clin. Nutr. 27:1277-1286. Fuller, R., and B. E. Brooker. 1974. Lactobacilli which attach to the crop epithelium of the fowl. Am. J. Clin. Nutr. 27:1305-1313. Kelly, R. D., R. A. F. Dekker, and J. G. Bluemink. 1973. Ligand-mediated osmium binding: its application for scanning electron microscopy (SEM). J. Cell. Biol. 59:165a. Larsen, B., A. J. Markovetz, and R. D. Galask. 1976. Quantitative alterations of the genital microflora of female rats in relation to the estrous cycle. J. Infect. Dis. 134:486-489. Larsen, B., A. J. Markovetz, and R. P. Galask. 1976. The bacterial flora of the female rat genital tract. Proc. Soc. Exp. Biol. Med. 151:571-574. Takeuchi, A., H. R. Jerris, H. Nakazawa, and D. M. Robinson. 1974. Spiral-shaped organisms on the surface colonic epithelium of the monkey and man. Am. J. Clin. Nutr. 27:1287-1296. Turner, C. D. 1960. General endocrinology, 3rd ed. W. B. Saunders, Philadelphia. Savage, D. C., and R. V. H. Blumershine. 1974. Surface-surface associations in microbial communities populating epithelial habitats in the murine gastrointestinal ecosystem: scanning electron microscopy. Infect. Immun. 10:240-250.
Vol. 33, No. 2 Printed in U.S.A.
NOTES Scanning Electron Microscopy of Vaginal Colonization BRYAN LARSEN, * A. J. MARKOVETZ, AND R. P. GALASK Department of Microbiology and Department of Obstetrics and Gynecology, University of Iowa College of Medicine, Iowa City, Iowa 52242 Received for publication 21 September 1976
Changes
in the appearance of the vaginal epithelium of rats during the were seen by scanning electron microscopy. Bacterial colonization
estrous cycle
of this tissue appeared to be influenced by these changes. A valuable contribution to understanding host-microbial interaction has been made by using scanning electron microscopy to examine the microbial communities of the gastrointestinal tracts of various species (1-3, 7, 9). We have been studying the microecology of the vaginal epithelium of rats, and, in addition to identifying the bacterial species associated with the cervix and vagina (6), we have found that this represents an interesting tissue, in that the epithelium undergoes cyclic changes during the estrous cycle as does bacterial colonization. During the estrus phase of the cycle, bacterial counts measured by vaginal lavage and plate count (5) are several orders of magnitude greater than during other phases of the cycle. Because of these previous findings, scanning electron microscopy was used to examine the vaginal epithelium from rats during estrus and at other times during the cycle. The estrous cycle stage was determined by vaginal cytologic evaluation (8). Eight rats were sacrificed during various cycle stages. Tissue was collected by exposing the viscera with a midline incision, pouring ice-cold sodium cacodylate-buffered (0.1 M, pH 7.4) glutaraldehyde (2.5%) into the abdominal cavity and then resecting the internal genitalia in toto. The tissue was immersed in glutaraldehyde, and specimens of suitable size were cut from the upper one-third of the vagina. After glutaraldehyde fixation, samples were postfixed with osmium tetroxide, treated with thiocarbohydrazide (4), and treated again with osmium tetroxide. Specimens were dehydrated in increasing concentrations of ethanol, and were finally dehydrated by the criticalPresent address: Department of Obstetrics and Gynecology, General Hospital, University of Iowa, Iowa City, IA 52242.
point method, using the Sorvall (Norwalk, Conn.) critical-point-drying system, which employed CO, as the solvent. Critical-point-dried specimens were coated with gold-palladium (10 nm thickness) and examined in a Kent-Cambridge Stereoscan S-4 electron microscope. Tissue obtained during the diestrus phase of the cycle is characterized by the presence of epithelial cells organized in a single intact layer (Fig. 1). The surface of these cells gives the appearance of microvilli or a similar surface irregularity. Bacteria are notably absent. Bacteria may be seen on the surface of diestrus epithelium, but this is a rare observation. When present on the diestrus epithelium, bacterial forms are widely dispersed (usually seen singly) and distribution appears random (Fig. 2). Although bacteriological evidence presented elsewhere (5) shows a substantial diminution in colonization during diestrus, the extreme paucity of bacteria on the diestrus epithelium was nevertheless surprising, since a large number of bacteria are recoverable by vaginal lavage even during times of minimal colonization. A possible explanation, which may reconcile these two findings, is that bacterial colonization during diestrus is predominantly associated with the cervix rather than the vagina. Some bacteria of cervical origin would be expected to contribute to the counts determined by vaginal lavage and plate count. Actually, the cervix at diestrus does contain a substantial collection of cellular debris and microcolonies of characteristic bacterial forms (Fig. 3). This finding is consistent with the concept that diestrus counts primarily originate from the cervix. Vaginal epithelium obtained during estrus was densely colonized in contrast to the diestrus tissue (Fig. 4). Many bacterial forms ap470
VOL. 33, 1977
NOTES
471
FIG. 1. Scanning electron micrograph of vaginal epithelium. Tissue was taken from the upper one-third of the vagina during the diestrus phase of the cycle. Note the characteristic surface irregularity of the epithelium and the absence of bacterial forms. Bar represents 5 ,um.
472
NOTES
APPL. ENVIRON. MICROBIOL.
FIG. 2. Vaginal epithelium collected during diestrus. Bacteria are rarely seen on the diestrus epithelium but, where present, occur singly as illustrated. Bar represents 5 ,um.
NOTES
VOL. 33, 1977
_
473
.
FIG. 3. Scanning electron micrograph of the uterine cervix during diestrus. (A) Entire cervix; approximate magnification, x30. Note the collection of cellular debris in the cervical os, magnified in (B) (ca. x160). The cellular debris contains may microcolonies of bacterial forms (C). Bar represents 1 pum.
474
APPL. ENVIRON. MICROBIOL.
NOTES
4~~~~4
.
L;:f .I
FIG. 3C.
VOL. 33, 1977
NOTES
475
FIG. 4. Appearance of the vaginal epithelium during estrus. Epithelial cells are flattened and detaching, and bacterial forms are more abundant than during the diestrus phase of the cycle. Bar represents 5 ,um.
476
APPL. ENVIRON. MICROBIOL.
NOTES
peared in each field examined. Bacteria frequently were distributed along the borders between cells or formed microcolonies. These findings have substantiated bacteriological studies presented previously (5), which indicate that the bacterial content of the rat genital tract is determined by events in the estrous cycle. The association of tissue changes with enhancement of colonization suggests the former may have some controlling influence on the genital flora. If the microflora of the vagina are a protective mechanism against genital tract infections, as is axiomatically believed by many, then it will be interesting to examine the relationship of fluctuations in the colonization density of the vaginal epithelium to the susceptibility of this tissue to infection. LITERATURE CITED 1. Akin, D. E. 1976. Ultrastructure of rumen bacterial attachment to forage cell walls. Appl. Environ. Microbiol. 31:562-568. 2. Erlandsen, S. E., and D. G. Chase. 1974. Morphological
3. 4.
5.
6. 7.
8. 9.
alterations in the microvillous border of villous epithelial cells produced by intestinal microorganisms. Am. J. Clin. Nutr. 27:1277-1286. Fuller, R., and B. E. Brooker. 1974. Lactobacilli which attach to the crop epithelium of the fowl. Am. J. Clin. Nutr. 27:1305-1313. Kelly, R. D., R. A. F. Dekker, and J. G. Bluemink. 1973. Ligand-mediated osmium binding: its application for scanning electron microscopy (SEM). J. Cell. Biol. 59:165a. Larsen, B., A. J. Markovetz, and R. D. Galask. 1976. Quantitative alterations of the genital microflora of female rats in relation to the estrous cycle. J. Infect. Dis. 134:486-489. Larsen, B., A. J. Markovetz, and R. P. Galask. 1976. The bacterial flora of the female rat genital tract. Proc. Soc. Exp. Biol. Med. 151:571-574. Takeuchi, A., H. R. Jerris, H. Nakazawa, and D. M. Robinson. 1974. Spiral-shaped organisms on the surface colonic epithelium of the monkey and man. Am. J. Clin. Nutr. 27:1287-1296. Turner, C. D. 1960. General endocrinology, 3rd ed. W. B. Saunders, Philadelphia. Savage, D. C., and R. V. H. Blumershine. 1974. Surface-surface associations in microbial communities populating epithelial habitats in the murine gastrointestinal ecosystem: scanning electron microscopy. Infect. Immun. 10:240-250.
