ANTIMICROBIAL AGENTS AND CHEMOrHERAPY, May 1975, P. 704-711 Copyright 0 1975 American Society for Microbiology
Vol. 7, No. 5 Printed in U.S.A.
Scanning Electron Microscopy of Candida albicans After In Vitro Treatment with Miconazole SONJA DE NOLLIN* AND M. BORGERS Department of Cell Biology, Janssen Pharmaceutica, Research Laboratories, 2340-Beerse, Belgium Received for publication 21 January 1975
A study was made on the in vitro action of the antimycotic miconazole on Candida albicans yeast cells with scanning electron microscopy, and the effects were compared with those seen on the yeast cells by means of transmission electron microscopy. It was found that cells exposed to fungistatic and minimal fungicidal doses of miconazole (10-7 M and 10-6 M) presented rough surfaces and had multiple, desoriented buds and bud scars. Whereas in control cultures the cells were well separated, the treated ones formed small clusters of interconnected cells. After exposure to a fungicidal concentration (10-' M) of the drug, most of the remaining cells showed smooth surfaces and were covered with numerous vesicular structures probably representing cytoplasmic remnants derived from broken cells. This has been substantiated by the presence of abundant fragments of cell walls and confirmed by examination of similarly treated cultures in the transmission electron microscope. Moreover, the cells with an apparently intact surface when examined with scanning electron microscopy were shown with transmission electron microscopical examination to be completely necrotic inside.
The structure of Candida albicans and other yeasts as observed by scanning electron microscopy has been described earlier by Barnes et al. (2), Joshi et al. (14), Carteaud et al. (6), and Garrison and Lane (10). An investigation into the effects of several antimycotics on C. albicans surface was done by Ansehn et al. (1). The antimycotic studied here, miconazole, is active against most pathogenic fungi as well as against gram-positive bacteria (4, 5, 11, 12, 20). Miconazole (R 14889) is the generic name for
CYG medium (casein hydrolysate 0.5%, yeast extract 0.5%, and glucose 0.5%) was used in all experiments as broth medium. For each experiment, a loopful of C. albicans was transferred to 4.5 ml of Sabouraud glucose broth and incubated stationary for 24 h at 37 C. Cells were inociilated at a concentration of 2 x 105 cells/ml in the CYG medium. Treatment with miconazole. Immediately after inoculation, the cultures were exposed to fungistatic (10-7 M), minimal fungicidal (10-6 M), and fungicidal (10-4 M) doses of miconazole and incubated as shake cultures in Erlenmeyer flasks at 37 C. Miconazole was dissolved in dimethylsulfoxide. The final concentration of dimethylsulfoxide in the cultures 2-(2,4-dichlorophenyl)-2[(2,4-dichlorophen1-f 0.05%. Control cultures containing the equal yl) methoxy]ethyll- lH-imidazole mononi- was amount of the solvent were incubated at the same trate. As shown previously in an ultrastruc- time. tural study (7), miconazole acts primarily Scanning electron microscopy (SEM). The cells by altering the plasmalemma and cell wall, were harvested after 24 h of incubation and washed inducing permeability changes. Depending on three times with physiological saline solution and dose and duration of the treatment, the yeast fixed for 10 min in a mixture of 6 volumes of 2% cells show progressive cytoplasmic deterioration osmiumtetroxide buffered to pH 7.2 with 0.05 M and prominent shape changes, finally resulting veronal-acetate and 1 volume of saturated aqueous mercuric chloride (18). After centrifugation, the cell in complete cell necrosis. pellets were washed thoroughly with veronal-acetate The purpose of the present investigation is to buffer (0.05 M) plus 0.22 M sucrose and dehydrated characterize with the aid of scanning electron in a graded ethanol-amylacetate mixture, until they microscopy the surface alterations and the gen- were finally impregnated with 100% amylacetate. The after in eral shape changes of C. albicans cells samples were dried by the critical-point method, vitro exposure to fungistatic and fungicidal coated with a thin layer of gold, and examined in a doses of the antimycotic miconazole. Cambridge Stereoscan electron microscope S4-10 at acceleration voltages of 10 and 20 kV. These experiMATERIALS AND METHODS ments were repeated seven times. Transmission electron microscopy (TEM). The Culture conditions. The C. albicans strain (57012) was maintained on Sabouraud glucose agar at 25 C. preparation procedure of C. albicans cultures for 704
VOL. 7, 1975
SCANNING ELECTRON MICROSCOPY OF C. ALBICANS
TEM was the same as described previously (3). For comparative reasons, some cell pellets were prepared by the SEM procedure until impregnated with 100% amylacetate, followed by embedding in epon and examination by TEM. Phase contrast microscopy. The unfixed cells were spread on a microscope slide and examined without staining.
705
RESULTS Control cells. The blastospores of C. albicans in SEM were generally smooth-walled bodies, spherical to elongate in shape (Fig. 1). Their mean diameter was 5 ,um, and under these in vitro circumstances they were mostly present in yeast form. Pseudomycelium forms were occa-
FIG. 1. Control cells, examined by SEM. Survey picture of C. albicans yeast cells. All cells lie apart, and show smooth surfaces and polar bud scars (arrows) (x35,400).
706
DE NOLLIN AND BORGERS
sionally found. All the yeast cells were lying apart, showing polar buds and bud scars (Fig. 1). The ultrastructure of C. albicans as seen by TEM is demonstrated later (see Fig. 8). The detailed description has been reported previously (3). Treated cells. The alterations of the surface micromorphology shown here were observed in most of the examined cells. Exposure to fungistatic (10-7 M) and minimal fungicidal (10-6 M) concentrations of miconazole. The surface of the miconazoletreated cells appeared uniformly rough because of a well-defined wrinkling of the cell wall. Moreover, the yeast cells were present in clusters of interconnected cells (Fig. 2). Single forms were rarely seen, and several buds and bud scars were present on one cell. The polar position of these buds was completely lost (Fig. 2 and 3). It was noted that the cells became round (Fig. 2). In some cells, and this mostly after treatment with minimal fungicidal doses, several small blebs appeared on the indented cell wall (Fig. 3). Some of these observations closely corroborate our previous TEM findings of frequent focal thickenings of the cell wall and deposition of irregular, dense inclusions in these walls (7; Fig. 4). Parts of the cell wall that protruded in the cytoplasm were commonly observed in TEM and were interpreted as an indication for a collapse of the wall. Exposure to a fungicidal (10-4 M) concentration of miconazole. SEM of cultures exposed to this high dose showed a completely different picture as compared with low-dose cultures. The variety and complexity of the alterations occurred in all the examined samples. The general survey (Fig. 5) demonstrates small groups of cells which are mostly covered with numerous vesicles of varying size. Large amounts of vesicular material were equally found distributed in between the grouped cells. This material, seen in more detail (Fig. 6), was presumably derived from broken cells and presented membrane-limited cytoplasmic remnants. This observation is strengthened by the frequent occurrence of irregular flattened surfaces which probably represent the remainders of the walls of the broken cells (Fig. 7). The remaining unbroken cells showed a smooth surface as observed in control cells. A control C. albicans cell as examined by TEM showed a regular outlined cell wall (Fig. 8). Pictures obtained by TEM indicated that the walls of such cells were completely regular in outline, without showing deposition of membra-
ANTIMICROB. AGENTS CHEMOTHER.
nous material in the wall as observed in the lower-dose cultures. However, the cell interior looked completely necrotic; internal subcellular organelles were barely identified. Large quantities of fat globules were present near the cell periphery, and only fragments of the plasmalemma were preserved (Fig. 9). Membranelimited cytoplasmic remnants probably corresponding to those observed with SEM were found sticking to the wall. It appeared, however, that the amount of vesicular material found in close proximity to the cell walls was not as large as expected from SEM observations. To exclude an artifact of the SEM procedure, cell pellets prepared for SEM, were examined with TEM. No difference was found between the two preparation procedures as far as the frequency in appearance of vesicles was concerned. Examination with phase contrast microscopy revealed that aggregations of cells were surrounded by a dark halo of vesicular and irregularly shaped material, probably derived from broken cells. Apart from these, a certain number of cells apparently underwent drastic shape changes.
DISCUSSION The SEM observations presented in this study clearly confirm the potent fungicidal action exerted by miconazole (20). The surface alterations are most probably due to a change in cell permeability, which is in agreement with earlier ultrastructural observations, showing that the first changes are localized at the plasmalemma and cell wall before any alteration can be detected in the cell interior (7). Studies with miconazole, done by Sreedhara Swamy et al. (19), showed that the permeability of the plasmalemma was altered, as evidenced by the leakage of cations, amino acids, and proteins. This was demonstrated also by Van den Bossche for miconazole (21) and other antimycotic and polyenic drugs (9, 13, 15, 16). It is obvious that this permeability change provokes an osmotic imbalance, which could explain the indentations of the walls in collapsed cells. The folds in the cell wall, as noted in most of the cells exposed to the lower doses, may correspond to the focal areas where membranous material is deposited between the plasmalemma and the cell wall or in the cell wall itself, thereby causing a bulged appearance of the cellular contour. In contrast to the observations of Ansehn et al. (1) of C. albicans after treatment with clotrimazole and amphotericin 8, cracks in the cell wall were not observed in all our samples, appearing only in one. In the latter sample (which was one of those exposed to 10' M
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Vol. 7, No. 5 Printed in U.S.A.
Scanning Electron Microscopy of Candida albicans After In Vitro Treatment with Miconazole SONJA DE NOLLIN* AND M. BORGERS Department of Cell Biology, Janssen Pharmaceutica, Research Laboratories, 2340-Beerse, Belgium Received for publication 21 January 1975
A study was made on the in vitro action of the antimycotic miconazole on Candida albicans yeast cells with scanning electron microscopy, and the effects were compared with those seen on the yeast cells by means of transmission electron microscopy. It was found that cells exposed to fungistatic and minimal fungicidal doses of miconazole (10-7 M and 10-6 M) presented rough surfaces and had multiple, desoriented buds and bud scars. Whereas in control cultures the cells were well separated, the treated ones formed small clusters of interconnected cells. After exposure to a fungicidal concentration (10-' M) of the drug, most of the remaining cells showed smooth surfaces and were covered with numerous vesicular structures probably representing cytoplasmic remnants derived from broken cells. This has been substantiated by the presence of abundant fragments of cell walls and confirmed by examination of similarly treated cultures in the transmission electron microscope. Moreover, the cells with an apparently intact surface when examined with scanning electron microscopy were shown with transmission electron microscopical examination to be completely necrotic inside.
The structure of Candida albicans and other yeasts as observed by scanning electron microscopy has been described earlier by Barnes et al. (2), Joshi et al. (14), Carteaud et al. (6), and Garrison and Lane (10). An investigation into the effects of several antimycotics on C. albicans surface was done by Ansehn et al. (1). The antimycotic studied here, miconazole, is active against most pathogenic fungi as well as against gram-positive bacteria (4, 5, 11, 12, 20). Miconazole (R 14889) is the generic name for
CYG medium (casein hydrolysate 0.5%, yeast extract 0.5%, and glucose 0.5%) was used in all experiments as broth medium. For each experiment, a loopful of C. albicans was transferred to 4.5 ml of Sabouraud glucose broth and incubated stationary for 24 h at 37 C. Cells were inociilated at a concentration of 2 x 105 cells/ml in the CYG medium. Treatment with miconazole. Immediately after inoculation, the cultures were exposed to fungistatic (10-7 M), minimal fungicidal (10-6 M), and fungicidal (10-4 M) doses of miconazole and incubated as shake cultures in Erlenmeyer flasks at 37 C. Miconazole was dissolved in dimethylsulfoxide. The final concentration of dimethylsulfoxide in the cultures 2-(2,4-dichlorophenyl)-2[(2,4-dichlorophen1-f 0.05%. Control cultures containing the equal yl) methoxy]ethyll- lH-imidazole mononi- was amount of the solvent were incubated at the same trate. As shown previously in an ultrastruc- time. tural study (7), miconazole acts primarily Scanning electron microscopy (SEM). The cells by altering the plasmalemma and cell wall, were harvested after 24 h of incubation and washed inducing permeability changes. Depending on three times with physiological saline solution and dose and duration of the treatment, the yeast fixed for 10 min in a mixture of 6 volumes of 2% cells show progressive cytoplasmic deterioration osmiumtetroxide buffered to pH 7.2 with 0.05 M and prominent shape changes, finally resulting veronal-acetate and 1 volume of saturated aqueous mercuric chloride (18). After centrifugation, the cell in complete cell necrosis. pellets were washed thoroughly with veronal-acetate The purpose of the present investigation is to buffer (0.05 M) plus 0.22 M sucrose and dehydrated characterize with the aid of scanning electron in a graded ethanol-amylacetate mixture, until they microscopy the surface alterations and the gen- were finally impregnated with 100% amylacetate. The after in eral shape changes of C. albicans cells samples were dried by the critical-point method, vitro exposure to fungistatic and fungicidal coated with a thin layer of gold, and examined in a doses of the antimycotic miconazole. Cambridge Stereoscan electron microscope S4-10 at acceleration voltages of 10 and 20 kV. These experiMATERIALS AND METHODS ments were repeated seven times. Transmission electron microscopy (TEM). The Culture conditions. The C. albicans strain (57012) was maintained on Sabouraud glucose agar at 25 C. preparation procedure of C. albicans cultures for 704
VOL. 7, 1975
SCANNING ELECTRON MICROSCOPY OF C. ALBICANS
TEM was the same as described previously (3). For comparative reasons, some cell pellets were prepared by the SEM procedure until impregnated with 100% amylacetate, followed by embedding in epon and examination by TEM. Phase contrast microscopy. The unfixed cells were spread on a microscope slide and examined without staining.
705
RESULTS Control cells. The blastospores of C. albicans in SEM were generally smooth-walled bodies, spherical to elongate in shape (Fig. 1). Their mean diameter was 5 ,um, and under these in vitro circumstances they were mostly present in yeast form. Pseudomycelium forms were occa-
FIG. 1. Control cells, examined by SEM. Survey picture of C. albicans yeast cells. All cells lie apart, and show smooth surfaces and polar bud scars (arrows) (x35,400).
706
DE NOLLIN AND BORGERS
sionally found. All the yeast cells were lying apart, showing polar buds and bud scars (Fig. 1). The ultrastructure of C. albicans as seen by TEM is demonstrated later (see Fig. 8). The detailed description has been reported previously (3). Treated cells. The alterations of the surface micromorphology shown here were observed in most of the examined cells. Exposure to fungistatic (10-7 M) and minimal fungicidal (10-6 M) concentrations of miconazole. The surface of the miconazoletreated cells appeared uniformly rough because of a well-defined wrinkling of the cell wall. Moreover, the yeast cells were present in clusters of interconnected cells (Fig. 2). Single forms were rarely seen, and several buds and bud scars were present on one cell. The polar position of these buds was completely lost (Fig. 2 and 3). It was noted that the cells became round (Fig. 2). In some cells, and this mostly after treatment with minimal fungicidal doses, several small blebs appeared on the indented cell wall (Fig. 3). Some of these observations closely corroborate our previous TEM findings of frequent focal thickenings of the cell wall and deposition of irregular, dense inclusions in these walls (7; Fig. 4). Parts of the cell wall that protruded in the cytoplasm were commonly observed in TEM and were interpreted as an indication for a collapse of the wall. Exposure to a fungicidal (10-4 M) concentration of miconazole. SEM of cultures exposed to this high dose showed a completely different picture as compared with low-dose cultures. The variety and complexity of the alterations occurred in all the examined samples. The general survey (Fig. 5) demonstrates small groups of cells which are mostly covered with numerous vesicles of varying size. Large amounts of vesicular material were equally found distributed in between the grouped cells. This material, seen in more detail (Fig. 6), was presumably derived from broken cells and presented membrane-limited cytoplasmic remnants. This observation is strengthened by the frequent occurrence of irregular flattened surfaces which probably represent the remainders of the walls of the broken cells (Fig. 7). The remaining unbroken cells showed a smooth surface as observed in control cells. A control C. albicans cell as examined by TEM showed a regular outlined cell wall (Fig. 8). Pictures obtained by TEM indicated that the walls of such cells were completely regular in outline, without showing deposition of membra-
ANTIMICROB. AGENTS CHEMOTHER.
nous material in the wall as observed in the lower-dose cultures. However, the cell interior looked completely necrotic; internal subcellular organelles were barely identified. Large quantities of fat globules were present near the cell periphery, and only fragments of the plasmalemma were preserved (Fig. 9). Membranelimited cytoplasmic remnants probably corresponding to those observed with SEM were found sticking to the wall. It appeared, however, that the amount of vesicular material found in close proximity to the cell walls was not as large as expected from SEM observations. To exclude an artifact of the SEM procedure, cell pellets prepared for SEM, were examined with TEM. No difference was found between the two preparation procedures as far as the frequency in appearance of vesicles was concerned. Examination with phase contrast microscopy revealed that aggregations of cells were surrounded by a dark halo of vesicular and irregularly shaped material, probably derived from broken cells. Apart from these, a certain number of cells apparently underwent drastic shape changes.
DISCUSSION The SEM observations presented in this study clearly confirm the potent fungicidal action exerted by miconazole (20). The surface alterations are most probably due to a change in cell permeability, which is in agreement with earlier ultrastructural observations, showing that the first changes are localized at the plasmalemma and cell wall before any alteration can be detected in the cell interior (7). Studies with miconazole, done by Sreedhara Swamy et al. (19), showed that the permeability of the plasmalemma was altered, as evidenced by the leakage of cations, amino acids, and proteins. This was demonstrated also by Van den Bossche for miconazole (21) and other antimycotic and polyenic drugs (9, 13, 15, 16). It is obvious that this permeability change provokes an osmotic imbalance, which could explain the indentations of the walls in collapsed cells. The folds in the cell wall, as noted in most of the cells exposed to the lower doses, may correspond to the focal areas where membranous material is deposited between the plasmalemma and the cell wall or in the cell wall itself, thereby causing a bulged appearance of the cellular contour. In contrast to the observations of Ansehn et al. (1) of C. albicans after treatment with clotrimazole and amphotericin 8, cracks in the cell wall were not observed in all our samples, appearing only in one. In the latter sample (which was one of those exposed to 10' M
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