A micromethod for the quantitative determination of the viability of Candida albicans hyphae

Journal of Microbiological Methods 1 (1983) 89-98

89

Elsevier

A micromethod for the quantitative determination of the viability of Candida albicans hyphae Tadayo Hashimoto Department of Microbiology, Stritch School of Medicine, Loyola University of Chicago, 2160 South First Avenue, Maywood, IL 60153 (U.S.A.)

(Received 23 November 1982) (Revised version received 7 February 1983) (Accepted 11 February 1983)

Summary A micromethod for the quantitative determination of the viability of Candida albicans hyphae was devised which takes advantage of the dimorphic nature of C. albicans which grows exclusively in the yeast form when incubated aerobicallyon Sabouraud dextrose agar at 30°C. When tested by this method, all viable C. albicans hyphae were recognized as microcoloniesconsisting of one hypha surrounded by several yeast form progeny. In contrast to this, no yeast form progeny emerged from nonviable hyphae. By counting appropriate total numbers (200--400)of microcolony-forminghyphae and infertile hyphae, it was possible to determine the ratio of viable to nonviable cells in a given hyphal suspension. This micromethod may be used for quantitative assessment of the candidacidal effects of various antimycotic agents or phagocytes on C. albicans hyphae whose viability could not have been determined by the conventional plating technique because of the species' high propensity to clump. Key words: Candida albicans hyphae - Viability of Candida - Micromethods, Candida viability test

Introduction Because of the general recognition of the important role played by phagocytes in the host resistance to C. albicans infections [1], many workers have investigated Candida-phagocyte interactions both in vitro and in vivo. In the majority of studies, however, p h a g o c y t e - C a n d i d a interactions were investigated by using only the yeast form of the fungus. Until recently, little or no attention has been paid to the reaction of the host to the hyphal form of C. albicans. Davies and Denning [2] were probably the first to suspect that C. albicans hyphae might be killed by an 'extraphagocytic' mechanism. Recently, this possibility has been more systematically investigated by D i a m o n d et al. [3, 4] who demonstrated that human polymorphonuclear leukocytes (PMNs) and monocytes could attack and damage C. albicans hyphae without total engulfment. The damage to C. albicans hyphae that had been exposed to human phagocytes was estimated by reduced [14C]cytosine incorporation into such hyphae. In their work [3, 4], however, the loss of viability of 0167-7012/83/$03.00 © 1983 Elsevier Science Publishers B.V.

90

C. albicans hyphae could not be firmly established by the conventional plating method because of the clumping problem. Presently no reliable method is available that can determine the viability of C. albicans hyphae exposed to the candidacidal action of drugs or phagocytes. In most studies, the viability of Candida cells has been estimated indirectly by such methods as dye exclusion [5-10], release of radiolabeUed compounds [11], incorporation of radiolabelled chemicals [3, 4, 12-14], and differential staining with acridine orange [15]. Only in one instance [7] was the conventional plating technique used in combination with these indirect methods. During the course of a study on macrophage-Candida hypha interactions in vitro, a reliable method has been developed which overcomes this difficulty. The method has been successfully used in studying the candidacidal activity of murine peritoneal macrophages activated by Listeria immunization and Listeria elicitation. (T. Hashimoto and S.F. Conti. In: Abstr. 20th Interscience Conference On Antimicrobial Agents and Chemotherapy, 1981, No. 586). In this paper, a novel microculture method for determination of C. albicans hyphae will be described and its accuracy and applicability will be assessed under various experimental conditions. Materials and Methods

Preparation of non-phagocytozable Candida albicans hyphae A strain of C. albicans (LUMC-101) serotype A obtained from the Clinical Microbiology Laboratory of Loyola University Medical Center was used throughout this investigation. The yeast form of C. albicans was obtained by growing this strain aerobically on Sabouraud dextrose agar (Difco Lab.) for 24 h at 37°C. Candida cells collected from isolated colonies were washed thoroughly in Hank's balanced salt (Grand Island Biological Co.) solution prior to inoculation into a tissue culture medium specified below. C. albicans cells were transformed into the hyphal form by incubating at 37°C in a synthetic serum-free tissue culture medium (Dulbecco's modified Eagle Medium: DMEM, Grand Island Biological Co.). With an inoculum of approximately 1 × 106 C. albicans yeast cells/ml, the medium consistently yielded 100% hyphae ranging in length from 40 to 50 ~tm when incubated for 3 h. Macrophages Peritoneal macrophages from outbred female mice (CD-1 strain, Charles River Breeding Laboratories, Inc., Wilmington, MA) weighing 20-25 g were used in this investigation. In a preliminary study, it was shown that resident macrophages from these mice had relatively low candidacidal activities for both the yeast and hyphal forms of C. albicans, and their non-specific candidacidal activities were shown to be significantly enhanced by in vivo activation treatment with live Listeria monocytogenes strain A4413 cells. The procedure used for activation of macrophages by L. monocytogenes vaccination was described by Harrington-Fowler et al. [16]. Peritoneal macrophages were harvested, purified and cultured in DMEM by the method described by Harrington-Fowler et al. [16]. In most experiments, a

91 circular cover slip (12 mm diameter, No. 1 thickness, Rochester Scientific Co.) was placed in each well (Coster chamber 3524, Coster, Cambridge, MA) prior to seeding. The macrophage viability was routinely determined by trypan blue dye (0.3%) exclusion [16], and the purity of the macrophage population was examined microscopically after staining cover slips with a Diff-Quick-system (Harleco, N J).

In vitro interaction of peritoneal macrophages with C. albicans hyphae Macrophage cultures containing approximately 1 x 1 0 6 macrophages per well (Coster wells) were inoculated with freshly prepared C. albicans hyphae, and incubated at 37°C for 30 min to 7 h (6 h in most experiments). The culture conditions were the same as those employed for culturing macrophages. Low multiplicities of infection (MOI), ranging from 0.1 to 0.02, were used routinely because a higher MOI resulted in poor killing of Candida hyphae due to early damage of macrophages. Samples taken at predetermined intervals (30-60 min) were processed for determination of the viability of C. albicans hyphae according to the method described below. Quantitative determination of the viability of non-phagocytozable C. albicans hyphae The rationale for the microculture method to determine the viability of C. albicans hyphae is illustrated in Fig. 1. Basically this method has taken advantage of the dimorphic nature of C. albicans. The viability of individual C. albicans hyphae recovered from the culture wells was determined as follows: Macrophages were first lyzed by 0.5% Nonidet P-40, (Sigma). Candida hyphae were then released into culture medium by gentle scraping of the bottom of culture wells with a rubber policeman (glass rod covered with amber pure gum rubber, Scientific Product R5110-1). Several gentle scrapings were sufficient to release the majority of hyphae in each well. The C. albicans hyphae collected by centrifugation (2000 x g, 5 min) of the medium were inoculated onto the flat surface (1.5 x 1.5 cm, 2 mm thick) of Sabouraud dextrose agar (Difco) placed on slide glass. Any excess fluid remaining on the surface of the agar after inoculation should be carefully removed by a piece of filter paper. The inoculated microcultures were incubated aerobically at 30°C for up to 6-8 h in a moist chamber. Then, the microculture surface was carefully covered with a thin cover glass (No. 1) and examined under a phase contrast microscope. Since the incubation conditions (30°C, aerobic, high glucose, neopeptone medium) strongly favor the growth of this dimorphic fungus in the yeast form, all viable hyphae inoculated on microculture agar would form a cluster of yeast cells along the hyphae (Fig. 1). Nonviable hyphae would not produce any yeast cells. Routinely, 200 hyphae were examined under the phase contrast microscope for each sample, and the percentage of killed hyphae in each sample was calculated. All samples were encoded in such a way that those who examined the microcultures had no knowledge of the treatment given to the hyphae. The viability could be more readily determined when C. albicans hyphae were exposed to macrophages cultured on the surface of a cover glass. In this case, the

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C

Fig. 1. The rationale behind the micromethod for determination of the viabilityof nonphagocytosable C. albicans hypha. C. albicans hypha (A) produced in a tissue culture medium is injured by macrophages (B) or antimyeotic agents. Viable C. albicans hypha can produce yeast form progeny on the microculture agar (Sabouraud dextrose) surface when incubated aerobically at 30°C resulting in a microcolonyformation (C). If a C. albicans hypha is killed, no microcolonyis formed under the same condition (D).

cover slips were retrieved from culture wells and soaked briefly (3-5 min) in Sabouraud dextrose broth containing 0.5% Nonidet, and subsequently rinsed gently in the broth before being placed on microculture agar with the cell side facing the agar surface. This procedure only partially lyzed the macrophage membrane allowing essentially all of the C. albicans hyphae to adhere to the glass surface via remnants of the macrophage membrane. The microculture was incubated at 30°C for several hours before determining the viability microscopically as described above. Apparently, the oxygen dissolved in Sabouraud dextrose agar was sufficient to produce only yeast form progeny from viable hyphae under our experimental conditions. Prior to applying this technique to the determination of the viability of C. albicans hyphae damaged by macrophages, a series of tests were performed with C. albicans hyphal suspensions in which known percentages of cells had been rendered nonviable by heat treatment. T o prepare a 1:1 mixture of viable and nonviable C. albicans hyphae, one half of a cell suspension (106 hyphae/ml) was heated at 100°C for 10 min, rapidly cooled, and then mixed with the second half (unheated) before testing. In some experiments, C. albicans hyphae killed by 0.1 M hydrogen peroxide or by ultraviolet light irradiation were also used. Determination o f viability o f C. albicans hyphae by the conventional plate count method For comparison with the above technique, the viability of C. albicans hyphae was also determined by the conventional plating method. One tenth ml samples of appropriately diluted hyphal suspensions were placed on Sabouraud dextrose agar plates spread evenly with a glass rod and then incubated at 37°C for 24 h before counting the number of colonies. All plate countings were performed in triplicate.

93

Dye exclusion test In certain comparative studies, the dye exclusion technique [9] using 0.01% methylene blue was employed as an indirect m e t h o d to determine the viability of C. albicans cells. Microscopy and photomicrography All light microscopy was p e r f o r m e d with a Zeiss Universal or Nikon light microscope fitted with phase contrast objectives. Photomicrographs were m a d e on panchromatic film (Plus-X, Kodak) with a Nikon c a m e r a equipped with an automatic exposure system. Results W h e n a suspension of viable C. albicans hyphae (average length 46 + 7 ~m) containing 1.1 x 106 viable cells per ml ( D M E M ) was serially diluted and plated out on Sabouraud dextrose agar plates, the numbers of colonies developing after incubation at 37°C for 24 h could account for only 7 . 9 - 6 2 % , depending on the type of diluent employed, of the actual viable cell counts (Table 1). These results confirm the difficulty of determining the viability of C. albicans hyphae by the conventional plating technique. Each viable hypha placed on the microculture agar produced there a microcolony consisting of one hypha surrounded by a cluster of yeast form progenies. A n example of such a microcolony is illustrated in Fig. 2A. Nonviable hyphae were, on the o t h e r hand, unable to reproduce; thus, no microcolonies were f o r m e d (Fig. 2B). T h e majority of viable hyphae produced discernible microcolonies within 4 h of incubation, and there was essentially no significant increase in the n u m b e r of microcolonies after 6 h (Fig. 3). With this microculture technique, it

TABLE 1 EFFECT OF DILUENT ON THE COLONY FORMATION BY C. ALBICANS HYPHAE AS DETERMINED BY THE CONVENTIONAL PLATING TECHNIQUE A suspension containing 1.1 x 106 viable C. albicans hyphae per ml of DMEM was prepared, diluted with specified diluents and plated out as described in the Materials and Methods section. Diluent used

Number of colonies formed

Recovery (%)

DMEM Distilled water 0.8% NaCI solution Sabouraud dextrose broth Na phosphate buffer (0.05 M, pH 7.2) Tris-maleate NaOH buffer (0.05 M, pH 7.2)

6.2 x 8.7 x 2.3 x 4.2 x 1.1 x

56.4 7.9 21 38 10

l0 s 104 los 105 105

6.8 x los

62

94

Fig. 2. A phase contrast photomicrograph of a microcolony developed from a single viable C. albicans hypha (A). If a hypha was dead, no microcolony could be formed (B). Both hyphae (A and B) were exposed to activated murine peritoneal macrophages for 3 h. Note that remnants of macrophage membranes are still discernible along the nonviable hypha (B).

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Fig. 3. The kinetics of C. albicans microcolony formation o n the microculture agar surface inoculated with all viable hyphae (A), all heat-killed hyphae (B) and the hyphae of which 50% were killed by heat prior to testing (C). Note that the maximum number of microcolonies was reached after 5-6 h of incubation. TABLE 2 EFFECTS OF SELECTED CHEMICAL, PHYSICAL, AND BIOLOGICAL TREATMENTS ON THE VIABILITY OF C. ALBICANS HYPHAE AS DETERMINED BY THE MICROCULTURE AND THE DYE EXCLUSION TECHNIQUES Treatment

Control (untreated) 0.5% Nonidet for 15 min Heating (boiling for 10 min) 0.1 M HzO2 for 30 min UV irradiation a Resident macrophages b Activated macrophages b Activated macrophage at 0°C for 3 h

% Viable hyphae as determined by: Microculture

Dye exclusion

98 98 0 0 2 89 29 92

96 97 0 94 95 93 56 94

a A thin layer of a hyphal suspension was placed under a UV light (20 cm from a Westinghouse Steril lamp, 782 L-20) and irradiated for 30 rain at 25°C. b See Materials and Methods for experimental detail. In this particular instance, C. albicans hyphae and macrophages were allowed to interact for 3 h. was possible to d e t e r m i n e th e viability o f n o t o n l y t h o s e h y p h a e w h i c h w e r e s e p a r a t e d by o n l y 10 Ixm o r so, b u t also i n d i v i d u a l cells within a h y p h a s e p a r a t e d by s e p t a ( d a t a n o t s h o w n ) . T h e d e t e r g e n t ( N o n i d e t ) u s e d f o r lyzing m a c r o p h a g e s h ad n o d e t r i m e n t a l effect on C. albicans h y p h a e at th e c o n c e n t r a t i o n e m p l o y e d ( T a b l e 2). T h e reliability o f this m i c r o c u l t u r e t e c h n i q u e was t e s t e d by using cell s u s p e n s i o n s w h i ch c o n t a i n e d k n o w n n u m b e r s o f v i a b l e a n d n o n v i a b l e C. albicans h y p h a e . A s e v i d e n t f r o m t h e

96 data presented in Fig. 3, the microculture method allowed a fairly accurate determination of ratios between viable and non-viable cells in such hyphal suspensions. Since the dye exclusion method has been often used as a convenient substitute for the plating technique to determine the viability of C. albicans cells [5-10], a series of experiments was performed in order to see whether the results obtained by the dye exclusion method were comparable to those derived from the microculture method. As seen in Table 2, there was a good correlation of the data when Candida cells were killed by certain treatments such as heating. However, Candida hyphae rendered nonviable by 0.1 M hydrogen peroxide or ultraviolet irradiation did not lose their ability to exclude dye immediately. In the latter case, the microculture method was able to determine the ratios of viable and nonviable hyphae accurately (Table 2). The results of the experiments in which the microculture method was applied to the assessment of the candidacidal activity of murine peritoneal macrophages are summarized in Table 2. To my knowledge, this is the first instance in which the actual viability of C. albicans hyphae subjected to the extraphagocytotic candidacidal activity of macrophages has been quantitatively determined. A more complete description of the candidacidal activity of murine peritoneal macrophages will be published elsewhere. Discussion

Because the dimorphic fungus C. albicans grows predominantly in the hyphal form under conditions prevailing in the host tissues, it seems important to assess the candidacidal effects of antimycotic agents or phagocytes on both its yeast and hyphal forms. Most previous studies, however, almost exclusively used the yeast form of C. albicans. One of the difficulties inherently associated with the use of C. albicans hyphae in such studies is related to its high propensity to clump. As pointed out by Diamond et al. [3] and as confirmed in this study (Table 1), the viability of C. albicans hyphae cannot be determined accurately with the conventional plating technique. There is no question that alternative methods such as the radiolabel incorporation technique [3, 4, 12-14] and the radiolabel release assay technique [11] may be used conveniently in assessing the damage of C. albicans hyphae. However, these techniques cannot determine the viability of C. albicans cells. In fact, a recent study by Bristoni et al. [11] presented evidence that the viability determined by a classical colony forming unit test did not correlate at all with the amount of radiolabelled chromium released from C. albicans cells damaged in vitro by murine adherent peritoneal exudate cells. The microculture method described in this paper allows one to determine the ratio of viable (colony forming) to nonviable (non-colony forming) cells in a given hyphal population (Table 2, Fig. 3). Furthermore, it is an advantage of this technique that results can be read several hours after each experiment. Under our experimental conditions, the majority of viable hyphae have developed microcolonies by 5-6 h. Further incubation of microcultures tended to cause coales-

97 cence of some adjacent microcolonies and is not recommended. It is possible that a small number of reversibly damaged hyphae may initiate microcolony formation after 6 h of incubation period. Once C. albicans cells growing on microculture media are killed by glutaraldehyde vapor and placed at 4°C, specimens can be examined at a later time. Although the proper use of this technique yields reproducible results it suffers from one major limitation. It is a time consuming technique in that one has to count randomly 200 hyphae for each microculture under the microscope. Since duplicate microcultures were routinely made, a total of 400 hyphae per single sample had to be examined under a phase contrast microscope. The use of a video scan system might alleviate this problem. To obtain reproducible results with this microculture method it is essential that technical procedures be strictly adhered to. It is especially important that vigorous scraping of culture wells with a rubber policeman be avoided during the release of C. albicans into supernatant fluid. Because of their fragility, such excessive physical stress often broke off hyphae, most often at the neck of a hypha (where the germ tube originally emerged). This undesirable effect can be completely avoided if C. albicans hyphae are allowed to interact with phagocytes on the surface of a cover glass. One interesting and perhaps significant observation made in this study is the fact that C. albicans hyphae rendered nonviable by certain agents, such as hydrogen peroxide, could retain their ability to exclude dye for some time after death (Table 2). These results seem important in view of the fact that hydrogen peroxide has been implicated in the non-specific candidacidal mechanism of phagocytes [10]. In any event, it would be most interesting to compare the results of an experiment in which the candidacidal activity of phagocytes is assessed simultaneously by several different techniques, including the microculture, radiolabel release [11], radiolabel incorporation [3, 4, 13, 14], autoradiography [12], dye exclusion [5-10] and other [15, 17] methods.

Acknowledgments This investigation was supported in part by Biomedical Research Support grant G0787. The excellent technical help of Ms. Debra Cahill in some aspects of this work is appreciatively acknowledged.

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Schmid, L. and Brune K. (1974) Assessment of phagocytic and antimicrobial activity of human granulocytes. Infect. Immun. 10, 1120-1126. 7 Lehrer, R.I. (1975) The fungicidal mechanisms of human monocytes. 1. Evidence for myeloperoxidase-tinked and myeloperoxidase-independent candidacidal mechanisms. J. Clin. Invest. 55,338-345. 8 Kernbaum, S. (1976) Effects of hyperosmolality on candidacidal activity of human neutrophil polymorphonuclear leukocytes and on clumping of Candida albicans by human serum. Biomedicine 25, 90-114. 9 Scheritz, C. and Martin, R. (1979) The phagocytosis of Candida albicans blastospores and germ tubes by polymorphonuclear leukocytes. Dermatologia 159, 12-23. 10 Sasada, M. and Johnston, Jr., R.B. (1980) Macrophage microbicidal activity. Correlation between phagocytosis-associated oxidative metabolism and killing of Candida by macrophages. J. Exp. Med. 152, 85-98. 11 Bistoni, F., Baccarini, M., Blastic, E., Puccetti, P. and Marconi, P. (1982) A radiolabel release microassay for phagocytic killing of Candida albicans. J. Immunol. Methods 52, 369-377. 12 Ozato, K. and Uesaka, I. (1974) The role of macrophages in Candida infection in vitro. Jpn. J. Microbiol. 18, 29-35. 13 Peterson, E.M. and Calderone, R.A. (1977) Growth inhibition of Candida albicans by rabbit alveolar macrophages. Infect. Immun. 15, 910-915. 14 Bridges, C.G., Dasilva, G.L., Yamamura, M. and Valdimarsson. H. (1980) A radiometric assay for the combined measurement of phagocytosis and intracellular killing of Candida albicans. Clin. Exp. Immunol. 42,226-233. 15 Smith, D.L., and Rommel, F. (1977) A rapid micromethod for the simultaneous determination of phagocytic-microbicidal activity of human blood leukocytes in vitro. J. Immunol. Methods 7, 241-247. 16 Harrington-Fowler, L., Henson, P.M. and Wilder, M.S. (1981) Fate of Listeria monocytogenes in resident and activated macrophages. Infect. Immun. 33, 11-16. 17 Zeligs, B.J. (1981) Bactericidal and fungicidal assay. Manual of Macrophage Methodology. (Herscowiz, H.B., Holden, H.T., Bellanti, J.A. and Ghaffar, A., eds.), pp. 271-281, Marcel Dekker, New York.