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A simple and inexpensive method for assessing in vitro candidacidal activity of leukocytes
Journal of Immunological Methods, 66 (1984) 27-33
27
Elsevier
J I M 02884
A Simple and Inexpensive Method for Assessing In Vitro Candidacidal Activity of Leukocytes J i m E. C u t l e r a n d B r e n t D. T h o m p s o n Department of Microbiology, Montana State University, Bozeman, M T 59717, U.S.A.
(Received 18 May 1983, accepted 3 August 1983)
Candidacidal activity of mouse neutrophils and macrophages was determined directly in microtiter plates. After a suitable period of interaction between phagocytic cells and C. albicans in the wells, the mouse cells were lysed with distilled water and corn meal agar was added to each well. Following incubation at 37°C, viability was assessed using an inverted microscope and counting the number of germ tubes or microcolonies which developed. This method does not use radioisotopes or vital stains and should be applicable to other genera of yeasts.
Key words: Candida albicans - neutrophils - macrophages - candidacidal activity
Introduction
Candida albicans is an important opportunistic pathogen of man (Rippon, 1982). Although host defense mechanisms are not well defined, it is presumed that neutrophils (Ruthe et al., 1978) and, possibly, macrophages (Lehrer and Fleischmann, 1982) are important in preventing candidiasis. It has even been suggested that natural killer (NK) cells may play a role (Murphy and McDaniel, 1982). At present, most leukocyte-C, albicans interaction studies are done using in vitro techniques. Methods usually used to determine viability of C. albicans following a suitable incubation in the presence of leukocytes include: determination of the amount of chromium-51 released from labeled yeasts (Yamamura et al., 1976); yeast uptake of 3H-labeled uridine (Yamamura et al., 1977; Bridges et al., 1980); uptake or exclusion of methylene blue by the yeast cells (Lehrer and Cline, 1969; Wood and White, 1978); and plating the yeasts for colony forming units (Poor and Cutler, 1981). All of these methods, however, are either cumbersome, yield unreliable results, are not amenable to use in microtiter plates, or are not very sensitive. In this report we present a new method for determining candidacidal activity which is simple and inexpensive, does not require staining or radioisotopes, and can be used in microtiter plates. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
28 Materials and Methods
Organisms and culture conditions Candida albicans 394 (R.P. Morrison, University of Oklahoma, Norman, OK), used throughout the experiments, was grown in the yeast phase in glucose (2%), yeast extract (0.3%), peptone (1%) broth (GYEP) at 37°C for 48 h under constant aeration by rotation of the flasks at 160 rpm (Gyrorotary incubator, New Brunswick Scientific, Edison, N J). Yeast cells were harvested by centrifugation and were washed three times with 0.15 N NaC1 (saline). Viability of cells was greater than 99% as determined by plating onto Sabouraud dextrose agar (Difco, Detroit, MI) and by viability staining with methylene blue as described elsewhere (Lehrer and Cline, 1969). Reagents Phosphate buffered balanced salts (PBBS) solution and PBBS + ethylenediamine tetraacetic acid (EDTA) were prepared as described elsewhere (Chesebro and Wehrly, 1976). Mouse blood obtained from retro-orbital bleeding was clotted on ice for 30-60 rain, centrifuged at 4°C, and the serum was referred to as fresh mouse serum (FMS) and used within 1 h of collection. Tissue culture medium 199 with 25 m M HEPES buffer and Hank's balanced salts (Grand Island Biological Supply Company, Grand Island, NY) was supplemented with 5% FMS just prior to use. Experimental animals and phagocytic cells B A L B / c B Y J mice from Jackson Laboratories (Bar Harbor, ME) were raised and maintained in our animal care facilities in which the bedding, cages, and water bottles were sterilized before use, and the water was acidified (McPherson, 1963). Neutrophil-rich populations of peritoneal exudate cells were obtained from mice 3 h after stimulation with 2.5 ml of 0.5% glycogen (Nutritional Biochemicals, Cleveland, OH) in saline given intraperitoneally (i.p.). Peritoneal cells rich in resident macrophages were obtained from unstimulated animals or elicited macrophages were harvested from the peritoneal cavity of mice 3 days after an i.p. injection of 2.5 ml of N I H thioglycollate broth (Difco). The cells from stimulated and unstimulated mice were harvested by washing the peritoneal cavity with PBBS + EDTA; the cells were washed three times in PBBS, and resuspended to the appropriate cell concentration in TC199 plus 5% FMS. Neutrophil-rich populations consisted of over 75% neutrophils, resident macrophage-rich populations consisted of over 80% mononuclear cells, and elicited cells consisted of 65-85% macrophages as evidenced by Giemsa differential staining (Cutler and Poor, 1981; Morrison and Cutler, 1981). Assay protocol Flat-bottomed wells in microtiter plates (Microtest II, Becton Dickinson, Oxnard, CA) were precoated with protein by adding either 10% FMS or 1% bovine serum albumin in saline for 1 h at 22-24°C followed by washing the wells with saline. A 0.2 ml aliquot of a yeast cell suspension (10 4 blastoconidia/ml saline) was added to
29 each well, the cells were rapidly settled by centrifugation at 360 x g for 5 rain, and the supernatant liquid was discarded by flicking the plate. The optimum number of blastoconidia (i.e. 2 x 103 per well) added to each well was arrived at empirically. Peritoneal cells in 0.2 ml medium (TC199 + 5% FMS) were added to wells to establish peritoneal cell to yeast cell ratios of 400 : 1, 100 : 1, 25 : 1, 6 : 1, 3 : 1 and 1.5 : 1. Each ratio was tested in triplicate and compared to triplicate control wells which received yeasts plus 0.2 ml of the tissue culture medium and FMS. The microtiter plate was incubated for 1 h at 37°C under 5% CO 2 (Hotpack CO 2 Incubator, Philadelphia, PA). Following incubation, the wells were washed 3 times with distilled water to lyse the phagocytic cells and the C. albicans cells were retained during each wash by centrifugation (360 x g, 10 min). Retention of the fungal cells in the wells was confirmed by the lack of growth which occurred following plating of the supernatant material onto Sabouraud dextrose agar (Difco) following each of the washes. Then 0.2 ml of molten (43°C) corn meal agar (CMA, Difco) was added to each well and the microtiter plate was incubated for an additional 1 h, 4 h, or 24 h under the above conditions. Results were assessed using a high dry power (200 x ) of an inverted microscope. By 1 h incubation over 95% of the yeasts had germinated in wells not containing phagocytes. At 1 h incubation the average number of germ tube forming units per high power field (hpf) was determined, whereas at 4 h and 24 h incubation periods the data were expressed as the number of microcolonies/hpf. The data reflect the mean of five hpf per well; each test was run in triplicate, and each experiment was run on at least three separate occasions.
Results
Candidacidal activity of neutrophils was evaluated at 1 h incubation, following addition of CMA to the wells, by counting the number of germ tubes which developed (Fig. 1). By this measure, candidacidal activity was detected at various phagocyte to yeast cell ratios (Fig. 2). Reduction in viability, compared to wells without phagocytes, ranged from 75% at neutrophil to yeast ratios of 25 : 1 to 30% at 3 : 1. Significant evidence of killing did not occur at a 1.5 : 1 ratio. Macrophage-rich populations, resident or elicited, did not inhibit C. albicans even at a ratio of 250 : 1 (phagocytes to yeast cells). If incubation time following addition of CMA to the wells was extended to 4 h or more, microcolonies developed which could be counted using the inverted microscope (Fig. 3). The number of microcolonies/hpf which occurred at 4 h and 24 h were essentially the same as germ tube counts which occurred at 1 h incubation (Fig. 4). These data support the idea that germ tube formation at 1 h is an indication of cell viability and not just a measure of germinative ability.
Discussion
Existing methods for evaluating candidacidal activity of leukocytes have many disadvantages. Plating for viability is laborious because homogenization of samples
30
Fig. 1. Viability of C. albicans as assessed by germination. Yeast cells were allowed to interact with phagocytes for 1 h; the phagocytes were osmotically lysed, corn meal agar was added; and the fungal cells released from the phagocytes were incubated for 1 h at 37°C and viewed microscopically (200 × ).
20
1
:':':':':':':4
,o E
10
!iiii!ii!!::~ i:i:i:i:i:i3
0
0
25
6
3
1.5
Neutrophils/yeast
Fig. 2. N e u t r o p h i l c a n d i d a c i d a l activity. Two t h o u s a n d yeast cells were added to various n u m b e r s of n e u t r o p h i l s and c a n d i d a c i d a l activity was assessed by inference from d e t e r m i n i n g the average n u m b e r of g e r m tubes per high power field (hpf). S t a n d a r d deviation m a r k e r s are i n d i c a t e d at the top of each bar.
31
J
't I
Fig. 3. Viability of C. albicans as assessed by microcolony formation. Yeast cells were interacted with phagocytes as indicated in Fig. 1, but incubation upon addition of corn meal agar was extended to 24 h and viewed microscopically (this picture was taken at 40 × , but counting of colonies was done at 200 x ).
20 A
¢n
>,
=10 .o
B
C
E1
,I o
1.5
3
1.5
3
0
1.5
3
Neutrophils/yeast
Fig. 4. Comparison of germ tube formation and microcolony development for assessment of candidacidal activity. Yeast cells were interacted with phagocytes as in Fig. 1. Candidacidal activity following addition of corn meal agar was assessed by germ tube formation at 1 h (A), or by microcolony formation at 4 h (B), and 24 h (C) incubation at 37°C. Standard deviation markers are indicated at the top of each bar.
32 is required to minimize cell clumping (Poor and Cutler, 1981). Chromium-51 release assays are not very sensitive since only about 1 cpm per 100 yeasts is detectable after labeling (Yamamura et al., 1976; and our unpublished observations) and the percentage release following death of yeasts is low. Methods which utilize uptake of [3H]uridine as a measure of yeast viability (Yamamura et al., 1977; Bridges et al., 1980) are not useful on a routine basis because of liquid scintillation quenching problems (our unpublished data). Both radioisotope procedures are relatively expensive and entail special handling precautions. Uptake of methylene blue as an indicator of yeast viability (Lehrer and Cline, 1969), has been adapted for use in microtiter plates (Wood and White, 1978). Interpretation of dye uptake is, however, subjective (Wood and White, 1978; and our unpublished data) and this procedure may not work with other genera of yeasts. Our method is sensitive in that only 2 x 103 fungal cells are required and the results can be ascertained 1 h following incubation of the leukocytes with C. albicans yeast cells. Germination seemed to correlate with cell viability because: incubation of the plates for 4 h or more gave rise to the same number of microcolonies as the number of germ tubes at 1 h; and the degree of candidacidal activity by neutrophils and lack of killing by peritoneal macrophages were in close agreement with our previous investigations using plating as a viability measure (Cutler and Poor, 1981). A potential technical problem, which was sometimes encountered, is that the yeast cells may not settle randomly in the wells. This problem was significantly resolved by pre-coating the plastic wells with mouse serum or bovine serum albumin and by using centrifugation to settle the yeast cells. Using these measures, the number of cells in 5 high power fields divided by five represented the average number of cells per field over the entire surface area of the well. Although we have not tried this method on other yeasts, microcolony formation should make the procedure amenable to non-germinating species or to strains of C. albicans which may germinate poorly.
Acknowledgements We wish to thank Craig Foss for help in initial experiments and Ruth Lloyd for excellent technical assistance. This work was supported by N I H Grant No. AI-13512 and N I H R C D A No. AI-00367 to J.E.C.
Refereces Bridges, C.G., G.L. Dasilva, M. Yamamura and H. Valdimarsson, 1980, Clin. Exp. lmmunol. 42, 226. Chesebro, B. and K. Wehrly, 1976, J. Exp. Med. 143, 73. Cutler, J.E. and A.H. Poor, 1981, Infect. Immun. 31, 1110. Lehrer, R.J. and M.J. Cline, 1969, J. Bacteriol. 98, 996. Lehrer, R.I. and J. Fleischmann, 1982, in: Microbiology-1982,ed. David Schlessinger (American Society for Microbiology,Washington, DC) p. 385. McPherson, C.W., 1963, Lab. Anim. Care 13, 737.
33 Morrison, R.P. and J.E. Cutler, 1981, J. Reticuloendothel. Soc. 29, 23. Murphy, J.W. and D.O. McDaniel, 1982, J. Immunol. 128, 1577. Poor, A.H. and J.E. Cutler, 1981, Infect. Immun. 31, 1104. Rippon, J.W., 1982, in: Medical Mycology, The Pathogenic Fungi and The Pathogoenic Actinomycetes (W.B. Saunders Co., Philadelphia) p. 484. Ruthe, R.C., B.R. Andersen, B.L. Cunningham and R.B. Epstein, 1978, Blood 52, 493. Wood, S.M. and A.G. White, 1978, J. Immunol. Methods 20, 43. Yamamura, M., J. Boler and H. Valdimarsson, 1976, J. Immunol. Methods 13, 227. Yamamura, M., J. Boler and H. Valdimarsson, 1977, J. Immunol. Methods 14, 19.
27
Elsevier
J I M 02884
A Simple and Inexpensive Method for Assessing In Vitro Candidacidal Activity of Leukocytes J i m E. C u t l e r a n d B r e n t D. T h o m p s o n Department of Microbiology, Montana State University, Bozeman, M T 59717, U.S.A.
(Received 18 May 1983, accepted 3 August 1983)
Candidacidal activity of mouse neutrophils and macrophages was determined directly in microtiter plates. After a suitable period of interaction between phagocytic cells and C. albicans in the wells, the mouse cells were lysed with distilled water and corn meal agar was added to each well. Following incubation at 37°C, viability was assessed using an inverted microscope and counting the number of germ tubes or microcolonies which developed. This method does not use radioisotopes or vital stains and should be applicable to other genera of yeasts.
Key words: Candida albicans - neutrophils - macrophages - candidacidal activity
Introduction
Candida albicans is an important opportunistic pathogen of man (Rippon, 1982). Although host defense mechanisms are not well defined, it is presumed that neutrophils (Ruthe et al., 1978) and, possibly, macrophages (Lehrer and Fleischmann, 1982) are important in preventing candidiasis. It has even been suggested that natural killer (NK) cells may play a role (Murphy and McDaniel, 1982). At present, most leukocyte-C, albicans interaction studies are done using in vitro techniques. Methods usually used to determine viability of C. albicans following a suitable incubation in the presence of leukocytes include: determination of the amount of chromium-51 released from labeled yeasts (Yamamura et al., 1976); yeast uptake of 3H-labeled uridine (Yamamura et al., 1977; Bridges et al., 1980); uptake or exclusion of methylene blue by the yeast cells (Lehrer and Cline, 1969; Wood and White, 1978); and plating the yeasts for colony forming units (Poor and Cutler, 1981). All of these methods, however, are either cumbersome, yield unreliable results, are not amenable to use in microtiter plates, or are not very sensitive. In this report we present a new method for determining candidacidal activity which is simple and inexpensive, does not require staining or radioisotopes, and can be used in microtiter plates. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
28 Materials and Methods
Organisms and culture conditions Candida albicans 394 (R.P. Morrison, University of Oklahoma, Norman, OK), used throughout the experiments, was grown in the yeast phase in glucose (2%), yeast extract (0.3%), peptone (1%) broth (GYEP) at 37°C for 48 h under constant aeration by rotation of the flasks at 160 rpm (Gyrorotary incubator, New Brunswick Scientific, Edison, N J). Yeast cells were harvested by centrifugation and were washed three times with 0.15 N NaC1 (saline). Viability of cells was greater than 99% as determined by plating onto Sabouraud dextrose agar (Difco, Detroit, MI) and by viability staining with methylene blue as described elsewhere (Lehrer and Cline, 1969). Reagents Phosphate buffered balanced salts (PBBS) solution and PBBS + ethylenediamine tetraacetic acid (EDTA) were prepared as described elsewhere (Chesebro and Wehrly, 1976). Mouse blood obtained from retro-orbital bleeding was clotted on ice for 30-60 rain, centrifuged at 4°C, and the serum was referred to as fresh mouse serum (FMS) and used within 1 h of collection. Tissue culture medium 199 with 25 m M HEPES buffer and Hank's balanced salts (Grand Island Biological Supply Company, Grand Island, NY) was supplemented with 5% FMS just prior to use. Experimental animals and phagocytic cells B A L B / c B Y J mice from Jackson Laboratories (Bar Harbor, ME) were raised and maintained in our animal care facilities in which the bedding, cages, and water bottles were sterilized before use, and the water was acidified (McPherson, 1963). Neutrophil-rich populations of peritoneal exudate cells were obtained from mice 3 h after stimulation with 2.5 ml of 0.5% glycogen (Nutritional Biochemicals, Cleveland, OH) in saline given intraperitoneally (i.p.). Peritoneal cells rich in resident macrophages were obtained from unstimulated animals or elicited macrophages were harvested from the peritoneal cavity of mice 3 days after an i.p. injection of 2.5 ml of N I H thioglycollate broth (Difco). The cells from stimulated and unstimulated mice were harvested by washing the peritoneal cavity with PBBS + EDTA; the cells were washed three times in PBBS, and resuspended to the appropriate cell concentration in TC199 plus 5% FMS. Neutrophil-rich populations consisted of over 75% neutrophils, resident macrophage-rich populations consisted of over 80% mononuclear cells, and elicited cells consisted of 65-85% macrophages as evidenced by Giemsa differential staining (Cutler and Poor, 1981; Morrison and Cutler, 1981). Assay protocol Flat-bottomed wells in microtiter plates (Microtest II, Becton Dickinson, Oxnard, CA) were precoated with protein by adding either 10% FMS or 1% bovine serum albumin in saline for 1 h at 22-24°C followed by washing the wells with saline. A 0.2 ml aliquot of a yeast cell suspension (10 4 blastoconidia/ml saline) was added to
29 each well, the cells were rapidly settled by centrifugation at 360 x g for 5 rain, and the supernatant liquid was discarded by flicking the plate. The optimum number of blastoconidia (i.e. 2 x 103 per well) added to each well was arrived at empirically. Peritoneal cells in 0.2 ml medium (TC199 + 5% FMS) were added to wells to establish peritoneal cell to yeast cell ratios of 400 : 1, 100 : 1, 25 : 1, 6 : 1, 3 : 1 and 1.5 : 1. Each ratio was tested in triplicate and compared to triplicate control wells which received yeasts plus 0.2 ml of the tissue culture medium and FMS. The microtiter plate was incubated for 1 h at 37°C under 5% CO 2 (Hotpack CO 2 Incubator, Philadelphia, PA). Following incubation, the wells were washed 3 times with distilled water to lyse the phagocytic cells and the C. albicans cells were retained during each wash by centrifugation (360 x g, 10 min). Retention of the fungal cells in the wells was confirmed by the lack of growth which occurred following plating of the supernatant material onto Sabouraud dextrose agar (Difco) following each of the washes. Then 0.2 ml of molten (43°C) corn meal agar (CMA, Difco) was added to each well and the microtiter plate was incubated for an additional 1 h, 4 h, or 24 h under the above conditions. Results were assessed using a high dry power (200 x ) of an inverted microscope. By 1 h incubation over 95% of the yeasts had germinated in wells not containing phagocytes. At 1 h incubation the average number of germ tube forming units per high power field (hpf) was determined, whereas at 4 h and 24 h incubation periods the data were expressed as the number of microcolonies/hpf. The data reflect the mean of five hpf per well; each test was run in triplicate, and each experiment was run on at least three separate occasions.
Results
Candidacidal activity of neutrophils was evaluated at 1 h incubation, following addition of CMA to the wells, by counting the number of germ tubes which developed (Fig. 1). By this measure, candidacidal activity was detected at various phagocyte to yeast cell ratios (Fig. 2). Reduction in viability, compared to wells without phagocytes, ranged from 75% at neutrophil to yeast ratios of 25 : 1 to 30% at 3 : 1. Significant evidence of killing did not occur at a 1.5 : 1 ratio. Macrophage-rich populations, resident or elicited, did not inhibit C. albicans even at a ratio of 250 : 1 (phagocytes to yeast cells). If incubation time following addition of CMA to the wells was extended to 4 h or more, microcolonies developed which could be counted using the inverted microscope (Fig. 3). The number of microcolonies/hpf which occurred at 4 h and 24 h were essentially the same as germ tube counts which occurred at 1 h incubation (Fig. 4). These data support the idea that germ tube formation at 1 h is an indication of cell viability and not just a measure of germinative ability.
Discussion
Existing methods for evaluating candidacidal activity of leukocytes have many disadvantages. Plating for viability is laborious because homogenization of samples
30
Fig. 1. Viability of C. albicans as assessed by germination. Yeast cells were allowed to interact with phagocytes for 1 h; the phagocytes were osmotically lysed, corn meal agar was added; and the fungal cells released from the phagocytes were incubated for 1 h at 37°C and viewed microscopically (200 × ).
20
1
:':':':':':':4
,o E
10
!iiii!ii!!::~ i:i:i:i:i:i3
0
0
25
6
3
1.5
Neutrophils/yeast
Fig. 2. N e u t r o p h i l c a n d i d a c i d a l activity. Two t h o u s a n d yeast cells were added to various n u m b e r s of n e u t r o p h i l s and c a n d i d a c i d a l activity was assessed by inference from d e t e r m i n i n g the average n u m b e r of g e r m tubes per high power field (hpf). S t a n d a r d deviation m a r k e r s are i n d i c a t e d at the top of each bar.
31
J
't I
Fig. 3. Viability of C. albicans as assessed by microcolony formation. Yeast cells were interacted with phagocytes as indicated in Fig. 1, but incubation upon addition of corn meal agar was extended to 24 h and viewed microscopically (this picture was taken at 40 × , but counting of colonies was done at 200 x ).
20 A
¢n
>,
=10 .o
B
C
E1
,I o
1.5
3
1.5
3
0
1.5
3
Neutrophils/yeast
Fig. 4. Comparison of germ tube formation and microcolony development for assessment of candidacidal activity. Yeast cells were interacted with phagocytes as in Fig. 1. Candidacidal activity following addition of corn meal agar was assessed by germ tube formation at 1 h (A), or by microcolony formation at 4 h (B), and 24 h (C) incubation at 37°C. Standard deviation markers are indicated at the top of each bar.
32 is required to minimize cell clumping (Poor and Cutler, 1981). Chromium-51 release assays are not very sensitive since only about 1 cpm per 100 yeasts is detectable after labeling (Yamamura et al., 1976; and our unpublished observations) and the percentage release following death of yeasts is low. Methods which utilize uptake of [3H]uridine as a measure of yeast viability (Yamamura et al., 1977; Bridges et al., 1980) are not useful on a routine basis because of liquid scintillation quenching problems (our unpublished data). Both radioisotope procedures are relatively expensive and entail special handling precautions. Uptake of methylene blue as an indicator of yeast viability (Lehrer and Cline, 1969), has been adapted for use in microtiter plates (Wood and White, 1978). Interpretation of dye uptake is, however, subjective (Wood and White, 1978; and our unpublished data) and this procedure may not work with other genera of yeasts. Our method is sensitive in that only 2 x 103 fungal cells are required and the results can be ascertained 1 h following incubation of the leukocytes with C. albicans yeast cells. Germination seemed to correlate with cell viability because: incubation of the plates for 4 h or more gave rise to the same number of microcolonies as the number of germ tubes at 1 h; and the degree of candidacidal activity by neutrophils and lack of killing by peritoneal macrophages were in close agreement with our previous investigations using plating as a viability measure (Cutler and Poor, 1981). A potential technical problem, which was sometimes encountered, is that the yeast cells may not settle randomly in the wells. This problem was significantly resolved by pre-coating the plastic wells with mouse serum or bovine serum albumin and by using centrifugation to settle the yeast cells. Using these measures, the number of cells in 5 high power fields divided by five represented the average number of cells per field over the entire surface area of the well. Although we have not tried this method on other yeasts, microcolony formation should make the procedure amenable to non-germinating species or to strains of C. albicans which may germinate poorly.
Acknowledgements We wish to thank Craig Foss for help in initial experiments and Ruth Lloyd for excellent technical assistance. This work was supported by N I H Grant No. AI-13512 and N I H R C D A No. AI-00367 to J.E.C.
Refereces Bridges, C.G., G.L. Dasilva, M. Yamamura and H. Valdimarsson, 1980, Clin. Exp. lmmunol. 42, 226. Chesebro, B. and K. Wehrly, 1976, J. Exp. Med. 143, 73. Cutler, J.E. and A.H. Poor, 1981, Infect. Immun. 31, 1110. Lehrer, R.J. and M.J. Cline, 1969, J. Bacteriol. 98, 996. Lehrer, R.I. and J. Fleischmann, 1982, in: Microbiology-1982,ed. David Schlessinger (American Society for Microbiology,Washington, DC) p. 385. McPherson, C.W., 1963, Lab. Anim. Care 13, 737.
33 Morrison, R.P. and J.E. Cutler, 1981, J. Reticuloendothel. Soc. 29, 23. Murphy, J.W. and D.O. McDaniel, 1982, J. Immunol. 128, 1577. Poor, A.H. and J.E. Cutler, 1981, Infect. Immun. 31, 1104. Rippon, J.W., 1982, in: Medical Mycology, The Pathogenic Fungi and The Pathogoenic Actinomycetes (W.B. Saunders Co., Philadelphia) p. 484. Ruthe, R.C., B.R. Andersen, B.L. Cunningham and R.B. Epstein, 1978, Blood 52, 493. Wood, S.M. and A.G. White, 1978, J. Immunol. Methods 20, 43. Yamamura, M., J. Boler and H. Valdimarsson, 1976, J. Immunol. Methods 13, 227. Yamamura, M., J. Boler and H. Valdimarsson, 1977, J. Immunol. Methods 14, 19.