APPLIED MICROBIOLOGY, July, 1966 Vol. 14, No. 4 Copyright @ 1966 American Society for Microbiology Printed in U.S.A. Modified Pagano Levin Medium to Isolate Candida Species' MICHAEL A. STEDHAM,2 DONALD C. KELLEY, AND EMBERT H. COLES Department of Pathology, Parasitology, and Public Health, College of Veterinary Medicine Kansas State University, Manhattan, Kansas Received for publication 14 January 1966 ABSTRACT STEDHAM, MICHAEL A. (Kansas State University, Manhattan), DONALD C. KEL- LEY, AND EMBERT H. COLES. Modified Pagano Levin medium to isolate Candida species. Appi. Microbiol. 14:525-528. 1966.-The use of Pagano Levin medium for primary isolation of Candida species from highly contaminated specimens proved unsatisfactory owing to overgrowth by bacteria and fungi. Addition of cyclohexi- mide and chloramphenicol to the medium greatly improved results by inhibiting overgrowth. The additional inhibitors also altered rate of growth and degree of pig- mentation reported for some of the Candida species. Complete Pagano Levin medium contains Pagano Levin base (Difco) plus 2,3, 5-triphenyl- tetrazolium chloride (TTC; Difco), 0.1 mg/ml, and neomycin (The Upjohn Co., Kalamazoo, Mich.), 0.5 mg/ml. It has been recommended as a diagnostic medium to differentiate species of Candida (4). The producing laboratory also recommends Pagano Levin medium for primary isolation of Candida species. It was used by Bixby (M.S. Thesis, Kansas State Univ., Manhattan, 1964) for primary isolation, and as one of the methods to differentiate Candida species from experimental swine. In preliminary trials in which the medium was used for primary isolation of C. albicans from fecal samples of swine, it was found that over- growth by other yeasts, bacteria, and especially molds made identification of yeast colonies diffi- cult. Additional inhibitors, chloramphenicol (Chloromycetin, Parke, Davis & Co., Detroit, Mich.) and cycloheximide (Acti-dione, The Upjohn Co.), were added to counteract the overgrowth. The antifungal properties of cyclo- heximide are well outlined by Georg, Ajello, and Papageorge (2) and Fuentes et al. (1). The anti- bacterial spectrum of chloramphenicol is covered by McLean et al. (3) and Welch et al. (5). Both antibiotics commonly are used in other media to 1 Contribution no. 154 from the Department of Pathology, Parasitology, and Public Health, Kansas Agricultural Experimental Station, Manhattan. 2 Present Address: U.S. Army Medical Research and Nutrition Laboratory, Fitzsimons General Hos- pital, Denver, Colo. isolate or propagate, or both, other pathogenic fungi. The two inhibitors were chosen, because, in combination, they inhibit a wide range of sapro- phytic and some pathogenic fungi and bacteria. Also, they may be added before the medium is heat-sterilized. Of the two added compounds normally in Pagano Levin medium, neomycin is included because of its broad antibacterial spectrum. TTC is used as an indicator, with the resultant colony color depending on the degree of reduction by the organism. At a concentration of 0.4 mg/ml, TTC also may inhibit the yeasts. Two trials were designed to compare Pagano Levin medium alone and with the additional in- hibitors. MATERIALS AND METHODS Trial 1. In trial 1, Pagano Levin medium was com- pared with Pagano Levin medium plus cycloheximide (0.5 mg/ml) and chloramphenicol (0.05 mg/ml). The following inocula were used: (i) a pure culture of C. albicans; (ii) three water samples from swine waterers with C. albicans added; (iii) three samples from the same waterers with C. albicans added after sampling; (iv) two fecal samples from swine; and (v) the same two fecal samples with C. albicans added. Serial dilutions of the inocula were pipetted into petri dishes, and 15 to 20 ml of the respective media was added. Two plates of each medium at each dilu- tion for each inoculum were poured. The plates were thoroughly mixed, and incubated for 3 days at 37 C. They were examined at 24, 48, and 72 hr. Thirty-four colonies typical of C. albicans from each of the two media inoculated with water plus 525 on July 2, 2020 by guest http://aem.asm.org/ Downloaded from
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APPLIED MICROBIOLOGY, July, 1966 Vol. 14, No. 4
Copyright @ 1966 American Society for Microbiology Printed in U.S.A.
Modified Pagano Levin Medium toIsolate Candida Species'
MICHAEL A. STEDHAM,2 DONALD C. KELLEY, AND EMBERT H. COLES
Department of Pathology, Parasitology, and Public Health, College of Veterinary MedicineKansas State University, Manhattan, Kansas
Received for publication 14 January 1966
ABSTRACTSTEDHAM, MICHAEL A. (Kansas State University, Manhattan), DONALD C. KEL-
LEY, AND EMBERT H. COLES. Modified Pagano Levin medium to isolate Candidaspecies. Appi. Microbiol. 14:525-528. 1966.-The use of Pagano Levin medium forprimary isolation of Candida species from highly contaminated specimens provedunsatisfactory owing to overgrowth by bacteria and fungi. Addition of cyclohexi-mide and chloramphenicol to the medium greatly improved results by inhibitingovergrowth. The additional inhibitors also altered rate of growth and degree of pig-mentation reported for some of the Candida species.
Complete Pagano Levin medium containsPagano Levin base (Difco) plus 2,3, 5-triphenyl-tetrazolium chloride (TTC; Difco), 0.1 mg/ml,and neomycin (The Upjohn Co., Kalamazoo,Mich.), 0.5 mg/ml. It has been recommended asa diagnostic medium to differentiate species ofCandida (4). The producing laboratory alsorecommends Pagano Levin medium for primaryisolation of Candida species. It was used by Bixby(M.S. Thesis, Kansas State Univ., Manhattan,1964) for primary isolation, and as one of themethods to differentiate Candida species fromexperimental swine.
In preliminary trials in which the medium wasused for primary isolation of C. albicans fromfecal samples of swine, it was found that over-growth by other yeasts, bacteria, and especiallymolds made identification of yeast colonies diffi-cult. Additional inhibitors, chloramphenicol(Chloromycetin, Parke, Davis & Co., Detroit,Mich.) and cycloheximide (Acti-dione, TheUpjohn Co.), were added to counteract theovergrowth. The antifungal properties of cyclo-heximide are well outlined by Georg, Ajello, andPapageorge (2) and Fuentes et al. (1). The anti-bacterial spectrum of chloramphenicol is coveredby McLean et al. (3) and Welch et al. (5). Bothantibiotics commonly are used in other media to
1 Contribution no. 154 from the Department ofPathology, Parasitology, and Public Health, KansasAgricultural Experimental Station, Manhattan.
2 Present Address: U.S. Army Medical Researchand Nutrition Laboratory, Fitzsimons General Hos-pital, Denver, Colo.
isolate or propagate, or both, other pathogenicfungi.The two inhibitors were chosen, because, in
combination, they inhibit a wide range of sapro-phytic and some pathogenic fungi and bacteria.Also, they may be added before the medium isheat-sterilized.Of the two added compounds normally in
Pagano Levin medium, neomycin is includedbecause of its broad antibacterial spectrum. TTCis used as an indicator, with the resultant colonycolor depending on the degree of reduction bythe organism. At a concentration of 0.4 mg/ml,TTC also may inhibit the yeasts.Two trials were designed to compare Pagano
Levin medium alone and with the additional in-hibitors.
MATERIALS AND METHODS
Trial 1. In trial 1, Pagano Levin medium was com-pared with Pagano Levin medium plus cycloheximide(0.5 mg/ml) and chloramphenicol (0.05 mg/ml).The following inocula were used: (i) a pure cultureof C. albicans; (ii) three water samples from swinewaterers with C. albicans added; (iii) three samplesfrom the same waterers with C. albicans added aftersampling; (iv) two fecal samples from swine; and(v) the same two fecal samples with C. albicans added.
Serial dilutions of the inocula were pipetted intopetri dishes, and 15 to 20 ml of the respective mediawas added. Two plates of each medium at each dilu-tion for each inoculum were poured. The plates werethoroughly mixed, and incubated for 3 days at 37 C.They were examined at 24, 48, and 72 hr.
Thirty-four colonies typical of C. albicans fromeach of the two media inoculated with water plus
TABLE 1. Pagano Levin medium compared with and without additional inhibitors
Additional C. albicans-like Other yeasts, molds,Inoculum inhibitors Candida albicans- C. albicans-like colonies plus other and/or bacteria, but(cycloheximide, like colonies only colonies plus mold yeasts, molds, no C. albicans-like
chloramphenicol) and/or bacteria colonies
C. albicans + xx
Water #1 + xx
Water #2 + Xa
xWater #3 + x
xWater #1 plus C. + X
albicans XWater #2 plus C. + x
albicans XWater #3 plus C. + x
albicans XFecal #1 + x
xFecal #2 + x
xFecal #1 plus C. + X
albicans XFecal #2 plus C. + Xb
albicans X
a Two mold colonies.b One mold colony.
C. albicans or feces plus C. albicans were randolymselected and cut into Chlamydospore Agar (Difco).The deep cuts were partially covered with sterilecover slips. A C. albicans control cut was made ineach plate. The plates were incubated in a candlejar at room temperature, and examined at 2 and 5days for chlamydospores and pseudomycelium typicalof C. albicans.
Trial 2. In trial 2, a comparison was made of thefollowing: (i) Pagano Levin medium; (ii) PaganoLevin medium plus cycloheximide (0.5 mg/ml) andchloramphenicol (0.05 mg/ml), but minus neomycin;and (iii) Pagano Levin medium plus cycloheximideand chloramphenicol. The following inocula wereused: (i) pure cultures of C. albicans, C. krusei, C.stellatoidea, C. tropicalis, Saccharomyces cerevisiae,Debaromyces species, Torulopsis species, and Han-seniaspora species; (ii) water from a swine waterer;and (iii) a fecal sample from a pig.
Dilutions were made and plates were poured as intrial 1. The plates were incubated at 37 C, and ex-amined at 36, 60, and 108 hr.
RESuLTS
Trial 1. The optimal time to identify C. albi-cans-like colonies was at 48 hr. In the pure-cultureC. albicans plates, the surface colonies on PaganoLevin medium were about 2 mm in diameter,cream-colored, raised, slightly moist, smooth-surfaced, and regular-edged. In the medium withthe additional inhibitors, the colony characteris-
FIG. 1. Water inoculum in Pagano Levin mediumwith inhibitors.
tics were the same, but average size was just over1 mm. In both media, the subsurface colonieswere slightly smaller and disc-shaped or discoidwith one or more fins.
FIG. 4. Fecal and Candida albicans inoculum inPagano Levin medium.
FIG. 3. Fecal and Candida albicans inoculum inPagano Levin medium with inhibitors.
The same colony size relationship of C. albi-cans-like colonies between the two media heldtrue in the other inocula, but interpretation wasmade difficult by overgrowth in the PaganoLevin medium (Table 1).At 24 hr, pinpoint colonies developed in the
additionally inhibited medium, and colonies witha diameter up to 1 mm developed in the PaganoLevin medium with the C. albicans inoculum.Competing colonies and small colonies madeinterpretation difficult in the Pagano Levin me-dium with water or fecal material.
TABLE 2. Growth of organisms in three mediawith different inoculaa
Inoculumn Medium Medium 2 Medium 3Inoculum (PL) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~(PL+2 MediumC'' Cc, - N) P c
a Symbols; PL, Pagano Levin medium; PL +Cc, -N, Pagano Levin medium plus cyclohexi-mide and chloramphenicol but minus neomycin;PL + Cc, Pagano Levin medium plus cyclohexi-mide and chloramphenicol; +, growth; -, nogrowth.
At 72 hr, larger C. albicans-like colonies wereevident in both media. Many types of contam-inants were growing well in the Pagano Levinmedium, and some of the yeast colonies werebecoming pinkish to deep-pink.At 90 hr, the additionally inhibited medium
contained only C. albicans-like colonies, com-pared with many colony types in Pagano Levinmedium (Fig. 1, 2, 3, and 4).Of the 34 randomly selected C. albicans-like
colonies cut into Chlamydospore Agar from theadditionally inhibited medium with fecal or waterinocula, 32 were identified morphologically asC. albicans. Of the 34 C. albicans-like coloniesfrom Pagano Levin medium, 17 were identifiedmorphologically as C. albicans.
Trial 2. Growth of the organisms in the mediais shown in Table 2.No C. albicans-like colony was seen from the
water of fecal samples in any medium. Only onetype of mold colony (Geotrichum by microscopicmorphology) and one type of yeast colony (un-identified, but not C. albicans) appeared in me-dium 2 and medium 3. Several types of mold andyeast colonies appeared in medium 1.The optimal time to examine plates in this trial
was 60 hr. Table 3 gives colony size differencesnoted among media at that time.At the 108-hr examination, some differences
among the medium pigmentation of pure culturecolonies were noted. C. albicans colonies were apaler cream in medium 3 than in medium 1 or 2.C. krusei lacked the pink center in medium 3,but the pink center was present in media 1 and2. C. tropicalis colonies were pink to red in me-dium 1, but cream in 2 and 3. Hanseniasporaspecies formed bright-red colonies in medium 1,but only pink in 2 and 3.
DISCUSSION
In both trials, fecal and water inocula inPagano Levin medium resulted in many morecolonies and more types of colonies than in me-dia with additional inhibitors. The colonies wereprimarily yeasts and molds. The neomycin ade-quately inhibited bacterial overgrowth in thePagano Levin medium in most cases. However,when highly contaminated inocula were used,such as feces and water, additional inhibition wasneeded.
C. albicans was isolated almost exclusively(32 of 34 colonies randomly selected) from the
additionally inhibited medium in trial 1. A muchlower yield (17 of 34) was obtained from PaganoLevin medium.
Variation in numbers of C. albicans-like colo-nies in different media in trial 1 did not exceed25%. Generally, the additionally inhibited me-dium yielded a higher count. This could havebeen due to less competition from other orga-nisms, or easier identification with lack of over-growth.The other organisms are included in trial 2,
not to establish the spectrum of the additionalinhibitors (already well documented in the litera-ture), but to illustrate colony size and pigmenta-tion differences with additional inhibitors. Thesefindings alter somewhat the color scale given byPagano et al. (4) for many species of Candida.Also, Torulopsis species, Debaromyces species,and Saccharomyces cerevisiae were inhibited bythe added antibiotics.When Pagano Levin medium was used for
primary isolation of Candida species from highlycontaminated material, more antifungal andperhaps more antibacterial activity is requiredthan neomycin furnishes. The use of cyclohexi-mide and chloramphenicol greatly improved theprocedure in trials reported here.
ACKNOWLEDGMENTThis investigation was supported by Public Health
Service Research grant AM05982-04 from the Na-tional Institute of Arthritis and Metabolic Diseases.
LITERATURE CITED1. FUENTES, C. A., F. TRESPALACIOS, G. F. BAQUERO,
AND R. ABOULOFIA. 1952. Effect of actidioneon mold contaminants and on human pathogens.Mycologia 44:170-175.
2. GEORG, L. K., L. AJELLO, AND C. PAPAGEORGE.1954. Use of cycloheximide in the selectiveisolation of fungi pathogenic to man. J. Lab.Clin. Med. 44:422-428.
3. McLEAN, I. W., JR., J. L. SCHWAB, A. B. HILLE-GAS, AND A. S. SCHLINGMAN. 1949. Suscepti-bility of micro-organisms to chloramphenicol(Chloromycetin). J. Clin. Invest. 28:953-963.
4. PAGANO, J., J. U. LEvIN, AND W. TREJO. 1958.Diagnostic medium for differentiation of speciesof Candida. Antibiot. Ann. 1957-1958, p. 137-143.
5. WELCH, H., W. A. RANDALL, R. J. REEDY, ANDJ. KRAMER. 1952. Bacterial spectrum of erythro-mycin, carbomycin, chloramphenicol, aureo-mycin and terramycin. Antibiot. Chemotherapy2:693-696.
