Comparative merits of eight popular media in aerometric studies of fungi

Although cultural surveys of airborne fungi are pursued widely, the comparative virtues of popular media have not been systematically tested. To facilitate an informed choice, eight different agar media were compared using paired single-plate exposures of identical, wind-oriented, Andersen samplers. Modified Mehrlich's (MM), Sabouraud's dextrose (SAB), malt extract (MALT), V8 juice (V8), and potato dextrose-rose bengal (PDAR) agar were compared initially; potato dextrose (PDA), MALT, rose bengal streptomycin (RBS), and casein hydrolysate (CH) agar were compared during a subsequent series, as were PDA and PDAR. Overall, SAB, MALT, V8, and PDA total recoveries were similar, while MM, CH, PDAR, and RBS were significantly low in paired comparisons with one or more media. MALT and SAB produced the highest frequencies of recovery of most colony types. Bacteria were infrequently recovered on any of the media. CH and MM excluded Epicoccum but are of potential use in surveys that focus sharply on high concentrations of Cladosporium. Rose bengal-containing media performed poorly in this study and must be approached with caution and under properly controlled conditions.     

The role of chlamydospores of Paracoccidioides brasiliensis.

The role of chlamydospores in the conversion process from a mycelial-to-yeast form using the slide culture method was studied. Three clinical isolates and two other isolates from armadillo, belonging to the fungal species Paracoccidioides brasiliensis, were cultured on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA) and brain heart infusion dextrose agar (BHIDA). Initially, the mycelial forms of each isolate were grown at 25 degrees C for 7, 14, 30 or 60 days on slide cultures and then the temperature was shifted to 35 degrees C. Interestingly, the slide cultures of all the isolates at 25 degrees C formed chlamydospores on either SDA or BHIDA, whereas, on PDA medium, aleurioconidia were formed. If the slide cultures on BHIDA were incubated at 35 degrees C for 7 to 14 days, multiple budding forms could be observed. This phenomenon was not evident in the slide cultures of SDA or PDA. The results of this morphological study indicate that in P. brasiliensis, chlamydospores may play an important role in the conversion process from a mycelial-to-yeast form.     

Allergens of Epicoccum nigrum grown in different media for quality source material

BACKGROUND: The Epicoccum nigrum (EN) extract used in allergy disorders exhibits batch-to-batch variations in protein composition and allergenic potency. In this study, the allergens of EN grown in different media were investigated. METHODS: EN was grown in five different nutrient media as stationary cultures at 25 degrees C for 5-23 days. The growth pattern was characterized by measuring dry weight, protein and carbohydrate content. The antigenic and allergenic content of EN extract was evaluated with EN-positive patients' sera and antibodies raised in rabbit. RESULTS: The growth of EN in Czapeck Dox medium yielded insufficient material, while Sabouraud's broth with yeast extract (SBY) gave maximum spore-mycelial mass and protein content. Potato dextrose broth (PDB) and potato dextrose agar (PDA) showed higher dry weight and protein in 7-9-day cultures. SDS-PAGE resolved 26, 22, and 21 protein bands in EN extracts from cultures of day-13 SBY, day-7 PDB, and day-9 PDA, respectively. IgE/IgG immunoblots showed more allergenic (25)/antigenic (25) bands in EN cultured in SBY than in the others. Specific IgE ELISA and intradermal tests showed EN extract from day-13 culture in SBY to be the most potent. CONCLUSIONS: The day-13 culture of EN in SBY was the most potent and may be selected for preparing EN extracts for diagnosis of allergy and future studies.     

Effects of amygdala lesions on body weight, conditioned taste aversion, and neophobia

Female rats with posterodorsal amygdala (PDA), basolateral amygdala (BLA), or sham lesions were compared regarding ad libitum food intake, weight gain, consumption of a novel food, and acquisition of a conditioned taste aversion (CTA). While only the rats with PDA lesions evidenced substantial weight gains at 10 days after surgery eating standard lab chow (25-45 g more than the other groups), only the rats with BLA lesions demonstrated significant deficits in the CTA and neophobia paradigms. Rats with basolateral lesions, on average, took less than 30 s to begin drinking the novel sweetened condensed milk after pairing with illness while the other groups took approximately 15 min to begin drinking. Also, rats with basolateral lesions ate, on average, 5 g of the novel Froot Loops while the other groups ate approximately 2 g. It is concluded that the changes in food-motivated behavioral tests frequently observed in animals with amygdala lesions do not coexist with the hyperphagia and weight gain of animals with PDA lesions.     

Identification of yeasts from clinical specimens by oxidase test

A total of 100 yeasts and yeast like fungi isolates from clinical specimens were negative for oxidase production on Sabouraud dextrose agar. When grown on Columbia agar, chocolate agar, tryptose agar, Mueller-Hinton agar, brain heart infusion and a medium resembling Sabouraud's dextrose agar but with starch instead of dextrose, all the isolate of Candida albicans (55), C. guilliermondii (6), C. parapsilosis (14), C. tropicalis (6), C. pseudotropicalis (6) and Crytococcus neoformans (2) were positive for oxidase producation. Torulopsis glabrata (2), Saccharomyces cervisiae (2) and two out of seven isolates of C. krusei were negative for oxidase test.     

Some physiological studies on fungi isolated from poultry feedstuffs

A total of 506 isolates of mesophilic, thermophilic and thermotolerant fungi isolated from the poultry feed ingredients included soybean meals, ground maize, cottonseed cake, wheat bran and fish meal, on glucose-Czapek's agar, Littman oxgall agar at 28 degrees C and yeast starch agar (YPSs) at 45 degrees C, were screened for their ability to produce hydrolytic protease enzyme on solid media. Most of the fungal isolates were able to produce such enzymes but with variable capabilities. The highest proteolytic activity was exhibited by some isolates of Penicillium chrysogenum, Aspergillus flavus, Thermoascus thermophilus and Rhizopus chizopodifarmis. Of all fungal isolates screened for proteolytic activity, Penicillium chrysogenum and Thermoascus thermophilus produced the highest amounts of proteases. These two isolates were used to study the effect of some environmental and nutritional factors on their proteolytic activity. It was found that the highest yield of protease by P. chrysogenum (12.5 units) was achieved 3 days after incubation at 30 degrees C. Marked reduction in protease activity was observed at 37 degrees C. The thermophilic fungus T. thermophillus exhibited maximum (18 units) proteolytic activity 6 days after incubation at 45 degrees C. The enzyme yield was reduced to 13 units at 50 degrees C. Among the seven carbon sources tested, sucrose was the most appropriate for maximum protease production by both P. chrysogenum and T. thermophilus (13.2 and 12.8 units, respectively). Of the sixteen nitrogen sources investigated, NaNO3 was the best inorganic additive nitrogenous salt which induced the highest proteolytic activity by P. chrysogenum and T. thermophilus, whereas DL-tryptophan was the most preferable organic nitrogen compound for maximum protease production by the two fungi tested.     

Microsporum equinum in North America.

Microsporum equinum was isolated in Ontario, Canada, from five human and two equine cases of ringworm infection. This dermatophyte was previously recovered from North American horses on several occasions, but was considered to be M. canis. We regard M. equinum as distinct from M. canis. It can be differentiated from M. canis by the smaller size of its macroconidia, its failure to perforate hair in vitro, its poor growth and sporulation on bromocresol purple casein dextrose agar, and its incompatibility with Nannizzia otae, the telemorph of M. canis.     

Vital growth factors of Malassezia species on modified CHROMagar Candida.

A comparison of several media, i.e., potato dextrose agar with olive oil (Oil-PDA), modified Dixon agar (mDIX) and variations of Leeming and Notman agar (LNA) for the isolation and growth of Malassezia and Candida species was examined. Since LNA supported the highest growth of Malassezia species its key components, i.e., ox bile, glycerol monostearate, glycerol and Tween 60, were added to CHROMagar Candida. All 7 species of Malassezia grew well on this modified medium (LN-CHROM) after incubation for 4 days at 30 degrees C and development was equal to that observed on LNA. Colonies on LN-CHROM were smooth and from pink to dark purple in color. Furthermore, the use of LN-CHROM did not alter the colony characteristics of Candida species as compared to that found on CHROMagar Candida. The results of the present investigation indicate that the use of LN-CHROM would make possible the simultaneous isolation and identification of Malassezia and Candida species.     

Comparison of Sabouraud dextrose and Pagano-Levin agar media for detection and isolation of yeasts from oral samples.

The sensitivities of Sabouraud dextrose agar and modified Pagano-Levin agar for the primary isolation of yeasts and the recovery of multiple yeast species from single clinical samples were compared by using oral-rinse samples. Although there was a highly significant positive correlation between the numbers of yeasts recovered from both media, modified Pagano-Levin agar was far superior in detecting multiple yeast species in a single sample. Of 150 oral samples containing yeasts, 23 (15.3%) contained more than one yeast species. The most frequent combination of different yeasts was Candida albicans and Torulopsis glabrata.     

Antifungal activity of dihydrodioscorine extracted from a wild variety of Dioscorea bulbifera L

The bulbils and tubers of a wild variety of Dioscorea bulbifera L. contained an alkaloid, dihydrodioscorine. When crystallized in its hydrochloride form and incorporated into potato dextrose agar at a final concentration of 0.1%, it was found to slow down the rate of growth of five plant pathogenic fungi namely: Sclerotium rolfsii, Curvularia lunata, Fusarium moniliforme, Macrophomina phaseolina and Botryodiplodia theobromae. Also sclerotia formation in S. rolfsii was delayed and the number was reduced. Conidia formation in C. lunata and F. moniliforme was also delayed and reduced but pycnidia formation in M. phaseolina and B. theobromae was not affected. There was no significant difference between the rate of growth of the fungi on PDA incorporated with 0.01% final concentration of dihydrodioscorine and on control plates. Abbreviations: PDA = potato dextrose agar, D.D. = dihydrodioscorine dihydrochloride.     

Principal component analysis of the biological characteristics of entomopathogenic fungi in nutrient-limited and cuticle-based media

Media formulated with insect cuticle (0.5% and 1%; Sphequit Sph®), with a reduction in nutrients (¼ Sabouraud dextrose agar + yeast [SDAY]) and commercial media (potato dextrose agar, Sabouraud dextrose agar) were evaluated for the cultivation of Beauveria bassiana, Cordyceps javanica (Isaria javanica [Bally] Samson & Hywel-Jones), and Metarhizium robertsii. By using principal component analysis, it was determined that the ¼ SDAY and Sph formulations have greater advantages than commercial media for the development of fungi. The ¼ SDAY and Sph (0.5% and 1%) improved hydrophobicity, radial growth rate, germination, conidia yield, and virulence in B. bassiana; in M. robertsii, they favored conidia yield, germination, and virulence, and in C. javanica, the ¼ SDAY and Sph 0.5% media enhanced conidia yield, germination, radial growth rate, and virulence. We suggest that these formulations are an alternative to commercial culture media as they are cheaper and appropriate to improve the growth characteristics and virulence of the three strains evaluated. Some applications of culture media are suggested, and the importance of multivariate analysis as an exploratory tool to carry out the choice of culture media in a suitable way for the development of mycoinsecticides is also discussed.     

Trichophyton eboreum sp. nov. isolated from human skin

An unusual dermatophyte was isolated from the plantar scales of a human immunodeficiency virus-positive man with tinea pedis. Morphology, physiology, and molecular data provided evidence to support the new species Trichophyton eboreum. This dermatophyte is characterized by rapid growth on common mycological media, a flat powdery off-white colony, formation of clavate microconidia, smooth- and thin-walled cylindrical or club-shaped macroconidia with two to nine cells, the presence of hook-shaped hyphae, the production of cleistothecium-like structures and spiral hyphae in older cultures, positive hair perforation, the absence of pigmentation on potato glucose agar, the absence of a requirement for vitamins, a weak positive urease reaction, no growth at 37 degrees C, resistance to 5% NaCl, resistance to fluconazole, good growth on human epidermal keratin, and the production of various enzymes on different media by the API-ZYM test. More than 5% divergence from any known species of dermatophyte was revealed by sequence analysis of the internal transcribed spacer of the rRNA gene.     

Evaluation of Bruker Biotyper and Vitek MS for the identification of Candida tropicalis on different solid culture media

BackgroundThe aim of this study was to investigate the performance of the Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Vitek MS systems for identification of genetically-confirmed blood isolates of Candida tropicalis that had been grown on several types of culture media commonly used for primary fungal isolation.MethodsIsolates included 105 from the National China Hospital Invasive Fungal Surveillance Net program (CHIF-NET) and 120 from National Taiwan University Hospital (NTUH). Culture media tested for CHIF-NET isolates included trypticase soy agar supplemented with 5% sheep blood (BAP), Sabouraud dextrose agar (SDA-C), CHROMagar, China blue agar (CBA), chocolate agar supplemented with vancomycin (CAP-VA), and MacConkey agar (MAC). Culture media used for NTUH isolates included BAP, SDA, CHROMagar, eosin methylene blue (EMB), inhibitory mold agar (IMA), Mycosel agar, and cornmeal agar (CMA).ResultsThe Bruker Biotyper correctly identified all CHIF-NET isolates to the species level on all six agar media tested and correctly identified the majority of NTUH isolates with the exception of isolates grown on SDA (85.8%) and CMA (52.5%). The Vitek MS system correctly identified all CHIF-NET isolates to the species level with the exception of isolates grown on CHROMagar (84.8%), and correctly identified the majority of NTUH isolates with the exception of isolates grown on SDA (51.7%), Mycosel agar (57.5%), and CMA (9.2%) for NTUH isolates.ConclusionClinical microbiologists should be aware that different culture media can affect the performance of the Bruker Biotyper MALDI-TOF MS and Vitek MS systems in identifying C. tropicalis.     

Comparison of colicine production and diffusion on different solid media

Using a freezing-thawing method for extracting colicine from solid media it has been shown that the choice of media for production and diffusion is important. Digest nutrient agar yielded the most colicine and peptone water agar the least. A factor in bacteriological peptone, but absent in a proteose peptone (Difco) and Neopeptone, was responsible for inhibiting production on peptone water agar. Dextrose reduced the diffusion of colicine. A minimum of six hours' incubation of the colicinogenic organism was required for satisfactory production of colicine.     

Identification of rare macroconidia-producing dermatophytic fungi by real-time PCR

To understand the pathogenicity and clinical significance of dermatophytes (also known as ringworms), the correct identification of these molds is essential. However, in routine practice they are notoriously difficult to classify and identify. The morphology of macroconidia, which are abundantly produced under suitable in vitro conditions, have provided useful criteria for the identification of many of the dermatophytes. However, several of them, including Microsporum audouinii, M. ferrugineum, Trichophyton concentricum, T. schoenleinii, T. verrucosum, and T. violaceum (including T. soudanense and T. yaoundei) rarely produce macroconidia and cannot be easily identified. The objective of this study was to design, optimize, and evaluate real-time PCR as a tool for identifying dermatophytic fungi in a laboratory setting. The performance of the assay was evaluated using 64 dermatophyte isolates, i.e., 35 rare macroconidia-producing reference strains, including the six species mentioned above, and 29 clinical isolates from our laboratory, including M. canis (4), T. mentagrophytes (2), T. rubrum (20), T. rubrum with the 'raubitschekii' morphotype (2), and T. tonsurans (1). Real-time PCR correctly identified 10 taxonomically distinct dermatophytes, particularly rare macroconidia-producing species, with excellent sensitivity (100%). The advantages of the assay include the provision of accurate and reliable diagnoses of dermatophytic fungi.     

High prevalence of oral colonization by Candida dubliniensis in HIV-positive patients in Argentina.

Candida dubliniensis is a recently described yeast species, closely related to Candida albicans. This work represents the first general survey of the carriage of C. dubliniensis in the oral cavities of HIV-positive patients in Argentina. We studied 133 strains isolated from 162 HIV-positive patients, using the following identification tests: chlamydospore production on corn meal agar with Tween 80; colony color on CHROMagar Candida media; differential growth at 45 degrees C on potato dextrose agar; D-xylose assimilation; chlamydospore formation on sunflower seed agar (SSA); carbohydrate assimilation profiles using the API 20 C Aux commercial kit and PCR using primers that hybridize to the class IV intron of the ACT1 gene. Out of the 133 strains, 21 were identified as C. dubliniensis, representing approximately 13% of the 162 patients in this study. From these data, we conclude that although the PCR assay is the most reliable method, clamydospore formation on SSA is an easier and less expensive test for the screening of C. dubliniensis in the routine laboratory. Our results show that C. dubliniensis has a high prevalence among HIV-positive patients in Argentina.     

Comparison of Guizotia abyssinica seed extract (birdseed) agar with conventional media for selective identification of Cryptococcus neoformans in patients with acquired immunodeficiency syndrome.

Growth of Cryptococcus neoformans from the sputum of patients with acquired immunodeficiency syndrome may be obscured by oral contamination with Candida albicans on conventional media. We prospectively compared direct plating of sputum and urine onto birdseed agar and compared birdseed agar plating with plating onto Mycosel and Sabouraud dextrose agar cultures. Thirty-two sputum and three urine specimens were compared. C. neoformans was isolated from five specimens. In two specimens, one of sputum and one of urine, C. neoformans was detected only on the birdseed agar plate because of overgrowth on the conventional media by C. albicans. C. neoformans produced dark colonies on birdseed agar, unlike C. albicans, which produces white colonies. The use of birdseed agar as the primary culture medium for sputum and urine specimens from patients with acquired immunodeficiency syndrome increases sensitivity for C. neoformans.     

Tween 40-based precipitate production observed on modified chromogenic agar and development of biological identification kit for Malassezia species.

We developed a simple identification kit for nine species of Malassezia (M. furfur, M. slooffiae, M. sympodialis, M. restricta, M. obtusa, M. globosa, M. pachydermatis, M. dermatis, and M. japonica) based on their biological features. This method utilizes Tween 40-based precipitate production on modified chromogenic agar (CHROMagar) Malassezia medium, growth on specific agars (Sabouraud's dextrose agar, Cremophor EL agar, Tween 60-esculin agar), and catalase reactions. This identification kit was verified with 11 type and reference strains of nine Malassezia species. An additional 26 clinical isolates were also successfully identified using the kit and the results were confirmed by molecular biological analysis.     

Clinical evaluation of the addition of gentamicin to commercially prepared mycological media.

A simple procedure is presented whereby an antibiotic solution can be added to prepared agar media for conversion to a selective medium to isolate fungi. Gentamicin solution was deposited onto slants of a variety of previously prepared agar media, allowed to diffuse overnight, and then the slants were inoculated with clinical specimens. Control media without gentamicin included a cycloheximide-chloramphenicol medium (CC), Sabouraud dextrose agar (SDA), and brain heart infusion agar (BHI). Of 75 specimens originating from the respiratory tract, the fungi isolated were predominantly yeast; 35, 39, and 43 were positive on CC, SDA, and SDA with gentamicin, respectively, incubated at 25 C. At 37 C, 32, 34, and 41 positive cultures were obtained with the same media, respectively. The same specimens, inoculated onto BHI with and without gentamicin, yielded 23 and 39 positive cultures, respectively. Of 90 specimens that were either urine, cutaneous, or mucocutaneous, the predominant flora again were yeasts, although on nine occasions dermatophytes were isolated. Positive cultures, 32, 34, and 41, were obtained with CC, SDA, and SDA containing gentamicin, respectively. Bacterial contamination was significantly reduced by the gentamicin, especially on BHI incubated at 37 C. None of the specimens was positive for systemically pathogenic fungi, other than species of Candida, Torulopsis, and Aspergillus. The effectiveness of varying concentrations of gentamicin was investigated by comparing growth of recently isolated bacteria. Of the bacterial isolates, 33% grew on CC, 16% grew on SDA containing gentamicin, 50 mug/ml, and 3% grew on SDA with a gentamicin concentration at 100 mug/ml. With BHI, 3% grew in the presence of 50 mug of gentamicin/ml and less than 1% grew at 100 mug of gentamicin/ml.     

Enhancement of IgE antibody production by ovalbumin aerosol in mice

The effect of aerosolized ovalbumin (OA) on the induction of IgE antibody production was investigated in BALB/c mice. Enhancement of anti-OA IgE antibody production was obtained after the administration of 10 micrograms OA in mice preexposed to 1% aerosolized OA for 6 or 30 min, and after the administration of 1 microgram OA in mice preexposed to aerosolized OA for 30 min. IgE antibody production could also be induced by preexposure to aerosolized OA at a concentration as low as 0.1%. A carrier effect was observed by preexposure to aerosolized OA using a hapten-carrier system. In the latter system, whole body irradiation (200 R) could not enhance IgE response, suggesting that irradiation-sensitive suppressor T cells might be generated in insufficient amount or not at all by aerosolized OA. Preexposure to aerosolized dinitrophenol-OA could not enhance anti-dinitrophenol IgE response. These results suggest that the exposure to aerosolized antigen primed preferentially T helper cells.