Identification of yeasts of clinical interest on CHROMagar Candida culture medium.

The efficiency of CHROMagar Candida was evaluated as a medium for the presumptive identification of yeasts. We tested 36 different yeast species, pertaining to 9 genera: one Blastoschizomyces, 20 Candida, five Cryptococcus, two Geotrichum, one Kloeckera, two Pichia, three Rhodotorula, one Saccharomyces and one Trichosporon, to determine the colony colors and characteristics on this medium. Afterwards, we identified 2,230 strains isolated directly on CHROMagar Candida from clinical samples by specific colouration and morphology of the colonies after 72 hours. Their results were compared with standard methods for the identification of yeasts. The sensitivity and specificity were both superior to 97% for all strains, 100% and 100% for Candida albicans, 97.3% and 99.9% for Candida glabrata, 92.3% and 99.6% for Candida krusei, 90.3% and 99.6% for Candida parapsilosis, and 100% and 100% for Candida tropicalis. CHROMagar Candida is a very useful medium for the culture of clinical samples; its use for identification of yeasts has an accuracy of 97.5%, close to 100% of conventional methods.     

Early Appearance of Colonies of Brucella Spp. on Solid Media containing Erythritol

S ummary . The incorporation of erythritol (1 μ M /ml) in Morris medium caused the earlier appearance of colonies of virulent strains of Brucella abortus and B. melitensis. On Albimi agar erythritol accelerated the growth of B. melitensis but not that of B. abortus. There was no acceleration of the growth of a virulent strain of B. suis on either medium.     

Scanning Electron Microscopy of Colonies of Six Species of Candida

Thirty strains of six species of Candida isolated from patients were cultured for 60 h on Sabouraud agar, freeze-dried, and examined with a scanning electron microscope. The colonies were circular (Candida albicans, C. guilliermondii) or oval (C. tropicalis, C. pseudotropicalis, C. krusei, C. parakrusei) in outline, and those of C. pseudotropicalis and C. krusei had an irregular outline due to a peripheral pseudomycelium. The morphology of individual microorganisms was examined at the margins and apex of those species which lacked a surface coat (C. pseudotropicalis, C. krusei, C. parakrusei, C. guilliermondii), and through cracks in the surface coating of those which showed a surface coat (C. albicans, C. tropicalis). All species showed buds, bud scars, and interconnecting intercellular processes, but were generally spherical (C. albicans, C. tropicalis) or ovoid (C. pseudotropicalis, C. krusei, C. parakrusei, C. guilliermondii) in fixed preparations. In unfixed material, individual organisms were almost invariably indented. Fixation with 3% glutaraldehyde and washing before freeze-drying caused partial removal of the surface coating of colonies of C. albicans and C. tropicalis, which persisted only as irregular sheets or as a filamentous meshwork. This filamentous meshwork was also present among the organisms of colonies of C. albicans, C. tropicalis, and C. pseudotropicalis. It is concluded that these filaments represent the precipitation or unmasking of some component of the intercellular matrix of these organisms.     

Simple new test for presumptive differentiation between genus Candida and genus Prototheca

A differential test was made between genus Candida and genus Prototheca using a new and very simple differential test.A total of 59 strains of Candida and 78 strains of Prototheca were used. The basis of the test was the differential use of a disc carrying 60 mcg of Rybostamicin to which all the Candida were resistant and the Prototheca inhibited.     

Appearance and properties of L-sorbose-utilizing mutants of Candida albicans obtained on a selective plate.

This is the first report that adaptive mutagenesis can arise by chromosomal nondisjunction, a phenomenon previously associated exclusively with DNA alterations. We previously uncovered a novel regulatory mechanism in Candida albicans in which the assimilation of an alternative sugar, l-sorbose, was determined by copy number of chromosome 5, such that monosomic strains utilized l-sorbose, whereas disomic strains did not. We present evidence that this formation of monosomy of chromosome 5, which is apparently a result of nondisjunction, appeared with increased frequencies after a selective condition was applied, i.e., by adaptive mutagenesis. The rate of formation of l-sorbose-utilizing mutants per viable cell per day ranged from 10(-6) at the initial time of detection to 10(-2) after 4 days of incubation on the selective plate.     

The Cytochromes of Prototheca zopfii

The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a₃. Cytochromes a and a₃ could be resolved spectrally in the α-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a₃ complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a₃, and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).     

The ultrastructure of Prototheca wickerhamii

Two clinical isolates of Prototheca wickerhamii were freeze-dried, fixed and studied by electron microscopy and were also examined growing in culture medium by phase contrast light microscopy. Resting spores placed on fresh medium developed cytoplasmic extensions which sequestrated before proliferation occurred. In the presence of adequate nutrients vegetative spores grew and subdivided to form up to 12 endospores within large sporangia which ruptured to release free spores every 5–6 hours. These proliferating or vegetative spores contained much more endoplasmic reticulum and mitochondria than the resting spores which contained more lipid, and often starch granules as well, but relatively few membranous organelles.     

Enzymatic profile of Prototheca species

The enzymatic profiles of algae of genus Prototheca were studied using 19 substrates included in the API ZYM system. Chymotrypsin and -glucuronidase were not detected. Of the enzymes detected the major percentages were alkaline phosphatase, esterase (C4), lipase esterase (C8), acid phosphatase and phosphoamidase.     

Identification of Candida albicans using the chromogenic medium Albicans ID

The correct identification of the microrganism is the base for epidemiological studies and treatment of infections. The aim of our study was to evaluate the efficacy of the chromogenic media Albicans ID (bioMerieux, France) in the identification of Candida albicans. A total of 190 yeasts strains were evaluated in the study. A rate of 100% of all C. albicans (80) and Candida dubliniensis (five) strains exhibited blue color. Nevertheless, the blue color was also observed with cultures of Candida rugosa (3/5) and Candida tropicalis (3/17). Albicans ID cromogenic media presented specificity rate of 90% and positive and negative predictive values of 88% and 100%, respectively, in the identification of C. albicans.     

New chromogenic agar medium for the identification of Candida spp

A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-(2-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3-methoxyphenyl]-vinyl)-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter(-1)). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37 degrees C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity of Candida detection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.     

Continuous culture of Candida utilis: influence of medium nitrogen concentration

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h^-1), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.     

Growth of Prototheca isolates on n-hexadecane and mixed-hydrocarbon substrate

Prototheca zopfii, an achlorphyllous alga, was capable of using hydrocarbons as sole carbon and energy source. The ability of P. zopfii to use hydrocarbons did not correlate with source of isolation. Seventy-five percent of the P. zopfii cultures recovered from sewage, plants, or animals utilized hydrocarbons. Other Prototheca species and P. zopfii that did not utilize hydrocarbons were isolated simultaneously from several sources with isolates that did use hydrocarbons. Species type rather than source of isolation was the predominant factor that determined hydrocarbon utilization.     

Electron microscopic study of Candida tropicalis yeasts grown on a medium containing selenium]

The yeast Candida tropicalis 303 was cultivated on a medium containing selenium, and studied by electron microscopy. The vacuoles of these cells contained electron-dense granules. The correlation between the increase in the number of the electron-dense granules in the cells of C. tropicalis 303 and the biomass suggests the presence of Se0 in the Granules.     

Emergence of Fungal-Like Organisms: Prototheca

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and animal intestines. However, this organism has forfeited its photosynthetic ability and switched to parasitism. In 1894, Krüger described two microorganisms isolated in Germany from mucous flux of Tilia and Ulmus spp., namely Prototheca moriformis and P. zopfii. Based on their yeast-like colony morphology, Krüger classified these organisms as fungi. The genus is now included within the class Trebouxiophyceae, order Chlorellales, and family Chlorellaceae. Historically, protothecosis and infections caused by green algae have been studied in the field of medical mycology. Prototheca spp. have been found to colonize human skin, fingernails, the respiratory tract, and digestive system. Although human infection by Prototheca is considered rare, an increase in infections has been noted among immunosuppressed patients, those on corticosteroid treatment, or both. Moreover, the first human outbreak of protothecal algaemia and sepsis was recently reported in a tertiary care chemotherapy oncology unit in 2018. Prototheca is also a causative pathogen of bovine disease. Prototheca zopfii and P. blaschkeae are associated with bovine mastitis, which causes a reduction in milk production and secretion of thin, watery milk containing white flakes. Economic losses are incurred either directly via reduced milk production and premature culling of affected animals or indirectly as a result of treatment and veterinary care expenses. Thus, knowledge of this fungal-like pathogen is essential in human and veterinary medicine. In this mini-review, I briefly introduce human and animal protothecoses.