Studies of the genus Pityrosporum in submerged culture

Differentiation betweenPityrosporum ovale andPityrosporum orbiculare is mainly based upon the morphological features of cells and on physiological characters such as the ability ofP. ovale of utilizing oleic acid (Marples, 1965). Our studies in submerged culture, however, have not shown remarkable differences between the two species: growth curves, nutritional requirements, lipid utilization and optimal temperature are practically the same. In some strains of both species (1 : 4) we did observe a limited growth capacity, associated with a lower utilization rate of lipids. But for the notably different morphological features of cells,P. ovale andP. orbiculare could be ascribed, as regards their cultural characteristics, to the same species. The search forP. orbiculare was successful in all the cases of Pityriasis versicolor, while all our attempts to isolateP. ovale from its usual areas (scalp, upper back and chest) from both healty and seborrhoic subjets, gave no results.P. orbiculare was found in the same areas in the greatest part (85 %) of these subjects. In order to explain these results, one could assume thatP. orbiculare is the only species ofPityrosporum widespread in our region.These studies were supported by grant of the Consiglio Nazionale delle Ricerche of Italy under contract n. 70.01023/115.2798.     

Prevalence of dermatophytes and yeasts (Candida spp., Malassezia furfur) in HIV patients

The prevalence of dermatophytes and yeasts (Candida spp. and Pityrosporum spp.) was studied in 40 former drug-addicts, all of whom were HIV seropositive but otherwise had no other symptoms (2nd Stage CDC Atlanta, 1987). We considered 7 skin areas for dermatophytes and Pityrosporum spp. (scalp, forehead, nose, back, chest, groin, toe webs) and the mouth for yeasts. Dermatophytes were found in 8 (20%) and tinea pedis was the most common dermatophytosis: Tricophyton rubrum was the fungus most frequently isolated (6 cases or 15%). The HIV + group showed almost the same rate of dermatophytes colonisation compared to a group of 121 athletes and to the control group. Candida spp. was present in 27 cases (67.5%) but clinical oral lesions were evident only in 5 patients (12.5%). Statistically significant differences were found in the presence of Candida spp. in HIV patients and controls (p<0.05). The lipophilic yeast Pityrosporum ovale was evaluated with quantitative and qualitative methods. Quantitative variations were evident between HIV patients and controls. P. ovale was present in 10 cases: 3 (7.5%) of them showed dischromic lesions while in 7 cases (17.5%) no clinical symptoms were evident.     

The diversity of Pityrosporum (Malassezia) yeasts in vivo and in vitro

Conclusions In considering the diversity of the lipophilic yeasts it has been shown that in vivo both spherical and oval yeasts may be found in normal conditions on the skin and also associated with hyphae in scales from pityriasis versicolor. There is however generally a different distribution pattern on the body for two forms. This may indicate a different ecology for two distinct varieties or the varying conditions at each site may influence changes in the cell shape of a single species. It is striking that the spherical yeasts (P. orbiculare) have not been found in animals. In vitro, several morphological variants can be maintained, but the change from one form to another which is the subject of a number of reports will be one deciding factor leading to the opinion that P. orbiculare and P. ovale are synonymous. However, physiological differences such as growth rate, their viability in subculture and the analysis of proteins, are all characteristics which alone would be insufficient to support a taxonomic division but when added together confirm the morphological separation of isolates. It remains to be seen if DNA studies, which have so far unified the anthropophilic, lipid dependant Pityrosporum yeasts, will in fact continue to show that they should be confined to a single species.This paper was presented at the X^th congress of the International Society for Human and Animal Mycology at Barcelona, Spain from June 27 to July 1, 1988.     

Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products.

A selective medium (LUSM medium) for the isolation of Leuconostoc spp. was developed. This medium contained 1.0% glucose, 1.0% Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25% sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 micrograms of vancomycin (Sigma) per ml, 0.20 microgram of tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco). LUSM medium was used successfully for isolation and enumeration of Leuconostoc spp. in dairy products and vegetables. Of 116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as members of the genus Leuconostoc. A total of 89 of these isolates were identified to species; 13.5% of the isolates were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp. mesenteroides, 11.2% were Leuconostoc mesenteroides subsp. dextranicum, 16.9% were Leuconostoc mesenteroides subsp. paramesenteroides, 10.1% were leuconostoc lactis, and 40.4% were Leuconostoc oenos. When we compared the counts obtained for two Leuconostoc strains, Leuconostoc dextranicum 181 and L. cremoris JLL8, on MRS agar and LUSM medium, we found no significant difference between the values obtained on the two media.     

Evaluation of Pseudosel Agar as an Aid in the Identification of Pseudomonas aeruginosa

Growth of Pseudomonas aeruginosa and thirty-five other species of gramnegative bacilli was observed on 0.03% cetrimide in heart infusion agar medium and Pseudosel agar (BBL). The 0.03% cetrimide agar was more selective for growth of P. aeruginosa than was Pseudosel agar; however, certain bacteria other than P. aeruginosa also grew on the former medium. Although Pseudosel agar was not a highly selective medium for P. aeruginosa, it was preferable to technicolor agar for detection of the pyocyanin and pyorubin pigments produced by P. aeruginosa.     

A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group

A new selective medium, Bacteroides fragilis ammonium-sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram-positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.     

Comparative media studies in the isolation ofCandida albicans from pregnant women

Summary Rice agar and corn meal agar, with and without Tween 80, were evaluated clinically as directly inoculable selective and differential media for the isolation ofC. albicans from vulvovaginal specimens taken from pregnant women. Chlamydospore formation on these media was investigated as a criterion for the identification ofC. albicans.Of 301 patients cultured, 118 (39.2 %) gave positive cultures for yeast-like fungi of the genusCandida. Of 118 strains for which fermentation patterns were determined, 69 (58.5 %) gave the pattern forC. albicans. Of these, 56 (81.1 %) formed chlamydospores.Tween 80 was found to exert a very stimulating influence on chlamydospore production. Rice agar with Tween 80 appeared to be the most efficient medium for eliciting chlamydospores. However, since strains of 4 out of 6 species ofCandida isolated were found to sporulate it was concluded that chlamydospore formation is not a reliable criterion for the speciation ofC. albicans.Each of the 4 media served satisfactorily as a directly inoculable selective medium for the isolation of yeast-like fungi of the genusCandida from vulvovaginal specimens. None of the media appeared to preferentially stimulate chlamydospore production inC. albicans.Dr.Smith is Associate Professor in the Department of Microbiology; Dr.Taubert is a Fellow in the Department of Obstetrics and Gynecology; Mr.Towns is Laboratory Assistant in the Department of Microbiology.Supported in part by a grant from the Lederle Medical Faculty Awards Committee and in part by United States Public Health Service Grant E-3068.     

Evaluation of guar gum derivatives as gelling agents for microbial culture media

Guar gum, a galactomannan, has been reported to be an inexpensive substitute of agar for microbial culture media. However, its use is restricted probably because of (1) its highly viscous nature even at high temperatures, making dispensing of the media to Petri plates difficult and (2) lesser clarity of the guar gum gelled media than agar media due to impurities present in guar gum. To overcome these problems, three guar gum derivatives, carboxymethyl guar, carboxymethyl hydroxypropyl guar and hydroxypropyl guar, were tested as gelling agents for microbial growth and differentiation. These were also evaluated for their suitability for other routine microbiological methods, such as, enumeration, use of selective and differential media, and antibiotic sensitivity test. For evaluation purpose, growth and differentiation of eight fungi and eight bacteria grown on the media gelled with agar (1.5%), guar gum (4%) or one of the guar gum derivatives (4%), were compared. All fungi and bacteria exhibited normal growth and differentiation on all these media. Generally, growth of most of the fungi was better on guar gum derivatives gelled medium than on agar medium. The enumeration carried out for Serratia sp. and Pseudomonas aeruginosa by serial dilution and pour plate method yielded similar counts in all the treatments. Likewise, the selective succinate medium, specific for P. aeruginosa, did not allow growth of co-inoculated Bacillus sp. even if gelled with guar gum derivatives. The differential medium, Congo red mannitol agar could not differentiate between Agrobacterium tumefaciens and Rhizobium meliloti on color basis, if gelled with guar gum or any of its derivatives However, for antibiotic sensitivity tests for both Gram-positive and -negative bacteria, guar gum and its derivatives were as effective as agar.     

Effects of essential oils on in vitro ruminal fermentation and ammonia release

Ruminal inoculum enriched with particle-associated microorganisms was collected from two lactating dairy cows fed an alfalfa hay/cereal silage/concentrate diet 1 h before feeding and used to evaluate effects of essential oils (EO) on ruminal fermentation in short-term in vitro incubations. Ruminal ammonia N was labeled with ¹⁵N and native and hydrolyzed casein were provided as sources of amino acids. Forty EO were tested at 10 and 100 mg/l final medium concentration. Monensin-Na, and sodium laurate were also incubated at 5 and 2000 mg/l, respectively. Compared with blanks (i.e., no addition of EO), sodium laurate increased medium pH and a number of EO reduced medium pH. Both sodium laurate and monensin reduced ammonia concentrations compared to the blank. Only one of the tested EO (i.e., Caraway) slightly reduced ammonia concentration, by 8%, compared with the blank. Monensin and sodium laurate resulted in higher (i.e., 9–34%, monensin, and 29–47%, sodium laurate) ¹⁵N enrichment of ammonia N, an indication of reduced deamination of amino acids in these treatments versus the blank. Several EO (i.e., FrankMyrrh, Gardenia, Hibiscus, Eucaliptus, and Peppermint) had similar effects, but of a smaller magnitude (i.e., 5–12%). Some EO increased medium total VFA concentration, primarily through an increase in acetate concentration. Overall, effects of EO on fermentation were subtle, and it is unlikely that these moderate in vitro effects would correspond to any substantive impact on ruminal fermentation in vivo.     

Growth of Desulfovibrio on the Surface of Agar Media

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.     

Gentamicin-Based Medium for the Isolation of Group D Streptococci and Application of the Medium to Water Analysis

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH₂PO₄, 2.0 g of NaHCO₂, 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO₃ (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

A reevaluation of the genus Malassezia by means of genome comparison

Results of a study of the genus Malassezia on the basis of genome characters confirm that two species should be maintained, M. furfur and M. pachydermatis. The two forms associated with skin disease, frequently referred to as Pityrosporum orbiculare and P. ovale, were found to be synonymous, the name M. furfur having priority. Malassezia pachydermatis, hitherto regarded as a strictly zoophilic species, may also be found on humans.     

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

The vitamin requirements of Staphylococcus cohnii

In heat-resistance studies with spores of Clostridium sporogenes BC-2, an improved recovery medium was needed for severely heat-damaged spores as the used previously--Wynne medium in Miller-Prickett tubes--did not allow accurate counts of spores because of gas formation and disruption of agar. Initial test with pour plates of Viande-Leyure medium containing egg-yolk gave much increased counts for spores previously heated for 50 min at 112 degrees C; this increase was attributed to the presence of egg-yolk. Addition of egg-yolk to Reinforced Clostridial Agar, All-Culture Medium and Trypticase Soy Agar showed that Trypticase Soy Agar with egg-yolk was the best recovery medium. For the final formulation, the value of supplementation with cysteine-HCl and methylene blue was also shown. The resultant Egg-yolk Trypticase Soy Agar is conveniently prepared from BBL Trypticase Soy Agar (40 g) with the addition of 0.4 g/l cysteine-HCl, 4 mg/l of methylene blue and 2% Oxoid egg-yolk emulsion aseptically to the melted basal medium. For optimal spore counts, pour plates are incubated anaerobically for 5--7 d at 30 degrees C.     

Somatic embryogenesis and plant development from immature zygotic embryos of seedless grapes (Vitis vinifera L.)

Summary Somatic embryo formation occurred from immature zygotic embryos within ovules of stenospermocarpic seedless grapes (Vitis vinifera L.), when cultured for two months on liquid Emershad/Ramming medium. Somatic embryos continued to proliferate after excision and transfer to Emershad/Ramming medium supplemented with 1 M benzylaminopurine and 0.65% TC agar. Plant development from somatic embryos was influenced by genotype, medium, phase (liquid, agar), stage (torpedo, mature) and their interactions. Optimal plant development occurred on Woody Plant Medium supplemented with 1.5% sucrose + 1 M benzylaminopurine + 0.3% activated charcoal and 0.65% TC agar.Abbreviations ABA abscisic acid - BAP benzylaminopurine - ER Emershad/Ramming - GLM general linear model - MGLH multivariate general linear hypothesis - MS Murashige/Skoog - NaClO sodium hypochlorite - TC tissue culture - WP woody plant     

In vitro susceptibility ofPityrosporum ovale (Malassezia furfur) to human androgenic steroids

Cells ofPityrosporum ovale that colonize human pilosebaceous units are constantly exposed to cutaneous androgenic steroids. The aim of our study was to find out whetherP. ovale is susceptible to these hormones. Three strains ofP. ovale were grown in vitro in the presence of various concentrations oftestosterone, dehydroepiandrosterone, androstenedione, androstanedione, 5--dihydrotestosterone andprogesterone (10, 100, and 1000 µg/ml; agar dilution assays). In addition, three strains ofCandida albicans were also exposed to equal concentrations of the same androgens. As a result, allP. ovale strains were suppressed by 1000 µg/mlandrostenedione, which was the strongest inhibitor. The other androgenic steroids also significantly reducedP. ovale growth at different concentrations, depending on the hormone used and the strain tested.Progesterone was inhibitory at the highest concentration for oneP. ovale strain only.Candida albicans was not affected by any of the androgens. These findings demonstrate an in vitro susceptibility ofP. ovale to high concentrations of human androgenic steroids. A relevance of this interaction for the in vivo fungus-host relation is not apparent.     

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca²⁺-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using ??-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T_1/2 was 1 h at 100°C. The K _m of the partial purified enzyme was 1 mM and the V _max was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca²⁺ and Hg²⁺ stimulated lipase activity, whereas Zn²⁺, Co²⁺, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.     

Comparison of Media for Detection of Fungi on Spacecraft

Five media, including Trypticase soy agar (TSA; BBL) pour plates, spread plates of TSA, Mycophil agar with chloromycetin, Mycophil agar with chloromycetin and Actidione, and cornmeal agar with chloromycetin were quantitatively and qualitatively compared for the detection of fungi on spacecraft. Cornmeal agar with chloromycetin yielded the highest number of fungal colonies, although not always significantly higher than Mycophil agar with chloromycetin or TSA spread plates. Cornmeal agar with chloromycetin also gave the best qualitative representation of fungi on the spacecraft, recovering 68% of the genera found from all media. This medium yielded 10 times the number of fungal colonies and 3 times the number of genera found on TSA pour plates as currently used for spacecraft assay.