Taxonomy and morphology of dematiaceous fungi isolated from nature

The Hughes system of classification was applied to the identification of dematiaceous fungi isolated from nature. The majority of the isolates encountered could be identified with this system using brightfield microscopy. However, phase contrast microscopy was needed to determine the mode of conidial ontogeny with some species of Phialophora, Exophiala and Wangiella. The identifications based upon phase contrast microscopy were confirmed using the scanning electron microscope (SEM). Scanning electron photomicrographs are presented for Bispora betulina, Cladosporium trichoides, Exophiala jeanselmei, Phialophora revens, P. verrucosa and Wangiella dermatitidis. SEM observations of W. dermatitidis support the present classification of this organism as proposed by other workers. It is suggested that the Hughes system of classification and phase contrast microscopy be used to facilitate identification of isolates of dematiaceous fungi encountered in the clinical laboratory.     

Novel aliphatic epoxide hydrolase activities from dematiaceous fungi

Abstract Epoxide hydrolases were found to be constitutively expressed in dematiaceous fungi coincident with secondary metabolite pigment production in stationary or idiophase. Washed-cell preparations of two fungi, Ulodadium atrum CMC 3280 and Zopfiella karachiensis CMC 3284, exhibited affinity for 2,2-dialkylated oxiranes, for which contrasting enantioselectivities were observed, but not for aromatic styrene oxide or alicyclic cyclohexene oxide type substrates. Lyophilised preparations of soluble epoxide hydrolase activities proved to be effective catalysts for the mild hydrolysis of aliphatic epoxides.     

Studies on pathogenic dematiaceous fungi

Two hundred and twenty-six samples of woody materials, vegetable matter and soil were processed by the direct plating and mouse inoculation technique for the isolation of pathogenic dematiaceous fungi. The species of fungi isolated were Fonsecaea pedrosoi — 13, Cladosporium carrionii — 7 and Phialophora verrucosa — 4 isolates. The mouse inoculation technique was found to be much better than direct plating for the recovery of these fungi. Woody plant materials proved to be a good sample source for pathogenic dematiaceous fungi contributing about 90% of the isolates. All the isolates were pathogenic for mice as evidenced by the presence of dark nodular lesions containing fungal elements in the organs of experimentally infected animals.     

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.     

Comparison of methods for the isolation of salmonella from bone meal

[This corrects the article on p. 711 in vol. 14.].     

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals.

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.     

Spectophotometric method for preparing the inocula of dematiaceous fungi.

The aim of this study was to adapt a spectrophotometric method for preparing the inocula of dematiaceous fungi used for in vitro susceptibility tests. Fifty-two isolates of 17 different species of dematiaceous fungi were used for this purpose. Homogeneous suspensions of conidia and hyphae of these isolates were obtained and adjusted for reading at 530 and 550 nm at 40% and 50% of transmittance. The suspensions were standardised to 1-5 x 10 e6 CFU/ml. Quality controls of the inocula were done by quantitative cultures on agar-Sabouraud plates. The inocula obtained by spectrophotometry showed little variability within all the isolates. This method can be useful for in vitro antifungal evaluation of dematiaceous fungi.     

几种直接从高温热泉沉积物中提取DNA方法之比较

用酶解法,化学裂解法和微波炉法,冻融法等物理裂解方法组合出5种方法直接从滇西一个高温热泉沉积物提取DNA。经常规的琼脂糖凝胶电泳检测,发现用溶菌酶,蛋白酶K酶解,SDS化学裂解结合微波炉法裂解不仅可以得到较大量的DNA。而且碎片段较少,提取出来的DNA经RNA酶和蛋白酶K处理后可直接进行PCR扩增。同时DGGE（变性梯度凝胶电泳）电泳检测,结果表明,此法不仅可以得到该种环境中较多微生物分类单位的DNA,而且还能够较好地体现出它们在量上的差异。     

Biotransformation of cycloalkenones by fungi Baeyer-Villiger oxidation of bicycloheptenone by dematiaceous fungi

Summary The biotransformation by ring expansion of a bicyclo [3.2.0]-alkenone has been demonstrated in 29 species of dematiaceous fungi. One of these biocatalysts,Curvularia lunata NRRL 2380 has been shown to produce synthetically-important chiral lactones in high yields.     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

Comparison of methods for the isolation of Salmonella from imported frog legs.

Four methods of sample preparation were compared for their relative efficiency in recovering Salmonella from imported frog legs. No significant difference (P greater than 0.10) was observed in the efficiency of submersion, blending, and stomaching methods, but rinsing recovered significantly fewer (P less than 0.01) Salmonella-positive frog legs than the other three methods. No significant difference (P less than 0.25) was observed in the number of positive frog legs recovered by selenite cystine or tetrathionate broth. Salmonella-Shigella agar, when streaked from either of these broths, gave significantly fewer (P less than 0.01) Salmonella-positive frog legs than brilliant green, bismuth sulfite, xylose lysine deoxycholate, and Hektoen-enteric agars. Use selective agars, resulted in detection of an additional 11 and 6 Salmonella-positive frog legs, respectively. A variety of serotypes, mostly uncommon, was recovered. One Salmonella serotype (6,14,24:r,i:e,n,z15), possessing a heretofore unreported antigenic formula, was isolated.     

Comparison of methods for the isolation of Salmonella from imported frog legs.

Four methods of sample preparation were compared for their relative efficiency in recovering Salmonella from imported frog legs. No significant difference (P greater than 0.10) was observed in the efficiency of submersion, blending, and stomaching methods, but rinsing recovered significantly fewer (P less than 0.01) Salmonella-positive frog legs than the other three methods. No significant difference (P less than 0.25) was observed in the number of positive frog legs recovered by selenite cystine or tetrathionate broth. Salmonella-Shigella agar, when streaked from either of these broths, gave significantly fewer (P less than 0.01) Salmonella-positive frog legs than brilliant green, bismuth sulfite, xylose lysine deoxycholate, and Hektoen-enteric agars. Use selective agars, resulted in detection of an additional 11 and 6 Salmonella-positive frog legs, respectively. A variety of serotypes, mostly uncommon, was recovered. One Salmonella serotype (6,14,24:r,i:e,n,z15), possessing a heretofore unreported antigenic formula, was isolated.     

Antimycotic therapy of experimental infections caused by dematiaceous fungi

Experimental infections of mice with Wangiella dermatitidis and Fonsecaea pedrosoi provided a model for evaluating new antifungal agents or new combination therapy. In our models flucytosine exerted a dose-related therapeutic effect on the acute and on the more chronic infection. In the acute Wangiella infection amphotericin B also showed therapeutic activity whereas in the Fonsecaea model the effect was weak. The azole derivative ICI 153066 was the most efficacious drug in the Wangiella model whereas ketoconazole was inactive. The effect on colony-forming units of fungi in the brain was stronger with all drugs tested than the effect on survival time. Combination therapy with flucytosine + amphotericin B showed reproducible potentiating effects whereas the combination of flucytosine + ketoconazole was only additive and amphotericin B + ketoconazole showed no synergistic effect.     

Comparison of two methods of high-molecular-weight DNA isolation from human leucocytes

Among the different methods for isolation of high-molecular-weight DNA from leucocytes, two of them were retained as being the most interesting and were compared. This choice was based on three criteria: the quality of the DNA obtained (high-molecular-weight DNA, RNA, and protein free), the efficiency of the method (in terms of the yield of DNA obtained), and the ease and length of the method. The first method described was chosen as a reference (100% efficiency); in comparison, the second one had a 70% efficiency, but was shorter and easier to handle.     

Comparison of two methods for the isolation of phytolith occluded carbon from plant material

Background and aims

Phytolith occluded carbon (PhytOC) is of interest for isotope studies, dating of sediments and the capture and storage of carbon. Many methodologies have been used for the isolation of phytoliths from plant material; however, there are wide disparities in the PhytOC contents when determined by different methodologies. In this study we examine the utility of the two main methods used for quantifying PhytOC.

Methods

These methods are: (1) a microwave digestion followed by a Walkley-Black digestion, and (2) H₂SO₄/H₂O₂.

Results

Method (1) produced PhytOC values over 50 times higher than those acquired by method (2). SEM examination indicated that the differences were likely due to shattering of the phytoliths by method (2) allowing consumption by the acid and peroxide of PhytOC .

Conclusion

These results indicate that for the samples analysed here: 1] the modified microwave method allowed the total PhytOC to be measured, 2] the H₂SO₄/H₂O₂ method allowed the PhytOC within the tightly packed silica matrix to be measured, and 3] the PhytOC retained within the phytolith cavities could possibly be calculated by subtracting 2] from 1]. For the samples analysed here most of the PhytOC resided in the phytolith cavities.     

The comparison of isolation techniques for nematophagous fungi from soil

Using a single soil, a comparison was made of six well-known techniques for the isolation of nematophagous fungi. Each technique was tested for its ability to isolate the fungi present with regard to the number of species recovered, the number of plates needed to be 95% sure of isolating all the species possible and the amount of soil required. The advantages and disadvantages of each method are discussed. The results show that specific techniques are required for the isolation of endoparasites and predators respectively. The soil sprinkling technique and the Baermann funnel technique are shown to be the most efficient methods overall, with six and seven replicates required for 95% probability of isolating all the predator and endoparasite species respectively.