Single strand conformation polymorphism analysis of PCR-amplified rDNA to differentiate medically important Aspergillus species

Aspergillus species are associated with allergic bronchopulmonary disease, mycotic keratitis, otomycosis, nasal sinusitis and invasive infection. In this study, we developed a PCR-Single Strand Conformational Polymorphism method to identify the most common Aspergillus species and we showed some advantages of this method comparing a PCR-Restriction Fragment Length Polymorphism with our designed restriction enzyme. We selected ITS2, as a short fragment within the rDNA region [length size: 330 bp] to be amplified as small size PCR product. We mixed 5 ml of the PCR product with an equal volume of loading buffer and followed by incubation for 5 min at 95°C and quenching in an ice bath. The mixture was applied to a 6%-12% Gradient Poly acryl amide gel to run in a vertical electrophoresis, then gel was stained with ethidium bromide and silver nitrate which followed by an ethidium bromide staining. Our results of restriction digestion showed a fine identification of 7 tested Aspergillus species during 5-6 hours after an overnight mycelial growth. As our results some of tested Aspergillus species: A. nidulans, A.fisheri, A. quadricincta, [A. fumigatus and A. niger] as a group and [A. flavus, A. tereus and A. ochraceus] as another group, can be discriminated. Moreover SSCP analysis enabled us to identify above Aspergillus species within 8-12 h after an over night growth without using an expensive restriction enzyme. It is concluded that Single Strand Conformational Polymorphism is a simple and rapid method for identification of some medically important Aspergillus

Colony-PCR is a rapid and sensitive method for DNA amplification in yeasts

Yeast infections are increasing cause of morbidity and mortality in immunocompromised patients. In order to perform a DNA-based diagnostic test, availability of a rapid and easy-to-perform DNA extraction protocol is essential. In the present study we evaluated colony-PCR as the easiest way to amplification of target DNA. Instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures. Serial cell dilution of three reference yeast strains including Candida albicans, Cryptococcus neoformans and Saccharomyces cerevisiae were used for determining the sensitivity of the colony-PCR. A total of one hundred yeast isolates were also tested. All reactions were performed using the universal fungal primers ITS1 and ITS4 complementary to the rDNA region. The colony-PCR resulted in a single band [with different sizes] for 106 cells or more for all reference species. Furthermore 98 out of 100 [98%] of samples showed a relevant single band after PCR. Directly application of the yeast cells obtained from culture colony for PCR reaction is a fast, reliable, cost-effective and simple method for performing any PCR-based protocol including diagnostic tests

Identification of aspergillus species using morphological characteristics

Although molecular methods continue to improve and become more rapidly available, microscopy and culture remain commonly used and essential tools for identification of Aspergillus spp. In this study we emphasize on morphological methods including; macroscopic and microscopic characteristics for identification of Aspergillus species isolated from environmental and clinical specimens. We used four differential media: czapek dox agar [CZ], czapek yeast agar [CYA], malt extract agar [MEA], and czapek yeast 20% sucrose agar. Morphological features of colonies on above culture media as well as microscopically characteristics for the major strains were studied and then compared with those of standard Aspergillus strains. Our major subjects were Iranian Aspergillus strains isolated from clinical and environmental specimens. Standard Aspergillus strains for study development included; A. fumigatus, [JCM 10253], A. flavus [JCM 2061], A. niger [JCM 10254], A. nidulans [JCM 02728], A. tereus [JCM 10227]. Morphological features of Aspergillus cultures were studied, the major and remarkable macroscopic features in species identification were the colony diameter, color [conidia and reverse], exudates and colony texture. Microscopic characteristics for the identification were conidial heads, stipes, color and length vesicles shape and seriation, metula covering, conidia size, shape and roughness also colony features including diameter after 7 days, color of conidia, mycelia, exudates and reverse, colony texture and shape. Finally we compared the morphological characteristics of tested Aspergillus isolates with those of the standard species Aspergillus isolates were identified in the level of species using the differential culture media. A total of 205 Aspergillus isolates studied included: 153[75%] environmental Aspergilli and 52 [25%] clinical isolates. Within 11 Aspergillus species identified, A.flavus [55%], A.niger [31.7%] and A. fumigatus [8.7%] were the most common Aspergillus isolates from all of the specimens. In our view morphological method using the differential media is the most reliable and sensitive assay to identify more medically important Aspergillus species

In Vitro antibacterial activity of Shallot [Allium ascalonicum] crude juice

Shallot [Allium hirtifolium Bosis] belongs to genus Liliaceae There is more than 500 species in this genus. Shallot produces a cluster of bulbs from a single planted bulb. This plant has been used as an additive in foods for many years, but there is little study about antibacterial activity of Shallot. The aim of this study was to determine antibacterial activity of Shallot against clinical isolated bacteria. Crude juice of shallot [Allium hirtifolium] was tested for it's growth inhibitory effect on 4 Gram-negative and 2Gram-positive species Minimal inhibitory concentration [MIC] was determined using dilution method. All test organisms were inhibited by shallot juice. The MIC for Gram-negative enteric rods including Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis ranged between 78-624 micro /ml. while the MIC for pseudomonas aeruginosa was 20-80mg/ml.The MIC for Gram -positive bacteria including Staphylococcus aureus strains and Staphylococcus epidermidis strains were 156-312 micro /ml and 19.5-78 micro /ml respectively. It is concluded that Crude juice of shallot has antibacterial activity against both gram positive cocci and Gram-negate rods. It is believed that antibacterial activity of shallot depends on its thiosulfinate components

Study of fungal contamination of indoor public swimming pools

Fungi are found in different environments with variable distribution patterns depending on various factors. The aim of this study was determination of fungal contaminants in public swimming pools in Uromia, Iran. The fungal contaminations of four indoor swimming pools were studied by using membrane filtration and swab sampling method. Samples were collected by a manual plastic pump, in a 200 ml sterilized bottle. All samples were collected within 2 hours and then transferred to the laboratory. A total of 384 samples including water and environmental surfaces were collected and tested for the presence of fungi in different seasons within one year. In addition to the above information, some physical and chemical parameters such as temperature, residual chlorine, pH, turbidity of water and the number of swimmers were studied. Findings indicated that, the average temperature, pH, residual chlorine and turbidity of water in the swimming pools within one year were: 29.9 [degree] C, 8.1, 0.6 ppm and 0.8 NTU respectively. The most common fungi recovered were as follows: Asepergillus Spp. 56.25%, Candida spp. 22.9%, Rhizopus spp. 4.16 %, other filamentous fungi 16.6% and other yeast species 2.8%. The fungi such as Alternaria, Cladosporium, Philophora and Trichophyton mentagrophytis were isolated from dressing room, bathing room and other places out of pools. According to these results and previous studies on pools, it has been indicated that contamination by fungi in the pools is not significant in water and environment. Presence of dermatophytic fungus from dressing room is probably due to human contact