Recovering Greater Fungal Diversity from Pristine and Diesel Fuel Contaminated Sub-Antarctic Soil Through Cultivation Using Both a High and a Low Nutrient Media Approach

Novel cultivation strategies for bacteria are widespread and well described for recovering greater diversity from the “hitherto” unculturable majority. While similar approaches have not yet been demonstrated for fungi it has been suggested that of the 1.5 million estimated species less than 5% have been recovered into pure culture. Fungi are known to be involved in many degradative processes, including the breakdown of petroleum hydrocarbons, and it has been speculated that in Polar Regions they contribute significantly to bioremediation of contaminated soils. Given the biotechnological potential of fungi there is a need to increase efforts for greater species recovery, particularly from extreme environments such as sub-Antarctic Macquarie Island. In this study, like the yet-to-be cultured bacteria, high concentrations of nutrients selected for predominantly different fungal species to that recovered using a low nutrient media. By combining both media approaches to the cultivation of fungi from contaminated and non-contaminated soils, 91 fungal species were recovered, including 63 unidentified species. A preliminary biodegradation activity assay on a selection of isolates found that a high proportion of novel and described fungal species from a range of soil samples were capable of hydrocarbon degradation and should be characterized further

Development of fluorescent in situ hybridisation for Cryptosporidium detection reveals zoonotic and anthrioponotic transmission of sporadic cryptosporidiosis in Sydney

Cryptosporidium, is the most common non-viral cause of diarrhea worldwide. Of the 5 described species that contribute to the majority of human infections, C. parvum is of major interest due to its zoonotic potential. A species-specific fluorescence in situ hybridisation probe was designed to the variable region in the small subunit of the 18S rRNA of C. parvum and labeled with Cy3. Probe specificity was validated against a panel of 7 other Cryptosporidium spp. before it was applied to 33 human faecal samples positive for cryptosporidiosis which were obtained during the period from 2006-2007. Results were compared to PCR-RFLP targeting the 18S rDNA. FISH results revealed that 19 of the 33 isolates analysed were identified as C. parvum. Correlation of PCR-RFLP and FISH was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. In this study, species identification by FISH and PCR-RFLP provided preliminary evidence to support both anthroponotic and zoonotic transmission of sporadic cases of cryptosporidiosis in the Sydney basin. In conclusion, FISH using a C. parvum-specific probe provided an alternative tool for accurate identification of zoonotic Cryptosporidium which will be applied in the future to both epidemiological and outbreak investigations

Biocleaning of starch glues from textiles by means of α-amylase-based treatments

Glues based on starch are widely used for the consolidation of brittle fibres in historic and archaeological textiles. Aging fabrics are affected by hydrolysis/oxidation and cross-linking of these glues, a decrease of glues’ solubility, the formation of cracks, and discoloration. The hydrolytic action of enzymes on starch-based glues is promising, as molecular recognition offers great selectivity. However, a systematic assessment of the best methods for applying enzymatic formulations has not been explored yet. Here, α-amylase was applied either by pipetting a solution or combining with gellan gel (embedded in the gel or spread on the gel surface). The effectiveness of the different formulations on the removal of potato and wheat starch was evaluated by Fourier Transform Infrared (FTIR) spectroscopy, Scanning Electron Microscopy (SEM) and colorimetric measurements. Enzymes dispersed in gel showed weak diffusion at the surface, resulting in poor starch breakdown and removal. On the contrary, amylase applied by pipette and spread on gel resulted in high starch removal selectivity and efficiency, with neither swelling nor damage to the fibres. These results validate protocols for the assessment of the enzymatic activity on glue-consolidated fibres, identify best application methods and confirm the excellent properties of amylase dispersions for the conservation of historic and archaeological textiles

Potential Use of Quantum Dots in Flow Cytometry

QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM) by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC) and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC

Out of thin air? Astrobiology and atmospheric chemotrophy

The emerging understanding of microbial trace gas chemotrophy as a metabolic strategy to support energy and carbon acquisition for microbial survival and growth has significant implications in the search for past, and even extant, life beyond Earth. The use of trace gases, including hydrogen and carbon monoxide as substrates for microbial oxidation, potentially offers a viable strategy with which to support life on planetary bodies that possess a suitable atmospheric composition, such as Mars and Titan. Here, we discuss the current state of knowledge of this process and explore its potential in the field of astrobiological exploration.An Australian Research Council Future Fellowshiphttps://home.liebertpub.com/publications/astrobiology/99am2023BiochemistryGeneticsMicrobiology and Plant Patholog

Enhancing student learning in science using virtual laboratories

INTRODUCTION AND SIGNIFICANCE Delivering an engaging and realistic laboratory experience for undergraduate biomedical and biological science students is difficult due to increasing class sizes, stringent health and safety requirements as well as costs, maintenance and availability of equipment and reagents. Flow cytometry is a core technology, widely used in research and diagnostic laboratories, but due to its complexity and cost, laboratory classes on the application of this technology are inadequate. Virtual laboratories provide students with the opportunity for lifelike, interactive, one-on-one learning, that simulates real-laboratory experiences. There are no suitable flow cytometry simulations available in the education space and with the essential role that flow cytometry plays in the biomedical and biological sciences, as well as industrial research, this project addressed this need. METHODOLOGY A virtual laboratory focused on flow cytometry was developed for undergraduate, honours and postgraduate research students. The simulation and tutorial were developed using the Smart SparrowTM Adaptive eLearning Platform. The tutorial was designed to be adaptable to multiple audiences, with several platform datasets incorporated into the simulation to cover different scientific fields that use the technology. RESULTS The simulation developed (Figure 1) provides students with a personal experience with the interface very similar to the latest in flow cytometry technology. Students are in control of a wide range of experimental variables and instrument parameters, most of which are not usually available to them in laboratory classes. It is accessible online, and allows students to repeat lessons in their own time and to their own satisfaction. It was deployed in an undergraduate pathology class and to honours and postgraduate students receiving training in flow cytometry. Students reported that the lesson improved their understanding, their confidence in their technical skills and was targeted to their level of understanding. The average amount of time students spent in the lesson was 47 minutes with some returning for further revision. The tutorial is made available by the Biomedical Education, Skills and Training Network (BEST Network) (https://www.best.edu.au/lesson/?id=49734), a community of educators that create and share teaching resources, including other virtual laboratories that have been accessed over 4700 times. CONCLUSIONS Our flow cytometry virtual laboratory facilitates the learning of technical skills in data collection and analysis. Such skills are important for both undergraduate and postgraduate students who require an understanding of the applicability of this technology in the real-world for future employment and/or research opportunities

Immobilized Enzymes on Gold Nanoparticles: From Enhanced Stability to Cleaning of Heritage Textiles

Enzyme-based treatments are used in heritage conservation for the effective removal of glues and other damaging organic layers from the surfaces of historic and artisitc works. Despite their potential, however, the application of enzymatic treatments is currently limited due to their poor efficiency, and low operational and environmental stability. We demonstrate the use of α-amylase immobilized on gold nanoparticles to improve the efficacy of enzymatic treatments enchancing both the reactivity and the stability of the formulations. Gold nanoparticles coated with α-amylase exhibit significant advantages compared to free enzymes. We report up to 5-times greater resistance to environmental changes, up to 2-times higher efficacy towards removal of starch-based glues from textile, and deeper penetration through the fibres, without causing damage or inducing salt precipitation. These results offer exciting prospects for the development of novel enzymatic formulations, both for heritage conservation and the wider application of enzymes, such as in medicine, the detergent industry and green chemistry

Microfluidic qPCR Enables High Throughput Quantification of Microbial Functional Genes but Requires Strict Curation of Primers

Quantification of microbial functional genes enhances predictions of soil biogeochemical process rates, but reliance on low-throughput quantitative PCR (qPCR) limits the scope of ecological studies to a handful of targets. Here, we explore whether microfluidic qPCR (MFQPCR) is a viable high-throughput alternative for functional gene quantification, by evaluating the efficiency, specificity and sensitivity of 29 established and 12 newly designed primer pairs targeting taxonomic, nitrogen-cycling, and hydrocarbon degradation genes in genomic DNA soil extracts, under three different sets of MFQPCR assay conditions. Without curation, commonly-used qPCR primer pairs yielded an extreme range of reaction efficiencies (25.9–100.1%), but when conditions were optimized, MFQPCR produced copy-number estimates comparable to traditional qPCR. To guide microbial soil ecologists considering adoption of MFQPCR, we present suggestions for primer selection, including omission of inosines, degeneracy scores of &lt; 9, amplicon sizes of ≤ 211 bp, and GC content of 32–61%. We conclude that, while the nanoliter reaction volumes, rapid thermocycling and one-size-fits-all reaction conditions of MFQPCR necessitates more stringent primer selection criteria than is commonly applied in soil microbial ecology, the ability to quantify up to 96 targets in 96 samples makes MFQPCR a valuable tool for monitoring shifts in functional community abundances. MFQPCR will particularly suit studies targeting multiple clade-specific functional genes, or when primer design is informed by previous knowledge of the environment