The total viable counts of aerobic mesophilic bacteria and fungi in dust samples were examined by plating dilution series from dust suspensions. Dust suspensions were prepared as described by Schulz et al. [20 (link)]. Briefly, 0.1 g dust was added to 10 mL phosphate buffer saline (PBS) with 0.01% TWEEN20 (v/v). Then, the suspension was shaken for 30 min in a water bath at 25 °C. Afterward, the suspension was vortexed for 4 min (Scientific Industries Inc., Bohemia, NY, USA). Aliquots (0.1 mL) of suspensions and serial dilutions from these suspensions were plated in triplicate on Tryptone Soya Agar plates (TSA, Oxoid Ltd., Basingstoke, UK) and on Dichloran-Glycerol (DG-18) agar supplemented with chloramphenicol (Oxoid Ltd) for counting colonies of mesophilic bacteria and fungi colonies, respectively. Negative controls were prepared by inoculating TSA and DG-18 media with 0.5 mL PBS. TSA plates were incubated for 48 h at 37 °C, while DG-18 agar plates were incubated at 25 °C for 5–7 d. After incubation, CFU was counted, and the results were expressed in CFU/g of dust.
Dichloran glycerol dg18 agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Dichloran-Glycerol (DG18) agar is a selective and differential culture medium used for the isolation and enumeration of xerophilic fungi. It is formulated with dichloran to inhibit spreading fungi and glycerol to reduce water activity, creating conditions favorable for the growth of xerophilic species.
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Lab products found in correlation
Chloramphenicol, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Ethanol, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Thermo Fisher Scientific (1 mentions)
Ammonium hydroxide, by Thermo Fisher Scientific (1 mentions)
Pyridine, by Thermo Fisher Scientific (1 mentions)
Butanol, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by ITW Reagents (1 mentions)
Bacteriological peptone, by Thermo Fisher Scientific (1 mentions)
Diethyl ether, by ITW Reagents (1 mentions)
Phytosphingosine, by Avanti Polar Lipids (1 mentions)
Dihydrosphingosine, by Avanti Polar Lipids (1 mentions)
Bag mixer 100 minimix, by Interscience (1 mentions)
Triton x 100, by GE Healthcare (1 mentions)
4 protocols using dichloran glycerol dg18 agar
1
Quantifying Airborne Bacterial and Fungal Loads
2
Analytical Reagents and Standards
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The salts and supplements used to prepare the culture media were in general purchased from Sigma-Aldrich; except Dichloran-glycerol (DG18) agar, yeast extract and bacteriological peptone that were purchased from Oxoid; sucrose from Alfa Aesar; casein hydrolysate from Fluka; agar and NaCl from Panreac AppliChem; NaOH from J.M.G. Santos and glycerol from Fisher Bioreagents. Molecular biology reagents used for PCR reactions were purchased from NZYTech. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich. The reagents used for sphingolipid extraction: pyridine, butanol, ammonium hydroxide and 40% methylamine in water were purchased from Acros Organics; ethanol, methanol and chloroform (and dimethyl sulfoxide used in MTT assays) were purchased from Fisher Chemical and diethylether from Panreac AppliChem. Sphingoid bases standards (phytosphingosine, sphingosine and dihydrosphingosine) were purchased from Avanti Polar Lipids. All solvents used in chromatographic analyses were of the highest analytical grade and water was obtained from a Milli-Q system (Millipore).
3
Fungal Load Determination in Food Samples
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The determination was performed based on the method described by SR ISO 21527-2/2008 standard [35 ], by Antoniewska et al., (2018) [36 (link)] and Naghy et al., (2017) [37 (link)]. Briefly, 5 g of each sample were mixed with 45 mL of 0.1% peptone water diluent (Oxoid Ltd., Basingstoke, Hampshire, England) in a stomacher (Bag Mixer 100 MiniMix, Interscience, St. Nom, France). Further decimal dilution (10−2) was prepared in 0.1% peptone water diluent. All dilutions were inoculated in duplicate. 100 µL of the initial dilution (10−1) were aseptically transferred to a Petri dish containing Dichloran-Glycerol (DG18) agar (Oxoid Ltd., Basingstoke, Hampshire, England) using a sterile pipette and spread immediately with a Drigalski-spatula.
The procedure was repeated with further decimal dilution (10−2). The inoculated dishes were inverted and incubated at 25 °C for 5 days. After incubation, the visible colonies on selected plates (less than 150 colonies) were counted using a colony counter Colony Star 8500, (Funke-Dr. N. Gerber Labortechnik, Berlin, Germany).
The procedure was repeated with further decimal dilution (10−2). The inoculated dishes were inverted and incubated at 25 °C for 5 days. After incubation, the visible colonies on selected plates (less than 150 colonies) were counted using a colony counter Colony Star 8500, (Funke-Dr. N. Gerber Labortechnik, Berlin, Germany).
4
Characterization of Tunisian Cork Oak Forest Soils
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Chemicals: If not explicitly stated otherwise, chemicals were of analytical grade and purchased from Sigma Aldrich. trans-Acetylacrylate (Alfa Aesar), malt extract agar (MEA) (HiMedia), dichloran-glycerol (DG18) agar (Oxoid) and triton X-100 (GE Healthcare) were also used. All Liquid Chromatography (LC) and Mass Spectrometry (MS) solvents, as well as those required in the fast-solvent extractions, were of the highest analytical grade. Chlorinated derivatives of resorcinol, hydroquinone and catechol were produced through an aqueous chlorination methodology (Heasley et al., 1989 ) and 2,3,5,6-tetrachloro-4-methoxyphenol (drosophilin A) was synthesised as described before (Hiebl et al., 2011) .
Collection and physicochemical-characterisation of soil samples: Soil samples were collected in three Tunisian demarked cork oak forests, namely Aîn Hamraia (AH), Fej Errih (FER) and Ras Rajel (RR) in February 2009, as previously described (McLellan et al., 2013) . In brief, three locations were chosen within each forest and a composite sample was collected from five sub-samples (0 -20 cm), sieved to < 2 mm in the field, and immediately conserved (dark, 4 ºC) until analysis. Total organic carbon content, total nitrogen
Collection and physicochemical-characterisation of soil samples: Soil samples were collected in three Tunisian demarked cork oak forests, namely Aîn Hamraia (AH), Fej Errih (FER) and Ras Rajel (RR) in February 2009, as previously described (McLellan et al., 2013) . In brief, three locations were chosen within each forest and a composite sample was collected from five sub-samples (0 -20 cm), sieved to < 2 mm in the field, and immediately conserved (dark, 4 ºC) until analysis. Total organic carbon content, total nitrogen
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