Various general methods of analysis used in the course of experimental investigations are detailed in this chapter. They include preparation of different agar media, isolation of fungi from seed samples using agar plate method and identification of fungi through morphological characteristics on different agar media. Extraction of aflatoxins and fumonisins from rice samples, clean-up and estimation by Enzyme linked immunosorbent assay (ELISA), High performance liquid chromatography (HPLC) and Liquid chromatography/mass spectrometry (LC/MS/MS).
A total of 96 rice samples consisting of paddy and milled rice intended for human consumption were collected from farmers's fields, different commercial markets and warehouses in Malaysia, which originating from Cambodia (4), India (7), Indonesia (4), Malaysia (56), Thailand (24) and Vietnam (1). The samples were consisting from paddy (4), white rice (52), brown rice (7), basmathi (2), white glutinous rice (11), black glutinous rice (5), fragrant rice (12), and parboiled rice (3). Each 1 kg composite sample was collected from one storehouse with a mixture of 10 sub-samples (200 g each) by collecting them from 5 diagonal on each of the upper, middle and lower layers of each storehouse to avoid the sampling error due to highly heterogeneous nature of fungal distribution (Liu et al., 2006).
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Half strength Potato dextrose agar media (PDA) (Reddy, 2008) was used to isolate fungi from rice samples. PDA medium was made using the pealed potato (10% w/v), dextrose (1% w/v) and agar (2% w/v). The peeled potatoes were cut in to small pieces and boiled for 20 minutes and the extract was collected and used. Potato extract and dextrose were dissolved in 500 ml of quartz distilled water. The pH of the medium was adjusted to 7.0 and agar-agar was added, boiled and the volume was made to 1 liter with quartz distilled water. Aliquots of 500 ml medium were dispensed into 1,000 ml borosil glass bottles and autoclaved at 15 lbs pressure, 1210C for 15 min. Similar procedure was followed for the other media used in this study.
3.2.2 Isolation of fungi without surface sterilization
This isolation technique was used (Reddy et al., 2009) to isolate surface contaminating fungal mycoflora from rice samples. Four hundred rice grains (ISTA, 1996) from each sample were directly placed on half strength PDA plates (40) by placing 10 grains in each plate. The plates were incubated at 25Â°C under two fluorescent day light tubes and one black light tube on/off for 12 hours and examined daily for seven days (Salleh & Sulaiman, 1984). After 7 days of incubation the fungal occurrence was recorded. The fungal isolates emerging out from rice samples were transferred to fresh PDA plates for further studies.
3.2.3 Isolation of fungi after surface sterilization
This technique was used to isolate internal fungal mycoflora from rice samples according to the method of Desjardins et al. (1999). About 20 g of each rice sample was surface sterilized with 0.05% sodium hypochlorite (NaOCl) solution for 1 min, washed with four rinses in sterile distilled water and blotted on sterile filter papers. Four hundred rice grains (ISTA, 1996) from each sample were placed on half strength PDA plates (40) by placing 10 grains in each plate. The plates were incubated at 25Â°C under two fluorescent day light tubes and one black light tube on/off for 12 hours and examined daily for seven days (Salleh & Sulaiman, 1984). After 7 days of incubation the fungal occurrence was recorded. The fungal isolates were transferred to fresh PDA plates for further studies.
3.2.4 Single spore isolation
Single spore isolation was employed according to Hansen and Smith (1932) to purify all the fungi that were isolated from rice grain samples. A small portion of mycelium from seven days old fungal culture was transferred into 10 ml sterile distilled water. Concentration of the conidial suspension were adjusted to 1-10 conidia in a drop viewed under a low power objective of a light microscope. Then a single loop of the spore suspension was streaked onto Â½ strength PDA plate. The inoculated plates were incubated under standard incubation conditions for 24 h to allow the germination of conidia. After incubation, single germinating conidia were picked and transferred with the aid of a compound microscope. Colonies initiated from a single conidium were considered as pure cultures that have uniform and consistent appearances for correct identification. This also useful for separating mixed cultures. All single conidial isolates were maintained on low nutrient medium for further studies.
3.3 Identification of fungi through morphological characteristics
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All the purified fungal cultures isolated from rice grain samples were identified through morphological characteristics. For studying the morphological characteristics of each fungi we used different media viz., potato dextrose agar (PDA), water agar (WA) (Burgess et al., 1994) and carnation leaf-piece agar (CLA) (Fisher et al., 1982) for Fusarium sp., for Aspergillus and Penicillium sp., we used Malt extract agar (MEA), Czapeks yeast agar (CYA), Czapeks yeast agar with 20% sucrose (CY 20S), and Czapeks Dox agar (CZ), czapek yeast autolysate with 5% NaCl (CYAS) agar, dichloran 18% glycerol agar (DG18), 25% glycerol nitrate agar (G25N), yeast extract sucrose agar (YES) and creatine sucrose agar (CREA) (Klich, 2002; Oh et al., 2008).
3.3.1 Composition of media
Write the composition of all media that you used for morphological study
3.3.2 Identification of Aspergillus spp.
The taxonomic systems of Aspergillus by Klich (2002) was followed for the identification of different species. All the Aspergillus spp. cultures were cultured on CYA (Czapeks yeast agar), CY 20S (Czapeks yeast agar with 20% sucrose), MEA (malt extract agar) and CZ (Czapeks Dox agar) at 25Â°C. Both macroscopical and microscopical characters were assessed when the cultures were 3, 7, and 14 days old. Colony colour were observed by naked eye and compared with the colour charts of Methuen Handbook of Colour (Kornerup & Wanscher, 1978). The material on the slide mounts were examined under light microscope. Observation of metulae, conidial diameter and conidial rough were determined under 1,000x magnification.
3.3.3 Identification of Penicillium spp.
The morphological charactristics of Penicillium spp. were studied by the method of Pitt (2000) and Oh et al. (2008). All the isolates were grown on different agar media were: 1) czapek yeast extract agar (CYA), 2) czapek yeast autolysate with 5% NaCl (CYAS) agar, 3) czapek dox agar (CZ), 4) malt extract agar (MEA), 5) dichloran 18% glycerol agar (DG18), 6) 25% glycerol nitrate agar (G25N) to test the ability of isolate to grow at low aw, 7) yeast extract sucrose agar (YES), and 8) creatine sucrose agar (CREA) which shows acid or base production by fungi. All media were incubated for 7 days at 250C when comparing the growth rates at different temperatures using CYAs incubated at 15, 25, and 300C. The diameters, overall colors, colors of conidia, reverse colors, and productions of sclerotia, exudates, and soluble pigments were compared within colonies on each medium. Microscopic features of isolates on MEA, such as conidial heads, stipes, shapes of conidia, roughness of conidial walls, existence of ascospores or cleistothecia, lengths of phialides, and metulae, were observed using a microscope (x1,000). The branching patterns and appressedness of conidiophores were also observed to determine if they are mono-, bi-, or ter-verticillate and whether or not they are pressed close.
3.3.4 Identification of Fusarium spp.
The morphological identification of Fusarium spp. were made using the criteria described in the Fusarium laboratory manual (Leslie & Summerell, 2006). All Fusarium isolates were transferred on PDA, Spezieller Nahrstoffarmer Agar (SNA) and Carnation Leaf Agar (CLA). Cultures grown on PDA were incubated at 25Â°C and Spezieller Nahrstoffarmer Agar (SNA) cultures were incubated at 25°C under near UV light for two to four weeks. Cultures grown on PDA were recorded for their colony morphology. The morphology of macroconidia, microconidia, conidiogenous cells and the chlamydospores were assessed from cultures grown on SNA and CLA.
3.3.5 Preservation of cultures
All the fungi isolated from the rice samples and successfully identified were preserved using different preservation techniques for further experiments.
3.3.6 Preservation through filter paper stock
It is one of the best methods to preserve the cultures alive for long time. The filter papers were punched into small circles by using puncher and were autoclaved to make them sterile. The sterilized filter papers discs (10 mm diameter) were arranged on PDA plates. A loopful of inoculum was transferred into the middle of the plate and the plates were incubated for seven days or until the fungi to overgrow on filter papers. Then, the filter papers along with culture were collected aseptically and stored in the cryo tube at -20Â°C.
3.3.7 Preservation through glycerol stock
This technique was adopted by Smith and Onion (1983) and Dhingra and Sinclair (1985) to preserve the cultures through glycerol stock. Briefly, 0.5 ml of 30% glycerol [C3H5(OH)3] was added into cryo-tubes (Nunc Cryo Tube Vials, 1.8 ml) and sterilized through autoclaving. Then, 0.5 ml of conidia suspension of each isolate was added into the cryo-tubes. The mixture was shaken and preserved in a deep-freezer at -80C.
3.4 Extraction, clean-up and estimation of mycotoxins from rice samples
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All solvents were of HPLC grade from VWR (West Chester, PA). All mycotoxin standards (aflatoxins B1, B2, G1, and G2, fumonisins B1 and B2) and other chemicals were obtained from Sigma (St. Louis, MO). The HPLC column was from Waters Corporation (Milford, MA), and the LC/MS column was purchased from Varian (Palo Alto, CA). Alltech frits and reservoirs for extraction as well as filter paper and scintillation vials were obtained from Fisher Scientific (Pittsburgh, PA).
3.4.2 Extraction of mycotoxins from rice samples
Samples of 1 kg were thoroughly mixed and 100 g sub-samples were ground to made powder using high speed blender for 5 min (particle size, <300 Âµm). Twenty g powders were extracted with 100 mL of methanol/water, 70:30. The samples were placed on a shaker table and shaken for 3-5 min at high speed and then filtered through Whatman no. 1 filters. Samples were collected in two scintillation vials each containing ~20 ml of extract each for a total of 40 ml, and stored in a freezer until ready for analysis. The percentage recovery was >80% for both toxins.
3.4.3 Enzyme-linked immunosorbent assay (ELISA) analysis of aflatoxin
ELISA analysis was conducted per included instruction in a commercially available ELISA kit (Neogen Corporation, Lansing, MI). Analysis was conducted for further confirmation of HPLC results and to determine ppb analysis range for HPLC.
3.4.4 Aflatoxin cleanup
Sample cleanup was carried out as described in detail by Sobolev and Donner (2002) with a minor modification. Briefly, 500 ÂµL aliquot of the extract was mixed with 500 ÂµL of acetonitrile. 800 ÂµL of the mixture was then pipetted into an Alltech 1.5 mL Extract-Clean reservoir packed with 200 mg of basic aluminum oxide (Scientific Adsorbents, Inc. Atlanta, GA.) After elution by gravity, the eluate was placed into HPLC vials for analysis. The limit of detection was 0.1 ngg-1.
3.4.5 High performance liquid chromatography (HPLC) analysis of aflatoxins
Using a method of Abbas et al. (2008), standards for this test were prepared using a Sigma aflatoxin mixture, and additional standard dilutions were carried out using a methanol/water/acetic acid solution (310/190/0.5ml). Samples and standards were programmed to inject and run for 13 min each at 0.9 mLmin-1 through a 150 mm Ã- 3.9 mm i.d. 4 Âµm Nova-Pak C18 column. Injections were made by a Waters 717 plus autosampler, and detection was achieved by a Waters 2475 multi Î» fluorescence detector set at 365 nm for excitation and 440 nm for emission in combination with a photochemical reactor for enhanced detection (PHRED) unit (Aura Industries Inc., New York, NY). The mobile phase used to obtain separation was isocratic water/methanol/1-butanol (700/360/12.5ml) delivered by a Waters 600 pump.
3.4.6 Fumonisin cleanup
Clean-up of the fumonisins was accomplished on Bond-Elute SAX columns (Varian, Harbor City, CA) by the method of Abbas et al. (2007). The SAX column was conditioned by applying 5 mL of methanol followed by 5 mL of methanol:water (3:1). A 10 mL aliquot of the extracted rice sample was applied followed by a 3 mL methanol wash step, followed by elution with 10 mL water containing 2% acetic acid. Samples were evaporated under a stream of nitrogen and stored at 5Â°C until further analysis by liquid chromatography/mass spectrometry (LC/MS/MS). The cleaned sample was reconstituted in 1 mL acetonitrile:water (1:1). The limit of detection was 1 ngg-1.
3.4.7 Liquid chromatography/mass spectrometry (LC/MS/MS) analysis of fumonisins
The analytical method used for detecting various fumonisins was liquid chromatography/mass spectrometry (LC/MS/MS) (Abbas et al. 2007). Fumonisin analysis was conducted using a MetaChem Intersil 5Î¼ ODS-3 column eluted with water:1% acetic acid:methanol (65:35:0) at 300 Î¼Lmin-1 for 10 min followed by a linear gradient to water:1% acetic acid in methanol:methanol (5:35:65), then held constant for 10 min. Between samples the solvent was returned to water:1% acetic acid in methanol:methanol (65:35:0) within 1 min and held constant for 4 min for column equilibration. Analysis was carried out using an LTQ XL linear ion trap mass spectrometer with a Surveyor pump and autosampler (Thermo Electron Corporation, West Palm Beach, FL). Quantitation of FB1, FB2 was carried out by the external standard method (Linearity ranged of standards were 0.99).