Immediately after decapitation, the ileum samples from 1 bird per pen of each treatment group were taken approximately 1 cm below Meckel's diverticulum and flushed with 0.1 M phosphate-buffered saline (pH 7.4). The samples were prepared through several gentle washing steps, then fixed in 3% buffered glutaraldehyde (in 100 mM phosphate buffer, pH 7.4), and further processed in the Center for Electron Microscopy at North Carolina State University. Samples were washed 3 times in the buffer, postfixed in buffered 2% osmium tetroxide (in 100 mM phosphate buffer, pH 7.4) for 2 h, and washed again in phosphate buffer. The samples were dehydrated in a graded ethanol series, dried in a liquid CO2 critical point dryer (Samdri-795, Tousimis, Rockville, MD), secured to stubs with silver paint, and sputter coated with approximately 50-nm gold or palladium (Anatech Hummer 6.2, Anatech USA, Hayward, CA). Observation was made at 15 KV using a JEOL JSM-5900LV scanning electron microscope (JEOL U.S.A., Peabody, MA). Electron micrographs were taken from different areas of the samples for estimating morphology of villi, amount of mucus, density of goblet cells (GC ), and bacterial colonization using a JEOL digital scan generator.
Digital scan generator
Manufactured by JEOL
The Digital Scan Generator is a laboratory equipment designed to produce controlled, precise electronic signals for various scientific and industrial applications. It generates digitally-controlled waveforms and patterns that can be used to drive or synchronize other electronic devices and systems.
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3 protocols using digital scan generator
1
Ultrastructural Analysis of Ileal Tissue
2
Scanning Electron Microscopy of Fungal Hyphae
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Scanning electron microscopy was performed at the North Carolina State University Center for Electron Microscopy, Raleigh, NC, USA. Agar blocks (~0.5 cm3) containing hyphae were excised and fixed in 0.1 M sodium cacodylate buffer (pH = 6.8) containing 3% glutaraldehyde at 4°C for several weeks. The agar blocks were rinsed with cold 0.1 M sodium cacodylate buffer pH 6.8 three times and then dehydrated in a graded series of ethanol to reach 100% ethanol. The blocks were subjected to critical-point drying with liquid CO2 (Tousimis Research Corp.) and sputter coated with 50 Å of gold/palladium using a Hummer 6.2 sputter coater (Anatech USA). The samples were viewed at 15 kV with a JSM 5900LV scanning electron microscope (JEOL) and captured with a Digital Scan Generator (JEOL) image acquisition system.
3
Microscopy Techniques for Fungal Mating Studies
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Inoculation and incubation conditions for microscopy specimens were the same as for the other mating crosses. For the light micrographs, a cross of DSM27421 × CBS6039 on V8 (pH = 5) mating medium was incubated for 39 days. The crosses were viewed with a Zeiss Scope.A1 microscope and photographed with a Zeiss Axiocam 105 color camera. For the scanning electron micrographs (SEM), a cross of DSM27421 × CBS6039 on V8 (pH = 5) mating medium was incubated for 11 days. SEM was performed at the North Carolina State University Center for Electron Microscopy, Raleigh, NC, USA. Agar blocks (approximately 0.5 cm3) containing hyphae on the edges of mating patches were excised and fixed in 0.1 M sodium cacodylate buffer (pH = 6.8) containing 3% glutaraldehyde at 4°C for several weeks. Before viewing, the agar blocks were rinsed with cold 0.1 M sodium cacodylate buffer (pH = 6.8) three times and then dehydrated in a graded series of ethanol to reach 100% ethanol. The blocks were subjected to critical-point drying with liquid CO2 (Tousimis Research Corp.) and sputter coated with 50 Å of gold/palladium using a Hummer 6.2 sputter coater (Anatech USA). The samples were viewed at 15 kV with a JSM 5900LV scanning electron microscope (JEOL) and captured with a Digital Scan Generator (JEOL) image acquisition system.
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