Lab_3_Optical_Microscopy

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University of British Columbia *

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373

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Chemistry

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Oct 30, 2023

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Wood 373 1 Lab 3 Optical Microscopy Lignin Determination Goal : During this lab, you will learn how to use a stain to visually quantify the presence of lignin in wood using an optical microscope. Experimental section: All samples (the original wood, partially delignified wood, fully delignified wood, and boiled wood) will be cut into thin sections [3 mm (longitudinal direction) x 3 mm (tangential direction) x 2 mm (radial direction)] using a microtome (or sharp knife). TA will demonstrate how to safely use the microtome. The thin sections will be placed on a microscope slide and a drop of appropriate stain* applied (prepared by the TA and the detail about the stain is listed below) and covered with a cover glass before viewing under the microscope. When observing original wood and delignified wood, draw sketches and pay attention to the similarities and differences between original wood and delignified wood. It is recommended that you also take photos for your report. Remove slides from microscope and dispose of slides in LAB GLASS WASTE container. Turn objective to lowest power, light intensity to lowest position and turn main switch OFF before unplugging microscope. Return microscope to cart and clean up before leaving the lab. Used slides must be disposed of in LAB GLASS WASTE containers and used blades must be disposed of in SHARPS WASTE containers. *Preparation of Phloroglucinol-HCl Stain (done by TA) There are various procedures to make up the staining solution but commonly it is prepared as a saturated solution of phloroglucinol in 20% hydrochloric acid. 2N hydrochloric acid used so please use care when handling. Prepare this solution in the fume hood. First dissolve phloroglucinol (about 2.0 g) in 80 ml of 20% ethanol solution and then add 20 ml of concentrated HCl (12 N) to it. The solution should be stored in an amber bottle and deteriorates with time, so it needs to be freshly prepared. Questions: 1. Why do you need to stain the wood to observe it under the microscope? 2. What is the reaction mechanism for staining? 3. Do you observe any differences with and without the stain and for samples before and after delignification?
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