Welcome to General Biology I Lab Fall Semester 2019-20 Week #1’s Schedule 1. Instructor introduction 2. Discussion of the syllabus 3. Review basic laboratory safety 4. Class introductions & Getting to know you activities 5. Prelab Discussion 6. Completion of designated laboratory activities Who is your instructor? • Course instructor: – Paul Shipman, PhD – Education: • BSE in Biology, Emporia State Universituy • MS in Biology, Emporia State University • PhD in Zoology, Oklahoma State University – Research: • Freshwater turtle communities • Email: – passbi@rit.edu • Office: – Bldg: Gosnell A354 • Office hours: 10-11am Mon, noon-1pm Wed & Fri – By appointment Week #1’s Schedule 1. Instructor introduction 2. Discussion of the syllabus 3. Review basic laboratory safety 4. Class introductions & Getting to know you activities 5. Prelab Discussion 6. Completion of designated laboratory activities Lab #1 – Scientific Method & Macromolecules Prelab Notes © RIT 2019 (Michelle Weatherell and Emily Coon-Frisch) Lab Objectives • Be able to identify three of the four major classes of macromolecules – What are their basic chemical structures? – Develop an understanding of the role that each of these plays in plants and animals? – Where exactly are the located in the body? And in what form? • Be able to identify which bio-indicator best identifies a particular class of macromolecules. – What property of that particular group of molecules makes this qualitative change notable? • Use methods learned to identify the properties of various macromolecules to evaluate an unknown Today’s Lab • Activity #1a: Benedicts Assay for the Presence of Simple Sugars • Activity #1b: Iodine Assay for the Presence of Complex Carbs • Activity #2a: Lipid Solubility • Activity #2b: Sudan IV Assay for the Presence of Lipids • Activity #3a: Ninhydrin Assay for the Presence of Amino Acids • Activity #3b: Biuret Assay for the Presence of Proteins • Activity #4: Identification of an Unknown What is Science? • Science is … – what scientists do – trying to explain the world around us – a way of thinking – a system of knowledge based on facts or principles • “The intellectual and practical activity encompassing the systematic study of the structure and behavior of the physical and natural world through observation and experiment.” Branches of Science Science Biological Science Physical Science living things matter and energy Earth Science systems of the earth What is Biology? • The study of life – “Bio” = life – “-logy” = study of • Understand basic concepts related to cells, development, and interaction • Build on the basics to understand more complex topics Let’s talk about cells • What is a cell? – the smallest structural and functional unit of an organism, typically microscopic. Water, small molecules, ions, buffers • What is a cell composed of? • Water • Ions, buffers • Various sized molecules What do macromolecules have to do with cells? • Definition: a molecule with a very large number of atoms; more than 100 component atoms. • The base elements of all macromolecules are: – Carbon (C), Hydrogen (H) and Oxygen (O). • Types of Macromolecules: – – – – Carbohydrates Lipids Proteins Nucleic acids What is a Carbohydrate? • Definition: – class of organic compounds that contain carbon, hydrogen and oxygen in a 1:2:1 ratio. • Function: – Fuel source, provide support and protection. • Classification: – Mono-, di- and polysaccharides. Monosaccharides • Simplest form of carbohydrates • Structure: – Comprised of 3-7 carbon atoms and their corresponding oxygen and hydrogen atoms • Function: – Fuel for organisms – Building blocks for larger molecules • Examples: – Ribose • (components of DNA/RNA) – Glucose & its isomers of it What is Benedicts Reagent? • Benedicts reagent - chemical compound made up of copper sulfate, which can detect the presence of glucose or fructose. – A reagent is a chemical that is applied to another substance in order to produce a chemical reaction that can give valuable information regarding the substance. • Benedicts reagent is blue...and when it is mixed with a solution containing reducing sugars and then heated, a color change will occur. – Blue à yellow/orange Activity #1a: Benedicts Assay for Simple Sugars • Test 4 substances for the presence of sugar using the following protocol: – Add 1 mL of each solution to a labeled test tube (1-4) – Then add 1 mL of Benedicts reagent and gently mix. – Place these test tubes in a preheated beaker of water for 5-10 minutes. – Remove and cool for 2 minutes. – Record your qualitative (color change) observations on the table provided at the end of your lab handout. How to read a graduated pipet • Identify the appropriate pipet for the job • What volume do you need to measure? • Once selected, measure and aliquot the appropriate volume. • Make sure you are using the correct pipet assistant. • Blue = 1.0 mL pipet • Green = 5.0 or 10 mL pipet • Largest volume increment • graduation indicated by the # and solid line • measured in mL • Smallest volume increment • graduation indicated partial lines • Actual value = largest graduation (mL) 10 • Measurement begins at the tip and migrates up Disaccharides (Double Sugars) • Structure: – 2 monosaccharides attached two one another • Uses dehydration synthesis to build molecules • Function: – Fuel for organisms • Examples: – Sucrose (table sugar) – Maltose Glucose + Glucose Dehydration Synthesis Maltose Polysaccharides (Complex carbohydrates) • Structure: – formed by linking shorter units together to form long or very long sugar chains • Function: – Energy Storage – Structural support • Examples: – – – – Cellulose = plant cell walls Starch = plant energy source Glycogen = stored animal energy Chitin = component of exoskeleton The Iodine Test for Complex Carbs • Iodine Test uses Iodine potassium-iodide (I2KI) to distinguish starch from other carbohydrates. Iodine + Starch Iodine + Water Note: I2KI does not react with non-coiled carbohydrates. Activity #1b: Iodine Assay for Complex Carbs • Test 4 substances for the presence of sugar using the following protocol: – Add 1 mL of each solution to a labeled test tube (4-8) – Then add 1 mL of Iodine reagent and gently mix. – Record your qualitative (color change) observations on the table provided at the end of your lab handout. • Color change should happen instantly in the case of positive results. • Don’t forget to test the paper of your actual lab handout as described in the protocol. What is a lipid? • Definition: – Oily or waxy molecules that are non-polar/hydrophobic (repel water; do not dissolve). • Function: – Fuel source à condensed energy storage. • Classification: – Triglycerides, phospholipids and steroids. 1 lb. of Fat What are Triglycerides? • Largest class of lipids; fats & oils • Structure: – Glycerol backbone – Three (tri) fatty acids (hydrocarbon chains) Largest Class of Lipids in Our Diet: Triglycerides • Distinguished by their structure and melting point. • Structure: – Glycerol backbone – Three (tri) fatty acids (hydrocarbon chains) • Examples: Fats & Oils • Types of: – Saturated Fats: contain all single bonds – Unsaturated Fats: contain some double bonds Sudan IV Assay for Lipids • A qualitative color-change test for lipids Sudan IV + Water Sudan IV + Oil • Sudan IV is non-polar…what does this mean? – It will selectively stain the lipids, as substances of similar polarity are drawn together Activity #2a: Lipid Solubility • Set-up 3 test tubes as directed in your lab handout. • Vigorously shake both test tubes and record your observations. – Oil and water don’t mix, but oil & acetone do! Why? • If you want……try to make an emulsion! Activity #2b: Sudan IV Stain Test for Lipids • Apply a small amount of 4 different samples to a designated spot on a piece of filter paper. – Don’t forget to label them J • Allow the spots to dry. (~10 mins) • Once dry, place a drop of Sudan IV on each spot and once again allow to dry. • After the paper has completely dried, make your qualitative observations and record them in the space provide in your lab handout. What are Proteins? • Definition: – Amino acids bound by peptide bonds • Function: – Necessary dietary component for animals that can’t synthesize all 20 amino acids needed to build protein structure within the cell – Enzymes = catalyze biochemical reactions within a cell – Structural = comprise muscle – Regulatory/signaling = hormones & receptors – Immune system = antibodies • Thus any process in a cell is more or less correlated with a protein. = • Classification: But wait a minute….what’s an amino acid? • Amino acids have a two-carbon bond. – One of the carbons is part of a group called the carboxyl group (COO-). – Amino means there is an NH2 group bonded to the carbon atom. In the image, you see a "+" and a "-". • Those positive and negative signs are there because, in amino acids, one hydrogen atom moves to the other end of the molecule. Ninhydrin Reagant • The reaction between alpha-amino acid and ninhydrin involved in the development of color are described by the following five mechanistic steps seen in your supplement handout. – In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. • Ninhydrin will react with a free alphaamino group, NH2C-COOH. – This is found in all amino acids. Activity #3a: Ninhydrin Assay for Amino Acids • Apply a small amount of 4 different samples to a designated spot on a piece of filter paper. Don’t forget to label them J • Allow the spots to dry. • Once dry, gently spray with 1% Ninhydrin. • Allow the filter paper to dry. • After the paper has completely dried, make your qualitative observations and record them in the space provide in your lab handout. Activity #3b: Biuret Assay for Proteins • What is Biuret Reagent? – made of sodium hydroxide (NaOH) and hydrated copper(II) sulfate. • What is a Biuret Assay? – a chemical test used for detecting the presence of peptide bonds. – The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. • Activity Itself • Obtain 4 test tubes and add 1 mL of sample and 1 mL of Biuret Reagent. • Thoroughly vortex and record the results. Biuret + Water Biuret + Protein Good Science & The Scientific Method Types of Variables • Constants • Portions of the experiment that you can control/keep the same (e.g. amounts of reagents, temperature, etc.) • Independent • Manipulated • Substance being tested (presence or lack of) • Dependent • Result; variable that is to be measured (e.g. color) • Responding variable (did a change occur) Types of Experimental Controls • What is a control? – Serves as a standard for comparison • Types of Controls – Positive = • Independent Variable is present; Reaction WILL occur – Negative = • Independent Variable is not present; Reaction will NOT occur • Why are they necessary? – It is the unchanged part of the experiment that detects the effects of hidden variables. Differences in Data Collection Activity #4– IDing an Unknown • Obtain 2 Unknown sample from your instructor. • Using your knowledge from the previous assays, conduct a series of tests to determine the contents on your sample. – Have your methodology (experimental design) approved by your instructor prior to completing your experimentation. • You should identify which macromolecule or combination of macromolecules are within the sample, as well as what specific “food product” you have. – Please refer to the tables provided within your lab handout. • Enter your results in the tables provided in your lab manual. Lab Clean-Up • All liquid chemical waste is disposed of in the organic collection containers in the back. • All used test tubes and pipets should be disposed of in the blue & white glass disposal bin found throughout the laboratory. • All paper items (gloves, pipet wrappers, filter paper, etc) should be placed in the general trash cans. • All electrical equipment should be turned off and unplugged. Next Week in Lab Regular lab schedule will resume during Week #3. Please be prepared for a quiz on Week #1’s material J Refer to announcements on MyCourses for specifics
