Cell Biology Laboratory Manual

Table of Contents

• Chapter 1: The Microscope
• Chapter 2: Histochemistry
• Chapter 3: Cell Fractionation
• Chapter 4: Electrophoresis
• Chapter 5: Enzymes
• Chapter 6: Membranes
• Chapter 7: Microsomes
• Chapter 8: Photosynthesis\Respiration
• Chapter 9: Tubules\Filaments
• Chapter 10: Chromosomes
• Chapter 11: Cell Cycles
• Chapter 12: Cell Cultures
• Chapter 13: Differentiation
• Chapter 14: Nucleic Acids
• Chapter 15: The Central Dogma
• Appendix A: Units and Measures
• Appendix B: Statistics
• Appendix C: Graphs
• Appendix D: Computers
• Appendix E: Image Analysis
• Appendix F: CentrifugationAppendix G: Spectrophotometry
• Appendix H: Radioactive tracers
• Appendix I: Photography
• Appendix J: Chemical Preparations
  

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  Chapter 1: The Microscope

• Introduction
• 1.1 The Bright Field Microscope
• 1.2 Use of Oil Immersion (100x)
• 1.3 Measurements: Ocular and Stage Micrometers
• 1.4 Measuring Depth
• 1.5 Measuring Area
• 1.6 Measuring Volume
• 1.7 Measurement of Cell Organelles
• 1.8 Use of Darkfield Illumination
• 1.9 The Phase Contrast Microscope
• 1.10 The Inverted Phase Microscope
• 1.11 The Transmission Electron Microscope
• 1.12 Comparison of Electron Micrographs
• Endnotes
  

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  Chapter 2: Histochemistry

• Introduction
• 2.1 Selective Staining: Prepared Slides
• 2.2 Basophilia
• 2.3 Periodic Acid Schiff (PAS) Reaction
• 2.4 Methyl Green-Pyronin Staining of DNA and RNA
• 2.5 The Feulgen Procedure for DNA
• 2.6 Alkaline Phosphatase Localization
• 2.7 Immunofluorescent Localization of Tubulin
• 2.8 Autoradiography
• Endnotes
  

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  Chapter 3: Cell Fractionation

• Introduction
• 3.1 Sucrose Fractionation of Castor Bean
• 3.2 Sedimentation Densities
• 3.3 Equilibrium Density Gradient - Percoll
• 3.4 Low Speed Separaton of Cells
• 3.5 Separation of Cells by Velocity Sedimentation
• Endnotes
  

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  Chapter 4: Electrophoresis

• Introduction
• 4.1 Preparation of SDS-polyacrylamide gels (SDS-PAGE)
• 4.2 Separation of protein standards: SDS-PAGE
• 4.3 Coomasie blue staining of protein gels
• 4.4 Silver staining of gels
• 4.5 Documentation
• 4.6 Direct densitometry of gels
• 4.7 Separation of LDH isozymes from serum
• 4.8 Western blots
• Endnotes
  

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  Chapter 5: Enzymes

• Introduction
• 5.1 Extraction of tyrosinase
• 5.2 Preparation of standard curve
• 5.3 Enzyme concentration
• 5.4 Effects of ph
• 5.5 Effects of temperature
• 5.6 Computer simulation
• 5.7 Kinetic analysis
• 5.8 Determination of Km and Vmax
• 5.9 Addition of enzyme inhibitors
• 5.10 Protein concentration/enzyme activity
• 5.11 Advanced analysis of enzyme parameters
• Endnotes
  

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  Chapter 6: Membranes

• Introduction
• 6.1 Lipid solubility of membranes
• 6.2 Osmosis
• 6.3 Pinocytosis
• 6.4 Computer simulation of membrane function
• 6.5 Extraction of brain lipids
• 6.6 Formation of lipid bilayer
• 6.7 Electrical properties of artificial membranes
• 6.8 Measurement of transmembrane potential
• 6.9 Chemical composition of myelin
• 6.10 Myelin lipid extraction
• 6.11 Quantitative analysis of lipid classes by TLC
• Endnotes
  

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  Chapter 7: Microsomes

• Introduction
• 7.1 Lysosome isolation in isotonic sucrose
• 7.2 Em observations of microsomes
• 7.3 Chemical characterization - acid phosphatase
• 7.4 Chromaffin granules from adrenal glands
• 7.5 Measurement of catecholamines
• Endnotes
  

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  Chapter 8: Photosynthesis\Respiration

• Introduction
• 8.1 Isolation of Chloroplasts from Spinach Leaves
• 8.2 ChlorophyII Content
• 8.3 The Hill Reaction
• 8.4 Isolation of Mitochondria
• 8.5 Respiration: Succinic Dehydrogenase
• 8.6 Cytochrome Oxidase Manometric Analysis
• 8.7 Respiration of Mitochondria
• 8.8 Photosynthesis by Respirometry
• Endnotes
  

  Chapter 9: Tubules\Filaments

• Introduction
• 9.1 Fluorescent Analysis of Microtubules
• 9.2 Actin & Myosin-Microscopic Observation of Muscle
• 9.3 Histology of Extracellular Matrix
• 9.4 Isolation of Microtubules (Bovine Brain)
• 9.5 Viscosity & Polymeriztion of Microtubules
• 9.6 Polymerization Induced Changes in Absorbance
• 9.7 TEM Visualization of Microtubules
• 9.8 SDS Gel Electrophoresis of Tubulin\MAPs
• 9.9 Isolation of Actin and Myosin Filaments
• 9.10 Further Studies
• Endnotes
  

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  Chapter 10: Chromosomes

• Introduction
• 10.1 Polytene Chromosomes of Dipterans
• 10.2 Salivary Gland Preparation (Squash tech.)
• 10.3 Extraction of Chromatin
• 10.4 Chromatin Electrophoresis
• 10.5 Extraction and Electrophoresis of Histones
• 10.6 Karyotype analysis
• 10.7 In Situ hybridization
• Endnotes
  

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  Chapter 11: Cell Cycles

• Introduction
• 11.1 Mitosis
• 11.2 Grasshopper Spermatogenesis
• 11.3 Ascaris Oogenesis
• 11.4 3H - Thymidine Uptake by Cultured Cells
• 11.5 Timing of Cycles
• 11.6 Vicia Faba and Chemical Damage
• Endnotes
  

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  Chapter 12: Cell Cultures

• Introduction
• 12.1 Aseptic Cell Transfers
• 12.2 Examination of Bacterial Colonies
• 12.3 Gram Stain (+\-)
• 12.4 Prokaryote Cell Number by Dilution Plating
• 12.5 Cell mass by Measurement of Turbidity
• 12.6 Transfer of Eukaryote Suspension Cultures
• 12.7 Viability Cell Count
• 12.8 Computation of Transfer Aliquots
• 12.9 Eukaryote Growth Dynamics
• 12.10 Establishment of a Primary Culture
• Endnotes
  

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  Chapter 13: Differentiation

• Introduction
• 13.1 Sea urchin embryology
• 13.2 Determinants and fate - C. elegans embryos
• 13.3 The hematopoietic system
• 13.4 Photomorphogenesis
• 13.5 D.discoideum growth on agar
• 13.6 Suspension cultures of D. discoideum
• 13.7 Isolation of fruiting bodies without slugs
• 13.8 Prestalk vs prespore effect
• Endnotes
  

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  Chapter 14: Nucleic Acids

• Introduction
• 14.1 Extraction of DNA from bovine spleen
• 14.2 Purification of DNA
• 14.3 Characterization of DNA
• 14.4 DNA - dische diphenylamine determination
• 14.5 Melting point determination
• 14.6 CsCl - density separation of DNA
• 14.7 Phenol extraction of rRNA (rat liver)
• 14.8 Spectrophotometric analysis of RNA
• 14.9 Orcinol determination of RNA
• 14.10 Sucrose density fractionation
• 14.11 Nucleotide composition of RNA
• 14.12 Other studies
• Endnotes
  

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  Chapter 15: The Central Dogma

• Introduction
• 15.1 Protein Synthesis In Cell Free Systems
• Endnotes
  

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  Appendices

• Appendix A: Units and Measures

• Appendix B: Statistics

• Appendix C: Graphs

• Appendix D: Computers
• Appendix E: Image Analysis

• Appendix F: Centrifugation

• Appendix G: Spectrophotometry

• Appendix H: Radioactive tracers

• Appendix I: Photography

• Appendix J: Chemical Preparations

Cell Biology Laboratory Manual