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Table of Contents

Cell Biology Laboratory Manual

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: Centrifugation Appendix G: Spectrophotometry
Appendix H: Radioactive tracers
Appendix I: Photography
Appendix J: Chemical Preparations

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

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

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

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

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

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

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

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

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

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

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

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

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

Chapter 15: The Central Dogma
Introduction
15.1 Protein Synthesis In Cell Free Systems
Endnotes

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

Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu

Cell Biology Laboratory Manual

Table of Contents

Appendices