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Cell Biology Laboratory Manual
Dr. William H. Heidcamp Biology Department, Gustavus Adolphus College, St. Peter, MN 56082
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
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