EBOOK

Tissue Culture Techniques


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August 1994

Beschreibung

Beschreibung

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xl II INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I STERILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Aseptic Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Physical manipulations - Use of the sterile cabinet (hood) Sterilization Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Heat - Radiation - Toxic gas - Filtration - Antibiotics Quality Control of Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Routine labeling Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 vi CONTENTS ROUTINE CELL CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Feeding Schedules and Media Components . . . . . . . . . . . . . . . . . . . . 29 General properties of media and salt solutions - water as a reagent· Establishingfeeding schedules Subcultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Solutions and methods for adherent cells - Common enzyme solutions - Inoculating (seeding) the cultures Cell Enumeration and Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Hemocytometer - Particle counter - Cell viability Putting Routine Methods to Work . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Normal cell growth characteristics Detecting and Disposing of Contamination . . . . . . . . . . . . . . . . . . . . 66 Bacteria and fUngi - Fungi - Mycoplasma - Viruses - Dealing with contamination Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Inadequate cell growth - Recurrent contamination - When to call your vendor Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Biological hazards - Chemical hazards Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Problem Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 EXPERIMENTS IN CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . 91 II Alterations of the Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Serum - Treatments of serum - Plasma-derived serum - Serum-free and low-protein media Substrata. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Coatingplasticware with solutions - Alterations with polymers - Using cells to coat the plasticware - Culturing cells on microcarriers Altering the Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Temperature changes - Gaseous changes Problem Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 CONTENTS vii PRIMARY CELL CULTURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Dissection - Enzymatic dissociation methods - Nonenzymatic isolation - Purification of cell suspensions - Consideringyield and survival Chatacterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Inhaltsverzeichnis

1 Introduction.
2 Sterility.
Aseptic Technique.
Physical manipulations . Use of the sterile cabinet (hood).
Sterilization Methods.
Heat . Radiation . Toxic gas . Filtration .Antibiotics.
Quality Control of Sterilization.
Routine labeling.
Suggested Readings.
Exercises.
3 Routine Cell Culture.
Feeding Schedules and Media Components.
General properties of media and salt solutions . Water as a reagent . Establishing feeding schedules.
Subcultivation.
Solutions and methods for adherent cells . Common enzyme solutions . Inoculating (seeding) the cultures.
Cell Enumeration and Cell Viability.
Hemocytometer . Particle counter . Cell viability.
Putting Routine Methods to Work.
Normal cell growth characteristics.
Detecting and Disposing of Contamination.
Bacteria and fungi . Fungi . Mycoplasma . Viruses . Dealing with contamination.
Troubleshooting.
Inadequate cell growth . Recurrent contamination . When to call your vendor.
Safety.
Biological hazards . Chemical hazards.
Suggested Readings.
Problem Set.
Exercises.
4 Experiments in Culture.
Alterations of the Media.
Serum . Treatments of serum . Plasma-derived serum . Serum-free and low-protein media.
Substrata.
Coating plasticware with solutions .Alterations with polymers .Using cells to coat the plasticware . Culturing cells on microcarriers.
Altering the Environment.
Temperature changes . Gaseous changes.
Problem Set.
Exercises.
5 Primary Cell Culture.
Isolation.
Dissection . Enzymatic dissociation methods . Nonenzymatic isolation . Purification of cell suspensions . Considering yield and survival.
Characterization.
Preparation . Fixation andpermeahilization . Identification.
Exercise.
6 Cell Preservation.
Freezing the Cells.
Thawing the Cells.
Equipment . Solutions.
Problem Set.
7 Cell Cloning.
Isolation.
Limiting dilution . Cloning cylinders.
Assisting Clonal Survival.
Using conditioned medium . Using feeder layers.
Problem Set.
8 Culture Changes.
The Loss of Differentiated Phenotype.
Immortality and Aging.
Cell Transformation.
Chemical transformation . Viral transformation . Transformed cells from tumors.
Transfection (Gene Transfer).
Methods . Evaluation.
Suggested Readings.
Problem Set.
Exercises.
9 Information for New Cell Studies.
Searching in the Literature.
Using Advertising and Catalogs.
References, Bibliographies, and Databases.
Appendices.
A The Cell Cycle.
Suggested Readings.
B Media and Salt Solutions.
Media.
Salt Solutions.
C Vendors.
D Glossary.
E Answers to Problem Sets and Exercises.
EAN: 9780817636432
ISBN: 0817636439
Untertitel: An Introduction. 1994. Auflage. Book. Sprache: Englisch.
Verlag: Birkhäuser
Erscheinungsdatum: August 1994
Seitenanzahl: 264 Seiten
Format: kartoniert
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