CONTENTS
Chapter 1. General Instruction and Laboratory Methods 1
General Instruction 1
General Laboratory Methods 2
Chapter 2. Tools and Techniques in Biological Studies 9
Spectrophotometry 9
Electrophoresis 13
Column Chromatography 18
pH Meter 22
Centrifugation 24
Ultrasonic Cell Disruption 29
Conductivity Meter 32
Radioactive Tracers 32
Autoradiography 37
Photography 39
Statistics 41
Graphs 44
Computers 47
Chapter 3. Biochemistry 51
Carbohydrates 51
Exercise 1. Qualitative Tests 52
Exercise 2. Qualitative Analysis 54
Exercise 3. Estimate the Amount of Reducing Sugars 56
Exercise 4. Estimation of Reducing Sugar by Somogyi’s Method 59
Exercise 5. Estimation of Sugar by Folin-Wu Method 60
vii
viii CONTENTS
Exercise 6. Estimation of Sugar by Hagedorn-Jenson Method 61
Exercise 7. Estimation of Reducing Sugars by the Dinitro
Salicylic Acid (DNS) Method 62
Exercise 8. Determination of Blood Glucose by Hagedorn-Jenson
Method 63
Exercise 9. Determining Blood Sugar by Nelson and
Somogyi’s Method 65
Exercise 10. Determination of Blood Glucose by the O-Toluidine
Method 66
Exercise 11. Estimation of Protein by the Biuret Method 67
Exercise 12. Estimation of Protein by the FC-Method 68
Exercise 13. Protein Assay by Bradford Method 69
Exercise 14. Estimation of Protein by the Lowry Protein Assay 70
Exercise 15. Biuret Protein Assay 71
Exercise 16. Estimation of DNA by the Diphenylamine Method 72
Exercise 17. Estimation of RNA by the Orcinol Method 73
Chapter 4. Enzymology 75
Enzymes 75
Exercise 1. Demonstrating the Presence of Catalase in Pig’s Liver 83
Exercise 2. Determining the Optimum pH for Trypsin 84
Exercise 3. Extraction of Tyrosinase 85
Exercise 4. Preparation of Standard Curve 87
Exercise 5. Enzyme Concentration 89
Exercise 6. Effects of pH 90
Exercise 7. Effects of Temperature 91
Exercise 8. Computer Simulation of Enzyme Activity 92
Exercise 9. Kinetic Analysis 93
Exercise 10. Determination of Km and Vmax 94
Exercise 11. Addition of Enzyme Inhibitors 95
Exercise 12. Protein Concentration/Enzyme Activity 97
Exercise 13. Studying the Action and Activity of Amylase
on Starch Digestion 98
Exercise 14. Determination of the Effect of pH on the
Activity of Human Salivary a-Amylase 99
CONTENTS ix
Exercise 15. Determining the Effect of Temperature on the
Activity of Human Salivary a-Amylase 101
Exercise 16. Construction of the Maltose Calibration Curve 102
Chapter 5. Electrophoresis 103
Electrophoresis 103
Exercise 1. Preparation of SDS-Polyacrylamide Gels 108
Exercise 2. Separation of Protein Standards: SDS-PAGE 110
Exercise 3. Coomassie Blue Staining of Protein Gels 111
Exercise 4. Silver Staining of Gels 112
Exercise 5. Documentation 114
Exercise 6. Western Blots 114
Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis
(SDS-PAGE) 117
Preparation of Polyacrylamide Gels 119
Agarose Gel Electrophoresis 121
Agarose Gel Electrophoresis 123
Chapter 6. Microbiology 125
Introduction 125
The Microscopy 127
Exercise 1. The Bright Field Microscope 134
Exercise 2. Introduction to the Microscope and Comparison
of Sizes and Shapes of Microorganisms 137
Exercise 3. Cell Size Measurements: Ocular and
Stage Micrometers 146
Exercise 4. Measuring Depth 147
Exercise 5. Measuring Area 148
Exercise 6. Cell Count by Hemocytometer or Measuring Volume 149
Exercise 7. Measurement of Cell Organelles 152
Exercise 8. Use of Darkfield Illumination 152
Exercise 9. The Phase Contrast Microscope 153
Exercise 10. The Inverted Phase Microscope 155
Aseptic Technique and Transfer of Microorganisms 155
Control of Microorganisms by using Physical Agents 163
x CONTENTS
Control of Microorganisms by using Disinfectants and
Antiseptics 172
Control of Microorganisms by using Antimicrobial Chemotherapy 180
Isolation of Pure Cultures from a Mixed Population 191
Bacterial Staining 201
Direct Stain and Indirect Stain 206
Gram Stain and Capsule Stain 211
Endospore Staining and Bacterial Motility 215
Enumeration of Microorganisms 221
Biochemical Test for Identification of Bacteria 233
Triple Sugar Iron Test 241
Starch Hydrolysis Test (II Method) 242
Gelatin Hydrolysis Test 244
Catalase Test 245
Oxidase Test 247
IMVIC Test 249
Extraction of Bacterial DNA 257
Medically Significant Gram–Positive Cocci (GPC) 259
Protozoans, Fungi, and Animal Parasites 263
The Fungi, Part 1–The Yeasts 264
Performance Objectives 267
The Fungi, Part 2—The Molds 269
Viruses: The Bacteriophages 274
Serology, Part 1–Direct Serologic Testing 279
Serology, Part 2–Indirect Serologic Testing 289
Chapter 7. Cell Biology and Genetics 299
Cell Cycles 299
Meiosis in Flower Buds of Allium Cepa-Acetocarmine Stain 306
Meiosis in Grasshopper Testis (Poecilocerus Pictus) 309
Mitosis in Onion Root Tip (Allium Cepa) 311
Differential Staining of Blood 313
Buccal Epithelial Smear and Barr Body 315
Vital Staining of DNA and RNA in Paramecium 316
CONTENTS xi
Induction of Polyploidy 317
Mounting of Genitalia in Drosophila Melanogaster 318
Mounting of Genitalia in the Silk Moth Bombyx Mori 319
Mounting of the Sex Comb in Drosophila Melanogaster 320
Mounting of the Mouth Parts of the Mosquito 321
Normal Human Karyotyping 322
Karyotyping 322
Black and White Film Development and Printing for Karyotype
Analysis 324
Study of Drumsticks in the Neutrophils of Females 327
Study of the Malaria Parasite 328
Vital Staining of DNA and RNA in Paramecium 330
Sex-Linked Inheritance in Drosophila Melanogaster 331
Preparation of Somatic Chromosomes from Rat Bone Marrow 333
Chromosomal Aberrations 334
Study of Phenocopy 336
Study of Mendelian Traits 337
Estimation of Number of Erythrocytes [RBC] in Human Blood 338
Estimation of Number of Leucocytes (WBC) in Human Blood 340
Culturing Techniques and Handling of Flies 342
Life Cycle of the Mosquito (Culex Pipiens) 343
Life Cycle of the Silkworm (Bombyx Mori) 344
Vital Staining of Earthworm Ovary 346
Culturing and Observation of Paramecium 347
Culturing and Staining of E.coli (Gram’s Staining) 348
Breeding Experiments in Drosophila Melanogaster 349
Preparation of Salivary Gland Chromosomes 354
Observation of Mutants in Drosophila Melanogaster 355
ABO Blood Grouping and Rh Factor in Humans 356
Determination of Blood Group and Rh Factor 357
Demonstration of the Law of Independent Assortment 358
Demonstration of Law of Segregation 360
xii CONTENTS
Chapter 8. Molecular Biology 363
The Central Dogma 363
Exercise 1. Protein Synthesis in Cell Free Systems 364
Chromosomes 366
Exercise 2. Polytene Chromosomes of Dipterans 367
Exercise 3. Salivary Gland Preparation (Squash Technique) 368
Exercise 4. Extraction of Chromatin 369
Exercise 5. Chromatin Electrophoresis 371
Exercise 6. Extraction and Electrophoresis of Histones 372
Exercise 7. Karyotype Analysis 375
Exercise 8. In Situ Hybridization 378
Exercise 9. Culturing Peripheral Blood Lymphocytes 380
Exercise 10. Microslide Preparation of Metaphases for
In-Situ Hybridization 382
Exercise 11. Staining Chromosomes (G-Banding) 384
Nucleic Acids 386
Exercise 12. Extraction of DNA from Bovine Spleen 387
Exercise 13. Purification of DNA 388
Exercise 14. Characterization of DNA 389
Exercise 15. DNA-Dische Diphenylamine Determination 390
Exercise 16. Melting Point Determination 391
Exercise 17. CsCl-Density Separation of DNA 393
Exercise 18. Phenol Extraction of rRNA (Rat liver) 394
Exercise 19. Spectrophotometric Analysis of rRNA 395
Exercise 20. Determination of Amount of RNA by the
Orcinol Method 396
Exercise 21. Sucrose Density Fractionation 397
Exercise 22. Nucleotide Composition of RNA 398
Isolation of Genomic DNA—DNA Extraction Procedure 400
Exercise 23. Isolation of Genomic DNA from Bacterial Cells 401
Exercise 24. Preparation of Genomic DNA from Bacteria 404
Exercise 25. Extraction of Genomic DNA from Plant Source 405
Exercise 26. Extraction of DNA from Goat Liver 407
Exercise 27. Isolation of Cotton Genomic DNA from Leaf Tissue 408
CONTENTS xiii
Exercise 28. Arabidopsis Thaliana DNA Isolation 411
Exercise 29. Plant DNA Extraction 412
Exercise 30. Phenol/Chloroform Extraction of DNA 414
Exercise 31. Ethanol Precipitation of DNA 414
Exercise 32. Isolation of Mitochondrial DNA 415
Exercise 33. Isolation of Chloroplast DNA 417
Exercise 34. DNA Extraction of Rhizobium (CsCl Method) 417
Exercise 35. Isolation of Plasmids 418
Exercise 36. RNA Isolation 422
Preparation of Vanadyl-Ribonucleoside Complexes
that Inhibit Ribonuclease Activity 422
Exercise 37. RNA Extraction Method for Cotton 423
Exercise 38. Isolation of RNA from Bacteroids 424
Exercise 39. Isolation of RNA from Free-Living Rhizobia 425
Estimation of DNA purity and Quantification 426
Exercise 40. Fungal DNA Isolation 427
Exercise 41. Methylene Blue DNA Staining 429
Exercise 42. Transformation 431
Blotting Techniques—Southern, Northern, Western Blotting 433
Preparing the Probe 440
Exercise 43. Southern Blotting (First Method) 444
Exercise 44. Southern Blotting (Second Method) 446
Exercise 45. Western Blotting 447
Exercise 46. Western Blot Analysis of Epitoped-tagged Proteins
using the Chemifluorescent Detection Method for
Alkaline Phosphatase-conjugated Antibodies 450
Southern Blot 452
Exercise 47. Southern Analysis of Mouse Toe/Tail DNA 453
Exercise 48. Northern Blotting 454
Restriction Digestion Methods—Restriction Enzyme Digests 457
Exercise 49. Restriction Digestion of Plasmid, Cosmid, and
Phage DNAs 460
xiv CONTENTS
Exercise 50. Manual Method of Restriction Digestion of
Human DNA 461
Exercise 51. Preparation of High-Molecular-Weight Human
DNA Restriction Fragments in Agarose Plugs 463
Exercise 52. Restriction Enzyme Digestion of DNA 465
Exercise 53. Electroelution of DNA Fragments from Agarose
into Dialysis Tubing 466
Exercise 54. Isolation of Restriction Fragments from Agarose
Gels by Collection onto DEAE Cellulose 469
Exercise 55. Ligation of Insert DNA to Vector DNA 471
PCR Methods (Polymerase Chain Reaction) 472
Exercise 56. Polymerase Chain Reaction 475
Exercise 57. DNA Amplification by the PCR Method 476
Chapter 9. Tissue Culture Techniques 477
Tissue Culture Methods 477
Plant Tissue Culture 487
Plant Tissue Culture 491
Many Dimensions of Plant Tissue Culture Research 493
What is Plant Tissue Culture? 496
Uses of Plant Tissue Culture 497
Plant Tissue Culture demonstration by Using
Somaclonal Variation to Select for Disease Resistance 498
Demonstration of Tissue Culture for Teaching 499
Preparation of Plant Tissue Culture Media 502
Plant Tissue Culture Media 505
Preparation of Protoplasts 512
Protoplast Isolation, Culture, and Fusion 514
Agrobacterium Culture and Agrobacterium—
Mediated transformation 518
Isolation of Chloroplasts from Spinach Leaves 520
Preparation of Plant DNA using CTAB 522
Suspension Culture and Production of Secondary Metabolites 525
Protocols for Plant Tissue Culture 529
Sterile Methods in Plant Tissue Culture 535
CONTENTS xv
Media for Plant Tissue Culture 538
Safety in Plant Tissue Culture 543
Preparation of Media for Animal Cell Culture 544
Aseptic Technique 546
Culture and Maintenance of Cell Lines 550
Trypsinizing and Subculturing Cells from a Monolayer 557
Cellular Biology Techniques 558
In Vitro Methods 574
Human Cell Culture Methods 577
Chapter 10. Tests 595
Starch Hydrolysis Test 595
Gelatin Hydrolysis Test 597
Catalase Test 598
Oxidase Test 599
Appendix A (Units and Measures) 603
Appendix B (Chemical Preparations) 617
Appendix C (Reagents Required for Tissue
Culture Experiments) 645
Appendix D (Chemicals Required for Microbiology
Experiments) 671
Appendix E (About the CD-ROM) 689
Index 691