Laboratory Exercises in Microbiology

PART ONE Microscopic Techniques

1. Bright-Field Light Microscope (Dark Image, Brighter Background) and Microscopic Measurement of Organisms

2. The Hanging Drop Slide and Bacterial Motility

3. Dark-Field Light Microscope: Bright Image, Dark Background

4. Phase-Contrast Light Microscope

5. Fluorescence Microscope

PART TWO Bacterial Morphology and Staining

6. Negative Staining

7. Smear Preparation and Simple Staining

8. Gram Stain

9. Acid-Fast Staining (Ziehl-Neelsen and Kinyoun) Procedures

10. Endospore Staining (Schaeffer-Fulton or Wirtz-Conklin)

11. Capsule Staining

12. Flagella Staining: West and Difco’s SpotTest Methods

PART THREE Basic Laboratory and Culture Techniques

13. Sterilization and Microbiological Culture Media Preparation

14. Culture Transfer Instruments, Techniques, and Isolation and Maintenance of Pure Cultures

15. Spread-Plate Technique

16. The Streak-Plate Technique: Selective and Differential Media

17. Pour-Plate Technique

18. Cultivation of Anaerobic Bacteria

19. Determination of Bacterial Numbers

PART FOUR Biochemical Activities of Bacteria

20. Carbohydrates I: Fermentation and ß-Galactosidase Activity

21. Carbohydrates II: Triple Sugar Iron Agar Test

22. Carbohydrates III: Starch Hydrolysis

23. Lipids: Lipid Hydrolysis

24. Proteins, Amino Acids, and Enzymes I: Hydrogen Sulfide Production and Motility

25. Proteins, Amino Acids, and Enzymes II: The IMViC Tests

26. Proteins, Amino Acids, and Enzymes III: Casein Hydrolysis

27. Proteins, Amino Acids, and Enzymes IV: Gelatin Hydrolysis

28. Proteins, Amino Acids, and Enzymes V: Catalase Activity

29. Proteins, Amino Acids, and Enzymes VI: Coagulase and DNase Activity

30. Proteins, Amino Acids, and Enzymes VII: Oxidase Test and Bioluminescence

31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity

32. Proteins, Amino Acids, and Enzymes IX: Lysine and Ornithine Decarboxylase Tests

33. Proteins, Amino Acids, and Enzymes X: Phenylalanine Deamination

34. Proteins, Amino Acids, and Enzymes XI: Nitrate Reduction

PART FIVE Rapid Multitest Systems

35. The API® 20E System

36. The EnteroPluri-Test System

PART SIX Unknown Identification

37. Using the First Edition of Bergey’s Manual of Systematic Bacteriology to Identify Bacteria

38. General Unknown

PART SEVEN Environmental Factors Affecting Growth of Microorganisms

39. Temperature

40. pH

41. Osmotic Pressure

42. The Effects of Chemical Agents on Bacteria I: Disinfectants and Other Antimicrobial Products

43. The Effects of Chemical Agents on Bacteria II: Antimicrobial Agents (Kirby-Bauer Method) and the Etest®

44. Hand Washing, Environmental Sampling, and Microbiological Monitoring

45. Determination of a Bacterial Growth Curve: Classical and Two-Hour Methods

PART EIGHT Environmental and Food Microbiology

46. Standard Coliform Most Probable Number (MPN) Test and Colilert®-18 Coliform Test

47. Membrane Filter Technique for Coliforms and Fecal Streptococci; KONFIRMTM Test for Fecal Coliforms

48. Isolation of Escherichia coli Bacteriophages from Sewage and Determining Bacteriophage Titers

49. Enumeration of Soil Microorganisms

50. Bacterial Count of a Food Product

51. Examination of Milk for Bacteria

PART NINE Medical Microbiology

52. Hemagglutination Reactions: Blood Groups

53. Isolation of Normal Microbiota from the Human Body

54. Staphylococci

55. Pneumococci

56. Streptococci

57. Neisseriae

58. Aerobic and Anaerobic Endospore-Forming Bacteria

PART TEN Survey of Selected Eucaryotic Microorganisms

59. Fungi (Ascomycota; also Ascomycetes) I: Yeasts

60. Fungi (Eumycota) II: Zygomycota (Rhizopus), Ascomycota (Penicillium), and Basidiomycota(Agaricus)

PART ELEVEN Microbial Genetics and Genomics

61. Bacterial Mutation

62. Bacterial Transformation

63. Bacterial Conjugation: The Transfer of Antibiotic-Resistant Plasmids

64. Isolation of Genomic DNA from Baker’s Yeast (Saccharomyces cerevisiae)

65. Isolation and Purifi cation of Genomic DNA from Escherichia coli

PART TWELVE Scientific Inquiry

66. Independent Group Projects

67. Case Studies

Appendix A Dilutions with Sample Problems

Appendix B Transmission-Absorbance Table for Spectrophotometry

Appendix C Logarithms; pH and pH Indicators

Appendix D Scientifi c Notation

Appendix E Identifi cation Charts

Appendix F Reagents, Solutions, Stains, and Tests

Appendix G Culture Media Appendix H Sources and Maintenance of Microbiological Stock Cultures

Appendix I Laboratory Skills and Laboratory Thinking Skills

Appendix J Orientation to the Laboratory: Rules of Conduct and General Safety

Appendix K Summary of Universal Precautions and Laboratory Safety Procedures

Appendix L Biosafety Level (BSL)

Index

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