Bio 411 Master Presentation

1. Bio 411 Master Presentation Dr. Rowe SH 222 X2521, j.rowe@notes.udayton.edu

2. Overview of Microbiology • The study of organisms and agents to small to be seen clearly by the unaided eye • Organisms involved are prions, viruses, bacteria, fungi, protozoa,and algae

3. Historical Perspectives • Spontaneous Generation. Living organisms can develop from non living matter. • Until the 17th century this was the primary hypothesis to explain how life arises.

4. Historical Perspectives • Francesco Redi (1626-1697) Experimented with decaying meats and showed maggots wouldn’t develop in the meat spontaneously if covered in gauze but flies laid eggs on top of gauze which developed into maggots thus temporarily disproving spontaneous generation.

5. Antony van Leeuwenhoek (1623-1723) Dutch. First to observe and describe microoganisms under amicroscope.

6. Historical Perspectives • With the discovery of microorganisms the idea of Spontaneous Generation resurfaced for the origin of microbes. • Needham (Catholic Priest), Schwann and Pasteur all performed experiments between 1780 and 1870 which proved microbes did not originate by spontaneous generation.

7. Pasteur disproves spontaneous generation by the famous swan necked flask experiment

8. Historical Perspectives • Joseph Lister -1867 • Developed the concept of antiseptic surgery. • Instruments are heat sterilized and phenol is used afterward to eliminate infection. • Pasteur correlated process of wine making and beer making with yeast by- products • Winogradsky and Biejerinck showed importance of bacteria in biogeochemical cycling (Carbon, sulfur, nitrogen)

9. Microbiology in the 20th Century • Robert Koch (1843-1910) Demonstrated the role of microbes in causing disease during his study of anthrax. • Koch established the relationship between Bacillus anthracis and anthrax by isolating the organisms from infected animals and injecting them into mice.

10. Microbiology in the 20th Century • Koch’s Postulates 1. Microbe must be present in every case of the disease but not in healthy animal 2.Suspected microbe must be isolated and grown in pure culture 3.Same disease must result when pure culture inoculated into healthy host 4. Same microbe isolated from infected host

11. Germ Theory • Idea that microbes caused disease was not well accepted until the late 1800’s • Pasteur was attributed to be the father of germ theory although Koch and Lister played major roles

12. IMMUNOLOGY Pasteur studied chicken cholera and found that if cultures were incubated for very long periods between transfers they would become ATTENUATED=NO LONGER ABLE TO CAUSE DISEASE. When the attenuated form was injected into healthy chickens they became resistant to infection Pasteur developed other means to attenuate a bacteria via heat treatment or chemical treatment.

13. Vaccine Production • The idea of injecting attenuated strains to protect healthy animals or humans from microbial infection was termed vaccination • Pasteur developed the first vaccine against anthrax and the viral disease rabies.

14. Vaccination • The acute contagious disease diphtheria caused by a toxin produced by the bacteria Corynebacterium diphtheriae was the first such disease identified • Von Behring and Kitasato responsible for identifying and creating a vaccine and antitioxin by injection of healthy animals with inaactivated toxin.

15. Koch’s Molecular Postulates • 1. The virulence trait under study should be associated much more with pathogenic strains of the species than non pathogenic strains • 2. Inactivation of the genes or genes (by mutation or knockout) associated with the suspected virulence trait should substantially decrease pathogenicity • 3. Replacement of the mutated gene (or knockout) with the normal wild-type gene should fully restore pathogenicity

16. Koch’s Molecular Postulates • 4. The gene should be expressed at some point during the infection or disease process • 5. Antibodies or immune system cells directed against the identified gene products should protect the host.

17. Microbiology in the 20th Century • Diseases such as diphtheria,yellow fever, malaria, were shown to be infections of microorganisms • Penicillin - 1st antibiotic • discovered by Flemming in 1945 • Waksman’s discovered streptomycin in 1952

18. Story of Louis Pasteur • Struggle of science and pseudoscience • Pasteurization process for wine 3. Story of establishment of germ theory 4. Story of scientific development of first human/animal vaccines, rabies, anthrax 5. Story of virus disease and bacterial disease 6. Story of antiseptic surgery 7. Story of modern culture technique and microscopy and disease

19. Microbiology in the 20th Century Microorganisms were used as experimental models for developing the area of genetics and molecular biology • 1962 - Watson and Crick discovered the genetic material DNA • 1965 - Jacob and Monod revealed how transcription of DNA is regulated

25. Important Techniques in Microbiology • Aseptic (sterile) techniques • Culture media • Solid Agar media for pure culture technique • Microscopy-light microscopy, scanning and transmission electron microscopy

26. The microbes • Domain Eucaryea • contain microbes in three kingdoms • Plants • Fungi • Protozoa

27. The microbes • Domain Bacteria -includes all procaryotes with bacterial ribosomal(rRNA) RNA and cell membranes with traditional ester bonds between glycerol and fatty acids.

28. The Microbes • Domain Archaea- All Procaryotes with membranes containing glycerol and fatty acids linked by an ether bond and a special Archaeal rRNA

29. Figure 19.3 p. 396

30. Figure 19.10 p 409

31. Light Microscopy

32. Microscope’s light path

33. Bright Field Microscope • Dark image against light background • Generally must stain bacteria to increase • visibility • Magnification is from 50X-1000X

34. Preparation and Staining • Fixation-Process whereby the internal and external structures of the cell and the microorganism itself are fixed in place • Two types for bacteriology-heat fix and chemical fix.

35. Simple Stains Basic Dyes are positively charged and bind to negatively charged macromolecules like nucleic acids. Bacterial surfaces are negatively charged so it basic stains are commonly used. • Crystal violet • Malachite green • safranin

36. Simple Stains Acid Dyes are negatively charged and thus bind to positively charged macromolecules such as calcium rich substances. • Acid fuchsin • eosin

37. Differential staining Gram stain Acid-fast-Use harsh treatment to allow penetration of dye. Heat, basic fuchsin and phenol. A rinse with acid alchohol. Acid fast cells retain the red fuchsin because of the presence of positively charged molecules. Used to stain mycobacterium

40. Transmission Electron Microscopy Figure 2.22 p 30

41. Transmission Electron Microscopy • Uses magnetic lenses • Uses electron beam in place of light • used for virus and cross sections of cells • specimen must be stained: osmium tetroxide or phosphotungstate are common stains.

42. Transmission pic

43. Proteus mirabilis and b)T4 bacteriophageShadowing process

44. THIOBACILLUS KABOBISFREEZE FRACTURE TECHNIQUE

45. Scanning Electron Microscopy • specimen scanned with primary electron beam and sample emits a shower of secondary electrons which can be detected • Visualizes surfaces or surface structures

46. Scanning Electron Microscopy Figure 2.26 p 32

47. a)Staphylococcus aureusb)Cristospira(spirochete)

48. Figure2.19 p 28

49. Parameters used in the phenotypic identification of microbes • Cell shape • Cell size • Colony morphology • Staining behavior • Physiological and biochemical characteristics

50. Cell Shape • Cocci (spherical) • Rod • spirochete