Basic Concepts of Microbiology

1. Basic Concepts of Microbiology Chapter 3

2. A Brief Survey of Microorganisms Pg. 78 to 81

3. Prokaryotes and Eukaryotes Have Some Similar Structures. • Both have Nucleic acids • Eukaryotes has its DNA in a protective compartment called the nucleus. • Prokaryotes DNA is free in the cell. • Cell membrane (plasma membrane) composed mainly of proteins and lipids (phospholipids). • Phospholipids form two layers referred to as bilayer. • Cytoplasm is everything inside the cell membrane • Cytosol the liquid of the cytoplasm without the structures ie. water, salts, ions, and organic compounds. • Ribosomes • RNA and protein bodies. • That are used to help synthesis protein. • The prokaryotic ribosomes are smaller than the eukaryotic.

4. Prokaryotes and Eukaryotes Have Some Different Structures . • Eukaryotic cells have distinct compartments called organelles. • Prokaryotic cells do not have organelles. • Eukaryotic Organelles • Endoplasmic reticulum (ER). • Rough ER is specific membrane with ribosomes attached. • Smooth ER has no ribosomes attached. • Both rough and smooth ER are for protein and lipid synthesis.

5. Eukaryotic Organelles • Endoplasmic reticulum (ER) • Rough ER is specific membrane with ribosomes attached. • Smooth ER has no ribosomes attached. • Both rough and smooth ER are for protein and lipid synthesis. • Golgi apparatus • Stacked flattened membranes. • This is were the proteins and lipids are processed, sorted and packaged. • Lysosomes • Sacs containing digestive enzymes • Kills pathogens once they have been taken up by white blood cells

6. Eukaryotic Organelles • Mitochondria • One or more • Used to convert chemical energy to cellular energy (powerhouse of the cell). • In bacteria this occurs on their cellular membrane. • Chloroplasts • Green algae has membrane bound chloroplasts. • Used in photosynthesis converts light to chemical. • Also some bacteria can convert light to energy • Cyanobacteria • Green sulfur bacteria

7. Cytoskeleton • Only found in eukaryotic cells. • Made up of a complex system of proteins not attached to membranes. • Proteins form into fiber and interwoven molecules. • This helps give structure to the cell. • Also moves material through the cell.

8. Motility • Many prokaryotes and eukaryotes have flagella or cilia. • Flagella are long thin protein projections that extend from the cell. • In prokaryotes, the flagella rotates like the propeller of a boat. • In eukaryotes, protozoa and algae the tail whips about. • The cilia are short and more numerous. • The cilia wave together like grass in the wind and this propels the protozoa.

9. Cell Walls • Many microorganisms have extra structure outside of their cell membranes. • The one most commonly seen is the cell wall. • The cell wall of different microorganism differ in composition. • Fungal have chitin (polysaccharide). • Algae has cellulose. • A number of bacteria have a cell wall made up of peptidoglycan. • Cell walls provide support. • Gives shape and protects from pressure.

10. Taxonomy and Nomenclature

11. Cataloging Microorganisms Pg 92 to 100 • Taxonomy – is the systematic classification of organisms based on their categories (taxa). • Taxonomy helps arrange related microorganisms and other living organisms into logical categories. • This is accomplished by arranging organisms together on basis of similar features.

12. History of Classification • Aristotle In 4 BC categorized over 500 plants and animal according to appearance and habits. • Linnaeus 1735 to 1759 (Two system of classification). • Covered the globe and classified thousands of plants and animals. • He developed the kingdoms Plant and Animal. • He was not interested in microorganisms so he classified them as Vermes (vermin) in the Chaos category. • Some microorganism a few bacteria, algae and fungi were put into plants. • Protozoa in animal.

13. Haeckel (1866) Three System of Classification • Proposed a new system of classification. • He separated the organisms into three groups not two. • Plants, Animals and Microorganisms. • Mainly due to mushrooms which did not have photosynthesis and was not an animal. • He found there was a lot of in-between organisms. • He classified these in a new group called protist. • Included protozoa, algae and bacteria

14. Five Kingdom System of Classification • Whittaker (1969) proposed a 5 kingdom system. • Monera – Bacteria. • Protista – Protozoa and unicellular algae. • Fungi – Molds, Yeasts and Mushrooms. • Plants – Multicellular use photosynthesis. • Animals – Multicellular, digest food through mouth.

15. Whittaker’s Five Kingdom System

16. Classification Using a Hierarchical System Taxonomy Humans Escherichia coli • Kingdom AnimaliaProtista (Monera) • Phylum ChordataSchizophyta (Protophyta) • Class MammaliaSchizomycetes • Order PrimatesEubacteriales • Family HominidaeEubacteriaceae • Tribe None Escherichiaceae • Genus HomoEscherichia • Species sapienscoli

17. Taxonomy • In this classification: • A species is the most specific category, comprises all organisms with characteristics similar enough to be considered of one and the same type. • A genus comprises all related species. • A tribe comprises all related genera. • A family comprises all related tribes. • An order comprises all related families. • A class comprises all related orders. • A tribe comprises all related genera. • A phylum comprises all related classes. • A kingdom, the broadest category, comprises all related phyla.

18. New System Three Domain System or Super-Kingdom • Proposed by Woese 1970 • Based on new techniques in molecular biology. • His system divides the Monera into two groups. • Archaea ( archae = “ancient”) • Most have the ability to survive in very extreme environments. • Eubacteria made up of “True Bacteria” • Why two • Woese showed that these two groups of bacteria had large differences in their base sequences of the 16S RNA in their ribosomes, and their composition of their cell walls and lipids in the cell membranes.

19. Domains • Eubacteria • Archaea • Eukaryotes –Protista, Fungi, Plantae and Animalia

20. Three Domain System

21. Bergey’s Bacterial Taxonomy • Bergey, 1923 one of the first systems for classification of bacteria. • The manual named after him “Bergey’s Manual of Determinative Bacteriology” is used to help researchers identify bacteria. • The first edition was released in 1984. • In 2001 a second addition was released and has more than 2,200 new species and 390 new genera.

22. Bergey’s Manual • Traditional criteria used for identification of bacteria are: • Color, Shape and Size • Oxygen, pH, temperature requirements • Staining reaction • Spore forming • Motility • Biochemical abilities photosynthesis • Ability to digest organic compounds

23. Molecular Taxonomy Now an Important Section in Classification • DNA sequence • RNA sequence • 16S rRNA sequence • Many researchers believe that the best method for classification is through the comparison of the DNA sequence of the 16S rRNA genes. • The reason for this is the importance of these genes in protein synthesis. • Any big changes in the DNA sequence due to mutations of the DNA will kill the bacteria so only small single base changes will occur over time. • This makes it easy to determine when species or geneses have diverged over time.

24. Nomenclature • Nomenclature is the process of assigning names to organisms following a formalized set of rules. • The names are given through set standards for each international groups committees for nomenclature, International Codes of Zoological Nomenclature, International Codes of Botanical Nomenclature and Bacteriological Code groups etc. • The names are based on identifying characteristics. • Organisms are then group into these categories based on similarities.

25. Naming of Microorganisms Through Binomial Nomenclature. • All organisms are named using the binomial system (binomial=“two names”) naming system. • Carolus Linnaeus was credited with the development of the binomial naming system. • The first name is the Genus to which the organism belongs. • Second name is the Species to which the organism belongs. • Genus and species names describe specific information about the organism. • The name can be derived from historical information, physical features, culture needs and diseases caused

26. Naming of Organisms • Name of Discoverer • Escherichia coli • Discovered by T. Escherich so genus name Escherichia • coli species name from were the bacteria inhabits large intestine, or colon. • Physical Features • Size, shape, color or odor etc. • Staphylococcus aureus genus Grape-like clusture and species golden color. • Streptococcus malodoratus organization of cocci into chains and causes a foul odor • Culture Requirements • Haemophilus aegyptius requires blood (heme) in the media to grow and was discovered in Egypt. • Disease • Propionibacterium acnes produces propionic acid and causes acne.

27. Naming • When a species is written only the first letter of the genus is capitalized. • The rest of the letter as well as the letters of the species name are written in lower case letters. • Both word are italicized. • Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes • Some cases only the first letter of the genus name is written E. coli.

28. Naming Microorganisms • All the microorganisms except viruses are named by binomial naming system (genus species). • The names given are determined by set standards for each international groups committees for nomenclature, International Codes of Zoological Nomenclature, International Codes of Botanical Nomenclature and Bacteriological Code groups etc.. • Viruses have no binomial naming system. • Virus names are determined by International Committee on Taxonomy of Viruses

29. MicroFocus • Read MicroFocus 3.4 • Pg 100

30. Microscopy Pg 101 - 113 • In order to determine size and shape a microscope has to be used. • Basic microscope used for microbiology is light microscope (bright-field microscope). • Read Pg 101 to 105. • Microorganisms must be stained to be viewed.

32. Size Comparisons Among Atoms, Molecules, and Microorganisms

33. Staining (Pg 105 to 108) • Microorganisms need to be colored by stains to be viewed. • Stains impart color to cells or their surroundings. • Labs 2 and 3 we will stain bacteria. • There are two general staining methods: • Simple staining • Differential staining

34. Staining • Simple staining • Uses one dye • Look at size, shape and arrangement • Two simple stains: • Positive • Negative • Differential staining • Two dyes • Differentiate types of bacteria and view special structures • Gram stain, Acid-fast stain, Flagella and Spore

35. Bacterial Smear Preparation • Take a little bacteria and smear on center of slide. • Allow cells to air dry. • Then briefly heat fix with Bunsen burner flame. • Three reasons for this: • Sticks cell to slide • Kills organism • Increases absorption of stain

36. Staining • Simple stain – a staining procedure that contrasts stained cells against bright background. • Positive Stain – negative charge of cell binds the positive charge of the dye. • Negative stain – negative charge of the cell repels the positive charge of the dye. • Stains background • No heat fixing used • Get a better idea of the shape and arrangement of cells

37. Differential Stains • Gram stain • Differentiates bacteria into two major groups. • Gram-negative and Gram-positive. • 4 step staining • crystal violet • iodine (mordent) • alcohol • Safranin • PPP purple, peptidoglycan, positive. • Gram negative counter stain pink with counter

39. Acid-fast Stain • Identify Mycobacterium. • Poorly stain with Gram stain. • Waxy substance (Mycolic acid) in the membrane prevents. • Heat carbolfuchsin into membrane. • Rinse with acid alcohol. • AF cells hold stain non-acid-fast cells lose stain. • Counter stain with blue counter stain.

40. Microscopy

41. Other Types of Microscopes • Phase-contrast • Dark-field • Fluorescence • Electron

42. Phase-contrast Microscope (Pg 109) • Prism splits light. • Increases contrast of images. • Image looks shadowed and three-dimensional. • Look at live bacteria.

44. Dark-field microscopy (Pg 109) • Background remains dark. • Light hits the side of the specimen. • Cause light reflection against the dark background. • Look at spiral bacteria hard to stain and at the limit of resolution 0.2 µm. • ie. Syphilis diameter 0.15 µm

46. Fluorescence Microscopy (Pg 109 – 110) • Some chemicals or object fluoresce a certain color after light of another color is shined on them. • Microorganism is coated with fluorescent dye (fluorescein) and illuminated with UV. • Fluorescent antibody technique. • Fluorescent dye is attached to antibodies. • The antibody is specific to one bacteria. • Attaches to the bacteria of interest. • Detect with UV

47. NeoMarkers Ab10 – EGF Receptor Zymed TAB250 – HER-2/neu Oncoprotein Immunohistochemistry with Antibodies Against EGF-Receptor & HER-2/neu Oncoprotein

48. Electron Microscope • Electrons are used instead of light to show image. • Ruska, 1933 electrons flow in a sealed vacuum tube. • Magnets rather then lenses focus the electron beam. • Image projected on screen on the bottom of the microscope. • Resolution 2 nm. • 100 x better than a light microscope

49. Electron Microscope

50. Electron Microscope • Two kinds: (Pg 111 – 112): • Transmission electron microscope (TEM) • Electron beam passes through a thin sliced section of the object . • Scanning electron microscope (SEM) • Electron sweeps across the gold coat object. • As the electrons hit the gold it causes shower of electrons to be released. • Detector picks up electrons. • Edges produces less electrons so shows depth (three-dimension).