Ch 3 & 4 Microscopy & Cell Components 1

Transcription

1 Objectives 1.White book: Read Chap 3 & p & Black book: Read Chap 3 & p75-96 & 106 Objectives: 1. List metric measurement units for microorganisms and convert to other metric units (m, mm, um, nm). 2. Identify parts & functions of the compound light microscope. 3. Define/calculate total magnification & resolution. 4. Compare, contrast, and identify uses (diseases/organisms) for brightfield, darkfield, fluorescent, electron-transmission, and electronscanning microscopy. 5. Differentiate, compare, and explain the appearance and uses of each of the following: acidic & basic dyes, simple, differential & special stains, capsule, endospore, acid-fast and flagella stains. 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 1 Objectives, cont d 6. List specific chemicals that are used for each type of stain in the objective above, primary stain, mordant, decolorizer, counterstain. 7. Gram stain: list the steps, purpose, and the appearance of GP & GN cells after each step. 8. Identify the 3 basic shapes of bacteria and secondary arrangements. 9. Describe the structure & function of the glycocalyx, flagella (including arrangement), axial filaments, fimbriae, pili. Identify flagellar arrangements. 10.Compare & contrast the cell walls of GP bacteria, GN bacteria, archaea, mycoplasmas, and mycobacteria. (Including composition, antibiotic & chemical resistance, presence of toxins, staining reactions, effect of penicillin, lysozyme, etc.) 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 2 Objectives, Cont d 11. Identify the functions of the cell/plasma membrane, chromatophores/thylakoids, nucleoid, ribosomes, endospores (including location), inclusions. 12. Transport: passive (simple diffusion, osmosis, facilitated diffusion), active transport, hypertonic, hypotonic, isotonic, osmotic lysis, plasmolysis 13. Discuss several pieces of evidence that support the endosymbiotic theory of eukaryotic evolution. 14. Describe the overall structure and defining characteristics of prokaryotes, as compared to eukaryotes. 15. On given slides identify shape, gram reaction, arrangement, type of stain. Measurement Units & Terms 1. Units A. Micrometer (µm) = B. Nanometer (nm) = i. Example: Convert 21.5 nm to m 2. Total Magnification 3. Resolution: Distance apart needed to see (Ability to see ) 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 3 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 4 Ch 3 & 4 Microscopy & Cell Components 1

2 Resolution & Refractive Index A. Resolving power = N.A. depends on: i. of material between lens & slide. ii. The of most divergent light ray B. To improve resolution: i.. ii.. C. Improve conditions but NOT resolution: i.. ii.. Fig 3.3 Refraction w/ & w/o Oil, p.59 Using oil does improve resolution, as it increases the numerical aperture, which will cause a better (smaller) resolving power number 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 5 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 6 Types of Scopes-3 subtypes of Light microscopes Scope Enhanced by Advantages Uses Light, Brightfield: Background Visible light Res: Mag: Light, Darkfield: Background & microbes Same Light, Fluorescent: Background & microbes Same & light N/A Fluorescent- dyes: Fluorescent dye on to microbe, microbe fluoresces Inexpensive Easy to use Easier to see microbes directly from specimen, w/o culture Detection of microbes compared to other light microscopy Live specimens (unstained) Stained specimens Bacteria, protozoa Live microbes: When immediate diagnosis needed When cultures aren t avail, or take long 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 7 Fig 3.6 Immunofluorescent Staining Technique Demo-Fluorescent marker drawings 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 8 Ch 3 & 4 Microscopy & Cell Components 2

3 Scopes-Electron Scope Enhanced by Advantages Uses Electron, Scanning Res; Mag; 3-D Book from U of I Surfaces structures - eukaryote to virus Fig 3.8 Transmission vs. Scanning Electron, Transmission Res Mag Scanned-Probe Res 1/100 of atom Stain w/+ salt of heavy metal res & mag DISADVANTAGE: Need slice as e- can t All e- scopes- due to killing, & fixing under vacuum Res No special prep Virus particles, bacterial flagella, cell structures, protein molecules Map atomic & molecular shapes & processes, ie. DNA, fibrin 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 9 (clot) formation 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 10 Stains-Slide Prep & Basic Stains Slide Prep: 1. Smear 2. Fix to slide (won t off) B.. C.. D. HOPEFULLY-preserves w/ Staining 1. Basic dye/ stain: Colored ( ) ion of a salt A. Attracted to ( ) bacterial cell; stains B. Crystal violet, methylene blue, safranin, malachite green Acidic Dye / Negative Stain 2. Acidic dye / stain: Colored ( ) ion A. & stains B. For cell, to detect C. Advantage: (no & stain so accurate size & shape) D. Examples: Acid fuchsin, nigrosin 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 11 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 12 Ch 3 & 4 Microscopy & Cell Components 3

4 Mordant, Simple Stain, Differential Stain 3. Mordant: Substance used to cause more staining NOTE: This is not the stain that gives color, only helps the stain be more intense color 4. Simple stain: basic dye A. All microbes - B. Only for 5. Differential Stain: Use of to groups of bacteria A. Examples: gram stain, acid fast stain Gram Stain 6. Gram Stain: - due to differences A. GP = gram positive,, retain stain i. Us. to penicillin B. GN = gram negative, red, stain & accepts i. to penicillin C. Staining problems i. Need cultures ii. Some bacteria stain iii. timing is iv. Potential -structures/distortions that appear due to prep or staining procedures NOTE: this is potential problem w/all stains 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 13 Most common stain in medical microbiology Know procedure-steps, purpose of each step/stain, appearance of cells after each step, how cell wall causes differential staining (Chap 4) 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 14 Gram Stain Diagram Stains: Acid Fast & Capsule 7. Acid Fast Stain A. Acid-fast positive = (due to in cell ) B. Acid-fast neg = C. ID species, 8. Capsule Stain (w/ stain) A. Capsule = covering on outside of bacteria B. Variation w/2 stains: i.. Shapes above? GN or GP? Combine? GNR/GNB & GPC 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 15 ii.. iii. of capsule left between the stains C. Problems: capsule may 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 16 Ch 3 & 4 Microscopy & Cell Components 4

5 Pictures-Acid Fast & Capsule Stains Stains: Endospore 9. Endospore Stain A. Endospore i. Position used to ID species A. Uses to force dye into endospores 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 17 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 18 Stains; Endospore Pictures Stains; Flagella 10. Flagella Stain A. Flagella = B. used to ID bacteria 1. Discuss vegetative vs. endospores. Free vs. still in cell. 2. Which of the 2 pictures above has been subjected to adverse conditions longer? Explain. 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 19 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 20 Ch 3 & 4 Microscopy & Cell Components 5

6 Chapter 4: Prokaryotic Cells Prokaryote 1.. Fig 4.5a Prokaryotic Cell Bacteria cell wall 6. Archaea 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 21 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 22 Fig 6.11a Binary Fission Arrangement Size of bacteria um vs. resolution of light microscope? Arrangement Review: Shapes/Morphology? Arrangements? Other morphology terms: /4/2013 Ch 3 & 4 Microscopy & Cell Componenets 23 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 24 Ch 3 & 4 Microscopy & Cell Components 6

7 Cell Wall - Bacteria Bacterial Cell Wall 1.. Diagram Cell Wall Diagrams Outside Cell Outside Cell 2. Clinical importance B Penicillin interferes Inside Cell Inside Cell 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 25 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 26 Table GP vs. GN Cell Wall Characteristics GP Wall GN Wall Contains 2. None Gram Stain & the Cell Wall Cell Wall & gram stain 1. Iodine = 2. Alcohol A. GP: 3. None 3. OUTER Wall Membrane A. Evades B. Contains C.. B. GN: C. GP falsely stain GN when cell wall damaged due to 4. None 4. Periplasm- (where peptidoglycan is) A. Contains 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets GPR/GPB only: A. : Bacillus & Clostridium B. : Mycobacterium (TB) 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 28 Ch 3 & 4 Microscopy & Cell Components 7

8 Chemicals & the Cell Wall Chemical Effects on Cell Wall 1. Lysozyme: A. Most effective on 2. Penicillin Atypical Cell Walls Atypical Cell Walls 1. Mycoplasma species: A. High amount in plasma membrane, from lysis 2. Mycobacteria- High in wall B.. 3. Archea; 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 29 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 30 Structures External to Cell Wall External Structures 1. Glycocalyx/Capsule: A. EPS (Extracellular polysaccharide) & polypeptide polymer B.. External Filamentous Structures 2. Table: Flagella Axial Filaments Fimbrae Pili C. Negative Stain, but uses 2 dyes i. Basic stains ii. Acidic stains iii. Monotrichous - Spiralled around cell within (AKA endoflagella) 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 31 Amphitrichous- Lophotrichous- Peritrichous- 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 32 Ch 3 & 4 Microscopy & Cell Components 8

9 Diagram-Axial Filament Photo-Fimbriae 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 33 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 34 Fig 8.26 Bacterial Conjugation Fig 8.27 Conjugation in E. coli 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 35 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 36 Ch 3 & 4 Microscopy & Cell Components 9

10 External Filamentous Structures, Cont d 3. NO 4. Taxis: A. Chemotaxis B. Phototaxis Discuss serovars Endospores Structure Internal to Cell Wall 1. Endospores: structures to adverse conditions B. Sporulation / Sporogenesis C. Germination return to state D.. E. Location: F. Survive G. Stains: i. Gram- ii. Endospore Stain: Primary: basic stain Rinse: removes stain from Counterstain: basic stain colors 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 37 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 38 Fig 4.20a Endospore Formation Endospore Stain Pictures 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 39 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 40 Ch 3 & 4 Microscopy & Cell Components 10

11 Plasma/Cytoplasmic Membrane 2. Plasma Membrane B.. C. Special: Alcohols, disinfectants, some antibiotics effective here Fig 2.11 Phospholipids 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 41 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 42 Fig 4.16 Diffusion D. Diffusion: i. Simple diffusion ii. Facilitated diffusion iii. Osmosis E. Active Transport: Diagram on the right: Which type of transport does it represent? Osmosis-Animal vs. Plant Special terms reflect % solute, and therefore affect net direction of osmosis. What do the following prefixes mean? Iso? Hypo? Hyper? Suffix is tonic = tension 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 43 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 44 Ch 3 & 4 Microscopy & Cell Components 11

12 Osmosis & Solution Types F. Osmotic Environments i. Isotonic/isoosmotic solution:».» Water movement». ii. Hypotonic solution:» Net H 2 O moves». Diagram from Bio-Osmosis & Plant vs. Animal Isotonic solution Hypotonic solution Hypertonic solution H 2O H 2O H 2O H 2O Animal cell (1) Normal (2) Lysed (3) Shriveled H 2O H Plasma H 2O 2O membrane H 2O iii. Hypertonic solution:» Net H 2 O movement». Plant cell (4) Flaccid (5) Turgid (6) Shriveled (plasmolyzed) 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 45 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 46 Fig 4.17 cde Osmosis in Varying Osmotic Environments Internal Cell Structures continued 3. Chromatophores/thylakoids: structures 4. Nucleoid/nuclear area: No nuclear membrane A. Contains 5. Plasmids: B. Conjugation: transfer through i. GN- ii. GP- C. Biotech: 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 47 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 48 Ch 3 & 4 Microscopy & Cell Components 12

13 Fig 8.27a Conjugation-Plasmid Transfer Internal Structures cont d-ribosomes 6. Ribosomes: A. 2 subunits: protein & RNA i. Prokaryotic size: ii. Euk: B. attach to subunit of size 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 49 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 50 Internal Structures cont d-inclusions 7. Inclusions: B.. Review Structures: Eukaryotes Eukaryotes If cell wall A. Algae: B. Fungi: 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 51 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 52 Ch 3 & 4 Microscopy & Cell Components 13

14 Endosymbiotic Theory Endosymbiotic Theory 1. Eukaryotes evolved from living inside Table 10.2 Prokaryotic Cells vs. Eukaryotic Organelles 2. Evidence B.. C.. i.. ii.. iii.. 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 53 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 54 Fig 10.2 Endosymbiotic Theory 2/4/2013 Ch 3 & 4 Microscopy & Cell Componenets 55 Ch 3 & 4 Microscopy & Cell Components 14