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Fundamental Techniques in Microbiology

Fundamental Techniques in Microbiology. Dr Paul D. Brown paul.brown@uwimona.edu.jm BC10M: Introductory Biochemistry. Fundamental Techniques. Microscopy Staining Aseptic technique Sterilization and waste disposal Media preparation. Microscopy. Measurement Microorganisms are very small

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Fundamental Techniques in Microbiology

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  1. Fundamental Techniques in Microbiology Dr Paul D. Brown paul.brown@uwimona.edu.jm BC10M: Introductory Biochemistry

  2. Fundamental Techniques • Microscopy • Staining • Aseptic technique • Sterilization and waste disposal • Media preparation

  3. Microscopy • Measurement • Microorganisms are very small • Use metric system • Metre (m) : standard unit • Micrometre (m) = 1 x10-6 m • Nanometre (nm) = 1 x10-9 m • Angstrom (Å) = 1 x10-10 m

  4. Terms Relevant to Microscopy • Total Magnification • Eyepiece x objective lens • Resolution • Ability of the lens to distinguish two points as separate • Optimal RP achieved with blue light • Theoretical limit for light microscope is 0.2 m • Refractive Index (η) • Measurement of relative velocity at which light passes through a material. • η= 1.0 in air • η (Oil) = η (glass) = up to 1.5

  5. Resolving Power

  6. Types of Microscopes • Simple: one lens • Compound: more than one lens

  7. ocular objective object condenser The Compound Microscope READ BOTTOM TO TOP! • enters the eye • sees virtual, inverted image • further magnif. by ocular • forms magnified real image • enters objective • focuses light on object • light enters condenser

  8. Objectives • 10X Scanning Find the object • 40X High-Dry Focus the object • 100X Oil immersion Fine focus (Course focus) (Fine focus)

  9. The Condenser • Functions • Focus light on object plane • Ensure adequate intensity • Height of condenser controls • Uniformity of brightness • Contrast (minimises “stray light”) • (Indirectly) angle of light entering objective

  10. Condenser Height stray light area  . 1 . intensity angle correct too low

  11. = 1, Air = 1.5, Immersion oil    > 41 , rays reflected inside glass (critical angle) max NA = 0.65 critical angle = 90, wide rays enter objective max NA = 1.5 Use of Immersion Oil R decreased •   •   • NA =  sin 

  12.  diaphragm aperture Condenser Iris Diaphragm • wide aperture  large , large NA, low R (good resolution), poor contrast (if too wide) • narrow aperture  small , small NA, high R (poor resolution), good contrast

  13. Bright-field Microscope • Contains two lens systems for magnifying specimens • Specimens illuminated directly from above or below • Advantages: convenient, relatively inexpensive, available • Disadvantages: R.P 0.2 m at best; can recognize cells but not fine details • Needs contrast. Easiest way to view cells is to fix and stain.

  14. Different magnifications

  15. Special Microscopy Applications • Dark Field • Phase Contrast • Fluorescence • Electron Microscope

  16. special condenser diaphragm occludes direct light, passes wide angle light angle too wide to enter objective diffracted light Dark Field Microscopy • diffracted light scattered • enters objective • objects light on dark background

  17. Phase Contrast Microscopy • light rays through objects of different   change in phase, not intensity • special ring-shaped condenser diaphragm • special glass disc in objective • change phase differences to intensity differences • can view transparent objects as dark on light background (without staining) • Right; human brain glial cells

  18. Fluorescence Microscopy • Illuminate specimen with UV  visible fluorescence (filter removes harmful UV) • View auto-fluorescent objects (e.g., chloroplasts) • Stain with specific fluorescent dyes, which absorb in region 230-350 nm & emit orange, yellow or greenish light • Images appear coloured against a dark background

  19. Electron Microscopy

  20. Stains and Staining • Bacteria are slightly negatively charged at pH 7.0 • Basic dye stains bacteria • Acidic dye stains background • Simple stain • Aqueous or alcohol solution of single basic dye

  21. Simple Stains

  22. Differential Stains • Gram stain • Crystal violet: primary stain • Iodine: mordant • Alcohol or acetone-alcohol: decolourizer • Safranin: counterstain • Gram positive: purple • Gram negative: pink-red Staphylococcus aureus Escherichia coli

  23. Gram stain – distinguishes Gram+ from Gram - Gm(+) and Gm(-) both take up CV-I equivalently CV-I is not readily removed from Gm(+) due to the reduced porosity of the thick cell wall CV-I is readily removed from Gm(-) thin peptidoglycan due perhaps to the discontinuities in the outer membrane structure introduced during the decolorization step. -removal of the cell wall (with lysozyme) from a Gm(+) bacterium results in a Gm(-) stain profile

  24. Differential Stains • Acid-fast stain • Used to detect Mycobacterium species

  25. Special Stains • Capsule stain • Klebsiella pneumonia

  26. Special Stains • Flagella stain

  27. Special Stains • Spore stain (Schaeffer-Fulton) Bacillus subtilis

  28. Aseptic Technique • First requirement for study of microbes • pure cultures, free of other microbes • Maintain a clean environment; work close to the flame

  29. Streak plate method of isolation

  30. Sterilization and Waste disposal • Sterilization ensures killing/removal of ALL life forms • Boiling kills most vegetative cells (Bacterial spores unaffected) • Tyndallisation (c.1880): heat, 24hr, heat • Dry heat (very high temperatures) • Moist heat • Autoclave: steam under pressure (121oC) • Filtration (0.45 mm or 0.22 mm filters) • Radiation (Gamma, UV, Ionizing) • Other methods

  31. Culture media formulation • C & energy source (e.g., glucose) • N source (organic or NH4+orNO3¯ ) • minerals (macronutrients, micronutrients) • Macronutrients • C, H, O, N, P, S - major • K, Ca, Mg, Fe - minor (as cations) • Micronutrients (trace elements) • Mn, Zn, Co, Mo Ni, Cu • (growth factors, vitamins) • (agar)

  32. Types of media • General purpose • Allows growth of most bacteria, e.g., Nutrient agar • Includes organic C, N, vitamins • May have undefined components e.g., yeast extract, peptone • Defined • All components are pure compounds, not mixtures such as yeast extract • E.g., glucose + (NH4)2SO4 + minerals for E. coli

  33. Types of media • Selective • Favours one organism and limits growth of others • Lacks some factor(s) • E.g., fixed N, to select for N2-fixing bacteria • Selective toxicity • E.g., bile salts to select for Enterobacteriaceae • Selective via incubation conditions • E.g., gas composition (e.g., N2, 5% CO2, O2), temperature

  34. Types of media • Differential • Different bacteria/groups give different responses • E.g., MacConkey agar: has lactose + peptone + indicator (neutral red) • lactose fermenters  acid  pink colour • non-lactose fermenters use peptone  neutral or alkaline  colourless

  35. Enrichment Techniques • Increase proportion of desired physiological class • E.g., N2-fixers; cellulose-decomposers; photosynthetic bacteria • Culture mixed population in selective medium and/or conditions • E.g., fixed N-free; cellulose as sole carbon, energy source; anaerobic conditions in light, without organic C • Sample treatment • E.g., boil to kill vegetative cells, leaving spores

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