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Fundamental Techniques in Microbiology Dr Paul D. Brown BC10M: Introductory Biochemistry.

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Presentation on theme: "Fundamental Techniques in Microbiology Dr Paul D. Brown BC10M: Introductory Biochemistry."— Presentation transcript:

1 Fundamental Techniques in Microbiology Dr Paul D. Brown 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 x 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 Optical InstrumentResolving Power R.P. in Angstroms Human eye0.2 mm2,000,000 Å Light microscope 0.2 m 2000 Å Scanning electron microscope 5-10 nm Å Transmission electron microscope 0.5 nm5 Å

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

7 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 ocular objective object condenser

8 Objectives 10XScanning Find the object 40XHigh-Dry Focus the object 100XOil 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 correct too low stray light area. 1. intensity angle

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

12 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 aperture diaphragm

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 Dark Field Microscopy special condenser diaphragm occludes direct light, passes wide angle light angle too wide to enter objective diffracted light 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 nm & emit orange, yellow or greenish light Images appear coloured against a dark background

19 Electron Microscopy


21 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

22 Simple Stains

23 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

24 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

25 Differential Stains Acid-fast stain Used to detect Mycobacterium species

26 Special Stains Capsule stain Klebsiella pneumonia

27 Special Stains Flagella stain

28 Special Stains Spore stain (Schaeffer-Fulton) Bacillus subtilis

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

30 Streak plate method of isolation

31 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 (121 o C) Filtration (0.45 m or 0.22 m filters) Radiation (Gamma, UV, Ionizing) Other methods

32 Culture media formulation C & energy source (e.g., glucose) N source (organic or NH 4 + or NO 3 ¯ ) 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)

33 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 + (NH 4 ) 2 SO 4 + minerals for E. coli

34 Types of media Selective Favours one organism and limits growth of others Lacks some factor(s) E.g., fixed N, to select for N 2 -fixing bacteria Selective toxicity E.g., bile salts to select for Enterobacteriaceae Selective via incubation conditions E.g., gas composition (e.g., N 2, 5% CO 2, O 2 ), temperature

35 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

36 Enrichment Techniques Increase proportion of desired physiological class E.g., N 2 -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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