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Fundamental Techniques in Microbiology
Dr Paul D. [email protected]
BC10M: Introductory Biochemistry
Fundamental Techniques
Microscopy
Staining
Aseptic technique
Sterilization and waste disposal
Media preparation
Microscopy
MeasurementMicroorganisms are very smallUse metric systemMetre (m) : standard unitMicrometre (m) = 1 x10-6 mNanometre (nm) = 1 x10-9 mAngstrom (Å) = 1 x10-10 m
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
Resolving PowerOptical Instrument Resolving
PowerR.P. in
Angstroms
Human eye 0.2 mm 2,000,000 Å
Light microscope 0.2 m 2000 Å
Scanning electron microscope
5-10 nm 50-100 Å
Transmission electron microscope
0.5 nm 5 Å
Types of Microscopes
Simple: one lensCompound: more than one lens
The Compound MicroscopeREAD 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
Objectives
10X Scanning Find the object
40X High-Dry Focus the object
100X Oil immersion Fine focus
(Course focus)
(Fine focus)
The Condenser
FunctionsFocus light on object planeEnsure adequate intensity
Height of condenser controlsUniformity of brightnessContrast (minimises “stray light”) (Indirectly) angle of light entering objective
Condenser Height
correct too low
stray light
area . 1 . intensity
angle
Use of Immersion Oil
R decreased NA = sin
> 41 , raysreflected inside glass (critical angle)max NA = 0.65
critical angle = 90, wide rays enter objectivemax NA = 1.5
= 1, Air
= 1.5, Immersion oil
NAR
5.0
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
Bright-field MicroscopeContains two lens systems for magnifying specimensSpecimens illuminated directly from above or belowAdvantages: convenient, relatively inexpensive, availableDisadvantages: R.P 0.2 m at best; can recognize cells but not fine detailsNeeds contrast. Easiest way to view cells is to fix and stain.
Different magnifications
Special Microscopy Applications
Dark Field
Phase Contrast
Fluorescence
Electron Microscope
Dark Field Microscopy
special condenser diaphragmoccludes direct light,
passes wide angle lightangle too wide to enter
objective
diffracted light
diffracted light scatteredenters objectiveobjects light on dark background
Phase Contrast Microscopylight rays through objects of different change in phase, not intensity special ring-shaped condenser diaphragmspecial glass disc in objective change phase differences to intensity differences can view transparent
objects as dark on lightbackground (without staining)
Right; human brain glialcells
Fluorescence MicroscopyIlluminate 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
Electron Microscopy
Stains and Staining
Bacteria are slightly negatively charged at pH 7.0Basic dye stains bacteria Acidic dye stains background
Simple stainAqueous or alcohol solution of single basic dye
Simple Stains
Differential Stains
Gram stainCrystal violet: primary stain Iodine: mordantAlcohol or acetone-alcohol:
decolourizerSafranin: counterstainGram positive: purpleGram negative: pink-red
Staphylococcus aureusStaphylococcus aureus
Escherichia coliEscherichia coli
Gram stain – distinguishes Gram+ from Gram -Gram stain – distinguishes Gram+ from Gram -
Gm(+) and Gm(-) both take up CV-I equivalentlyGm(+) and Gm(-) both take up CV-I equivalently
CV-I is not readily removed from Gm(+) due to the reduced CV-I is not readily removed from Gm(+) due to the reduced porosity of the thick cell wallporosity of the thick cell wall
CV-I is readily removed from Gm(-) thin peptidoglycan due CV-I is readily removed from Gm(-) thin peptidoglycan due perhaps to the discontinuities in the outer membrane perhaps to the discontinuities in the outer membrane structure introduced during the decolorization step.structure introduced during the decolorization step.
-removal of the cell wall (with lysozyme) from a Gm(+) -removal of the cell wall (with lysozyme) from a Gm(+) bacterium results in a Gm(-) stain profilebacterium results in a Gm(-) stain profile
Differential Stains
Acid-fast stainUsed to detect Mycobacterium species
Special Stains
Capsule stain Klebsiella pneumonia
Special Stains
Flagella stain
Special Stains
Spore stain (Schaeffer-Fulton)
Bacillus subtilis
Aseptic Technique
First requirement for study of microbespure cultures, free of other microbes
Maintain a clean environment; work close to the flame
Streak plate method of isolation
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 m or 0.22 m filters)Radiation (Gamma, UV, Ionizing)Other methods
Culture media formulationC & energy source (e.g., glucose)
N source (organic or NH4+ or NO3¯ )
minerals (macronutrients, micronutrients)Macronutrients
C, H, O, N, P, S - majorK, Ca, Mg, Fe - minor (as cations)
Micronutrients (trace elements)Mn, Zn, Co, Mo Ni, Cu
(growth factors, vitamins)(agar)
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
Types of mediaSelectiveFavours one organism and limits growth of othersLacks some factor(s)
E.g., fixed N, to select for N2-fixing bacteria
Selective toxicityE.g., bile salts to select for Enterobacteriaceae
Selective via incubation conditionsE.g., gas composition (e.g., N2, 5% CO2, O2),
temperature
Types of media
DifferentialDifferent bacteria/groups give different responsesE.g., MacConkey agar: has lactose + peptone +
indicator (neutral red) lactose fermenters acid pink colournon-lactose fermenters use peptone neutral or
alkaline colourless
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