Fundamental Techniques in Microbiology

Fundamental Techniques in Microbiology
Presentation lecture by:Dr Paul D. Brown

Introductory Biochemistry
Fundamental Techniques
* Microscopy
* Staining
* Aseptic technique
* Sterilization and waste disposal
* Media preparation

Microscopy
* Measurement
o Microorganisms are very small
o Use metric system
o Metre (m) : standard unit
o Micrometre = 1 x10-6 m
o Nanometre = 1 x10-9 m
o Angstrom = 1 x10-10 m

Terms Relevant to Microscopy
* Total Magnification
o Eyepiece x objective lens
* Resolution
o Ability of the lens to distinguish two points as separate
o Optimal RP achieved with blue light
o Theoretical limit for light microscope is 0.2 m
* Refractive Index (η)
o Measurement of relative velocity at which light passes through a material.
o η= 1.0 in air
o η (Oil) = η (glass) = up to 1.5
Resolving Power
Transmission electron microscope
Scanning electron microscope
Light microscope
Human eye
R.P. in Angstroms
Resolving Power
Optical Instrument

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

The Compound Microscope
* enters the eye
o sees virtual, inverted image
* further magnif. by ocular
* forms magnified real image
* enters objective
* focuses light on object
* light enters condenser

Objectives

* 10X Scanning Find the object
* 40X High-Dry Focus the object
* 100X Oil immersion Fine focus

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

Use of Immersion Oil
Condenser Iris Diaphragm
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.

Different magnifications
Special Microscopy Applications
* Dark Field
* Phase Contrast
* Fluorescence
* Electron Microscope
* special condenser diaphragm
o occludes direct light, passes wide angle light
o angle too wide to enter objective
Phase Contrast Microscopy
Fluorescence Microscopy
Electron Microscopy
Stains and Staining
Simple Stains
Differential Stains
* Gram stain
o Crystal violet: primary stain
o Iodine: mordant
o Alcohol or acetone-alcohol: decolourizer
o Safranin: counterstain
o Gram positive: purple
o Gram negative: pink-red

Staphylococcus aureus
Escherichia coli
Gram stain – distinguishes Gram+ from Gram -
* Acid-fast stain
Special Stains
* Capsule stain
* Flagella stain
* Spore stain (Schaeffer-Fulton)

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

Streak plate method of isolation
Sterilization and Waste disposal
Culture media formulation
Types of media

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

* Selective
o Favours one organism and limits growth of others
o Lacks some factor(s)
+ E.g., fixed N, to select for N2-fixing bacteria
o Selective toxicity
+ E.g., bile salts to select for Enterobacteriaceae
* Selective via incubation conditions
o E.g., gas composition (e.g., N2, 5% CO2, O2), temperature
* Differential
o Different bacteria/groups give different responses
o E.g., MacConkey agar: has lactose + peptone + indicator (neutral red)
+ lactose fermenters - acid - pink colour
+ non-lactose fermenters use peptone - neutral or alkaline - colourless

Enrichment Techniques

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

Fundamental Techniques in Microbiology.ppt