Professional Documents
Culture Documents
Fundamental Techniques
Microscopy Staining Aseptic technique Sterilization and waste disposal
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 () = 1 x10-10 m
Resolution
Ability of the lens to distinguish two points as separate Theoretical limit for light microscope is 0.2 m
Types of Microscopes
Simple: one lens Compound: more than one lens
further magnif. by ocular forms magnified real image enters objective focuses light on object light enters condenser
Objectives
10X Scanning Find the object
(Course focus)
40X
High-Dry
100X
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
diffracted light
change phase differences to intensity differences can view transparent objects as dark on light background (without staining)
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
Electron Microscopy
Types of stains
Simple stains Negative staining Differential staining Special stains
Simple stains
Simple stain
Aqueous or alcohol solution of single basic dye Stains bacteria Background unstained
Simple Stains
Negative staining
background is stained, leaving the actual specimen untouched
Differential Stains
Stains both background and bacteria
Differential Stains
Acid-fast stain
Special Stains
Capsule stain
Klebsiella pneumonia
Special Stains
Flagella stain
Primary staining
Decolorisation
Counter staining
Alberts stain
For C. Diphtheria Granules black Body green