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Fundamental Techniques in Microbiology

Microscopy and Staining

Dr. Ashish V. Jawarkar (M.D.) Parul Sevashram Hospital

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

Terms Relevant to Microscopy


Total Magnification

Eyepiece x objective lens

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

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

Objectives
10X Scanning Find the object
(Course focus)

40X

High-Dry

Focus the object


(Fine focus)

100X

Oil immersion Fine focus

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

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

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

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

Stains and Staining


Staining produces contrast

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

Used to detect Mycobacterium species

Special Stains
Capsule stain

Klebsiella pneumonia

Special Stains
Flagella stain

Gram stain procedure

Primary staining

Gram positive violet Gram negative red

Decolorisation

Counter staining

Acid fast stain / Ziehl Neelsen Stain

Alberts stain
For C. Diphtheria Granules black Body green