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Fundamental Techniques inMicrobiologyMicroscopy and Staining

Dr. Ashish V. Jawarkar (M.D.)

Parul Sevashram Hospital

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Fundamental Techniques

Microscopy

Staining

Aseptic techniqueSterilization and waste disposal

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Microscopy

Measurement Microorganisms are very small

Use metric system

Metre (m) : standard unit

Micrometre (m) = 1 x10-6 m

Nanometre (nm) = 1 x10-9 mAngstrom () = 1 x10-10 m

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Terms Relevant to Microscopy

Total Magnification Eyepiece x objective lens

Resolution

Ability of the lens to distinguish two points asseparate

Theoretical limit for light microscope is 0.2 m

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Types of Microscopes

Simple: one lensCompound:more than onelens

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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

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Objectives

10X Scanning Find the object

40X High-Dry Focus the object

100X Oil immersion Fine focus

(Course focus)

(Fine focus)

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Bright-field Microscope

Contains two lens systems for magnifyingspecimensSpecimens illuminated directly from aboveor below

Advantages: convenient, relativelyinexpensive, availableDisadvantages: R.P 0.2 m at best; can

recognize cells but not fine detailsNeeds contrast. Easiest way to view cells isto fix and stain.

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Different magnifications

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Special Microscopy

ApplicationsDark Field

Phase Con trast

Fluorescence

Elect ron Microscope

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Dark Field Microscopy

special condenserdiaphragm occludes direct light,

passes wide angle light angle too wide to enter

objective

diffractedlight

diffracted light scattered

enters objective

objects light on dark background

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Phase Contrast Microscopylight rays through objects of different changein phase, not intensityspecial 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 glial

cells

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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 orgreenish light

Images appear coloured against a dark background

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Electron Microscopy

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http://www.lyon.edu/webdata/users/dthomas/microbiology/labweb/microtelescope.jpg

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Stains and Staining

Staining produces contrast

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Types of stains

Simple stains

Negative staining

Differential stainingSpecial stains

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Simple stains

Simple stainAqueous or alcohol solution of single basic dye

Stains bacteria

Background unstained

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Simple Stains

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Negative staining

background is stained, leaving the actualspecimen untouched

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Differential Stains

Stains both backgroundand bacteria

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Differential Stains

Acid-fast stain Used to detect Mycobacterium species

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Special Stains

Capsule stain Klebsiel la pneumon ia

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Special Stains

Flagella stain

Gram

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Gram

stain

procedureGram positive violet

Gram negative red

Primary

staining

Decolorisation

Counter

staining

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Acid fast stain /

Ziehl NeelsenStain

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Alberts stain

For C. Diphtheria

Granules black

Body green