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 (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
 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
ocular
further magnif. by ocular
objective
forms magnified real image
enters objective object
focuses light on object condenser
light enters 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 diffracted
 occludes direct light, light
passes wide angle light
 angle too wide to enter
objective
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 Primary

procedure
staining

Gram positive –
violet
Gram negative –
red Decolorisation

Counter
staining
Acid fast stain /
Ziehl Neelsen
Stain
 Albert’s stain
For C. Diphtheria
Granules black
Body green