This is a series of lectures on microbiology useful for undergraduate medical and paramedical students

1. Fundamental
Techniques in
Micr obiolog y
Micr oscopy and Staining
Dr. Ashish V. Jawarkar (M.D.)
Parul Sevashram Hospital

2. Fundamental Techniques
Microscopy
Staining
Aseptic technique
Sterilization and waste disposal

3. Microscopy
Measurement
 Microorganisms
are very small
 Use metric system
 Metre (m) : standard unit
 Micrometre (µm) = 1 x10-6 m
 Nanometre (nm) = 1 x10-9 m
 Angstrom (Å) = 1 x10-10 m

4. 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 µm

5. Types of Microscopes
Simple: one lens
Compound:
more than one
lens

6. 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

7. Objectives
10X
Scanning
object
Find the
(Course focus)
40X
High-Dry
(Fine the object
Focus focus)
100X
Oil immersion Fine focus

8. 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 µm at best; can
recognize cells but not fine details
Needs contrast. Easiest way to view cells is
to fix and stain.

9. Different magnifications

10. Special Microscopy
Applications
Dark Field
Phase Contrast
Fluorescence
Electron Microscope

11. Dark Field Microscopy
special condenser
diaphragm
 occludes
direct light,
passes wide angle light
 angle too wide to enter
objective
diffracted light scattered
enters objective
objects light on dark background
diffracted
light

12. Phase Contrast Microscopy
light rays through objects of different η → 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

13. 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

14. Electron Microscopy

16. Stains and Staining
Staining produces contrast

17. Types of stains
Simple stains
Negative staining
Differential staining
Special stains

18. Simple stains
Simple stain
 Aqueous
or alcohol solution of single basic dye
 Stains bacteria
 Background unstained

20. Simple Stains

21. Negative staining
background is stained, leaving the actual
specimen untouched

22. Differential Stains
Stains both background
and bacteria

23. Differential Stains
Acid-fast stain
 Used
to detect Mycobacterium species

24. Special Stains
Capsule stain

Klebsiella pneumonia

25. Special Stains
Flagella stain

26. Gram
stain
procedure
Gram positive –
violet
Gram negative –
red
Primary
staining
Decolorisation
Counter
staining

27. Acid fast stain /
Ziehl Neelsen
Stain

29. Albert’s stain
For C. Diphtheria
Granules black
Body green