Basic Techniques

In Microbiology
Course Teacher Zebun Nahar

Course Code 0511MB113

Microscopes
and Microscopy
 INTRODUCTION
• Microorganisms are too small to be seen with the naked
eye, so they must be observed with microscope
• The word microscope was derived from Latin word micro,
which means small and the Greek word skopos, to look at.
• Microorganism was first discovered by Scientist Antony Van
Leeuwenhoek in 17th century. He discovered them through
his simple singe lens microscope (magnifying lenses). The
highest magnification with his microscope was about 300X
(times).

Fig: Antony Van Leeuwenhoek’s

microscope and some of his drawing
of bacteria in 1683.
 INTRODUCTION (con….)
Units of Measurement
• Because microorganisms and their component parts are very
small, they are measured in units that are unfamiliar to many of
us in everyday life.
• The metric system is generally used and standard unit of length
in metric system is meter.
Table: Metric Units of Length
Metric unit meaning of prefix Metric Equivalent
1 kilometer kilo=1000 1000m = 103 mm
1 meter (m) standard unit of length
1 decimeter (dm) deci = 1/10 0.1m = 10-1 m
1centimeter (cm) centi = 1/100 0.01m = 10-2 m
1millimeter (mm) milli = 1/1000 0.001 m = 10-3 m
1 micrometer (µm) micro = 1/1,000,000 0.000001 = 10-6 m
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Resolution, or Resolving power (RP)
RP is the ability of the lenses to distinguish fine detail and
structure. It refers that it has the ability to distinguish images
of two close objects as separate distinct entities. For e.g. if a
microscope has a resolving power of 0.4 nm, it can
distinguish two points as separate objects if they are at least
0.4 nm apart.
The shorter the wave length of light used in microscope, the
greater the resolution. The white light used in compound
light microscope has a relatively long wave length and
cannot resolve structures smaller than 0.2 micro miter.
Resolution, or Resolving power (RP)
 For two points to be seen as separate the light has to be
able to pass between them
 If the points are very close and the light is not able to pass
between, the image will appear as fuzzy.
 Resolution is determined by the wavelength of light used
in the microscope.
 Total magnification
 The total magnification of the system is
determined by multiplying the objective lens
magnification (power) by the ocular lens
magnification (power).
 Generally, in compound light microscope an
eyepiece (ocular lens) contain a magnification of
10X, although eyepieces of higher or lower
magnifications are available. And several
objective lenses which includes 10X (low power)
40X (high power) and 100X (oil immersion).
 Total magnification (contd..)

Objective Objective Ocular Total

designation magnification Magnification Magnification
Low power x10 X10 X100

High Power x40 X10 X400

Oil Immersion x100 x10 x1000

 Categories of Microscope
 Several different kinds of microscopes are available, and
many techniques have been developed.

1. Light Microscope
a. Bright-field
b. Dark-field
c. Phase-contrast microscopy
d. Differential interference contrast
e. Fluorescence

2. Confocal
 Categories of Microscope
3. Electron

a. Transmission or

b. Scanning electron microscopy.

4. Scanning tunneling

5. Atomic force.
1. Bright-field or compound microscope
 Objects are visible against a bright background.
 A modern compound light microscope has a series
of lenses and uses visible light as its source of
illumination which forms a clearly image that is
many times larger than the specimen itself.
 Light rays pass through the objective lenses, the
lenses closest to the specimen. The image of the
specimen is magnified again by the ocular lens, or
eyepiece.
 Generally, an ocular lens contain a magnification of
10X and several objective lenses (4X, 10X, 40X and
100X )
1. Bright-field or compound microscope
(contd..)

 To achieve the high magnification (100X) with good

resolution immersion oil is placed between the glass
slide and objective lens.
 The immersion oil has the same refractive index as
glass which improves the resolving power of lens.
 Refraction and Refractive index: light is refracted
(bent) when passing from one medium to another
which is Refraction. Refractive index means it’s a
measure of the relative velocity at which light passes
through a material .
Relationship between Oil immersion and
refractive index
Because the refractive
indexes of the glass
microscope slide and
immersion oil are the same,
the light rays do not refract
when passing from one to
the other when the oil
immersion objective lens is
used.

This method produces

images with better resolution
at magnification greater than
100X.
1. Bright-field Microscopes: Distinguishing
Features
 Use visible light as a source of illumination
 Cannot resolve structures smaller than about 0.2µm
 Specimen appears against a bright background
 Bright field illumination shows internal structures and
the outline of the transparent sheath.
 Inexpensive and easy to use.
1. Bright-field Microscopes: Principal Uses

 To observe various stained specimens and to count

microbes.

 Does not resolve very small specimens, such as

viruses.
2. Dark-field Microscopy
A dark-field microscope is used for examining live microbes that either
are invisible in the ordinary light microscope,
cannot be stained by standard methods, or
are so distorted by staining that their characteristics cannot be
identified.
Light objects are visible against a dark background.
Light reflected off the specimen cannot enters the objective lens.
Pathways of light in bright-field and
dark-field microscopy
 2. Dark-field Microscopes: Distinguishing
Features

 Use a special condenser with as opaque disc

that blocks light from entering the objective
lens directly.
 The only light that reaches the specimen
comes in at an angle; thus, only light reflected
by the specimen reaches the objective lens.
 Light reflected by specimen enters the
objective lens.
 Specimen appears light against a black
background.
2. Dark-field Microscopes: Principal Uses

 To examine living microorganisms that are

invisible in bright field microscopy.
 This technique is frequently used to examine
unstained m.os suspended in liquid.
 Frequently used to detect Treponema pallidum
in the diagnosis of syphilis.
3. Phase-Contrast Microscopy
 Phase-contrast microscopy is especially useful because it
permits detailed examination of internal structures in living
microorganisms.
 The principle of phase-contrast microscopy is based on
slight variations in refractive index.
 As rays pass from the light source through the specimen,
their velocity may be altered by differences in the
thickness and physical properties of various portions of
the specimen.
3. Phase-Contrast Microscopy (contd…)

 Light rays passing through the specimen are

diffracted (bent) differently and travel different
pathways to reach the eye of the viewer. These
phase differences are seen through the
microscope as different degrees of brightness.
 Details of the internal structures of the
specimen also become sharply defined in
phase-contrast microscopy.
 3. Phase-Contrast Microscopy:
Distinguishing Features
Use a special condenser containing an annular (ring
shaped) diaphragm
The diaphragm allows direct light to pass through the
condenser, focusing light on the specimen and a
diffraction plate in the objective lens.
Direct and reflected or diffracted light rays are brought
together to produce the image of the specimen on the
ocular lens, containing areas that are relatively light (in
phase), through shades of gray, to black (out of phase).
 3. Phase-Contrast Microscopy:
Principal Uses
 To facilitate detailed examination of the internal
structures of living specimen
4. Differential Interference Contrast (DIC)
Microscopy

DIC microscopy is
similar to phase-
contrast microscopy
in that it uses
differences in
refractive indexes.
 4. Differential Interference Contrast
Microscopy: Distinguishing Features
 Like phase-contrast, uses differences in refractive
indexes to produce images.
 DIC microscope use two beams of light instead of one
separated by prisms. Prisms split each light beam,
adding contrasting colors to the specimen. Therefore,
the resolution of a DIC microscope is higher than that
of a standard phase-contrast microscope.
 The specimen appears colored as a result of the prism
effect. The image is brightly colored and appears
nearly three-dimensional.
 No staining required.
Differential Interference Contrast Microscopy:
Principal Uses
 To provide brightly colored nearly three dimensional
images.
 Excellent way to observe living cells.
Reference

General microbiology: Tortora, Funke, Latest

edition
Madigan et al. (1999) Brock’s Biology of
Microorganisms. 9th Ed
General Microbiology- Pelczar International ed.