2. INTRODUCTION
Discovery of Microorganisms
Antony van Leeuwenhoek
(1632-1723)
•First person to observe and describe
micro-organisms accurately
Microorganisms are too small to be seen with the naked eye, so
they must be observed with microscope
micro = small, skopein = to see
2
3. INTRODUCTION
The microscope is the instrument most characteristic
of the microbiology laboratory.
The magnification it provides enables us to see
microorganisms and their structures otherwise
invisible to the naked eye.
The magnifications attainable by microscopes range
from X100 to X400000.
Several different kinds of microscopy are available,
and many techniques have been developed.
3
4. Microscopes and Microscopy
Microscopes are of two
categories depending upon
the principle on which
magnification is based:
Light (or optical) and
Electron.
4
5. Light microscopy
Light microscopy, in which magnification is
obtained by a system of optical lenses using light
waves, includes:
1. Bright-field
2. Dark-field
3. Fluorescence and
4. Phase-contrast microscopy
5
6. Electron Microscopy
The Electron Microscope, as the name
suggests, uses a beam of electrons in place of
light waves to produce the image.
Specimens can be examined by either
Transmission or
Scanning electron microscopy.
6
7. Units of Measurement
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
1 nanometer (nm) nano = 1/1,000,000,0000.000000001 m = 10-9 m
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.
7
8. A simple microscope has
only one lens, whereas a
compound microscope
has more than one lens.
Microscopy: The Instruments
8
11. Bright-field Microscopy (Compound
light microscopy)
In bright-field microscopy, the microscopic field
(the area observed) is brightly lighted and
microorganisms appear dark because they absorb
some of the light.
Ordinarily, microorganisms do not absorb much
light, but staining them with dyes greatly
increases their light-absorbing ability, resulting in
greater contrast and color differentiation.
11
12. Dark objects are visible against a bright
background.
Light reflected off the specimen does not enter
the objective lens.
A modern compound light microscope has a
series of lenses and uses visible light as its
source of illumination.
Bright-field Microscopy (Compound
light microscopy)
13. Bright-field Microscopy (Compound light
microscopy)
A series of finely ground lenses forms a clearly focused
image that is many times larger than the specimen itself.
This magnification is achieved when light rays from an
illuminator, the light source, are passed through a
condenser, which has lenses that direct the light rays
through the specimen.
From here, light rays pass into the objective lenses, the
lenses closest to the specimen.
Most lab microscopes are equipped with 4 objectives, each
capable of a different degree of magnification.
These are referred to as the oil-immersion (X100), high-
dry (X40), low-power (X10) and scanning (X4)
objectives.
14. The image of the specimen is magnified again by the
ocular lens, or eyepiece. Generally, an eyepiece having a
magnification of X10 is used, although eyepieces of
higher or lower magnifications are available.
In a compound microscope the image from the objective
lens is magnified again by the ocular lens.
Total magnification = objective lens ocular lens
Microscopy: The Instruments
14
15. 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
17. Lenses and the Bending of Light
Refraction: light is refracted (bent) when passing from one
medium to another
Refractive index: a measure of how greatly a substance
slows the velocity of light. OR
The refractive index is a measure of the light-bending
ability of a medium.
refractive index of specimens can be changed by
staining them.
Working distance: distance between the front surface of
lens and surface of cover glass or specimen
Direction and magnitude of bending is determined by the
refractive indexes of the two media forming the interface.17
18. Focus light rays at a
specific place called the
focal point
distance between center
of lens and focal point is
the focal length
strength of lens related to
focal length
short focal length(f)
more magnification
LENSES
18
19. The basic limitation of bright field microscopy is not
magnification, but resolving power.
Resolving power (RP) is the ability of the lenses to
distinguish fine detail and structure. It refers to the
ability of the lenses to distinguish between two points at
a specific distance apart.
Resolution or RP of a microscope is a function of
the wavelength of light and
the numerical aperture of the lens system.
Resolution, or Resolving power (RP)
19
20. The numerical aperture is a measure of the light that is
collected and directed through the microscope oculars.
The angle subtended ( ortho ja) by the optical axis and the
outermost rays still covered by the objective is the measure of the
aperture of the objective; it is the half -aperture angle.
The magnitude of this angle is expressed as a sine value.
The sine value of the half aperture angle multiplied by the
refractive index, n of the medium filling the space between the
front lens and the cover slip gives the NA.
NA = n sin
Where, n of air is 1 and of immersion oil is 1.56.
Numerical Aperture (NA)
20
22. Numerical Aperture (NA)
• The degree to which microscope objectives can be
altered to increase the NA is limited (maximum NA of
dry objective is less than 1 and oil immersion objective
is greater than 1).
• The wavelength of light used in optical microscope is
also limited (the visible light range is between 400 nm
(blue light) and 700 nm (red light), or 0.4 m to 0.7 m).
• Thus it is apparent that the resolving power of the optical
microscope is restricted by the limiting values of the NA
and the wavelength of visible light.
22
23. Limit of resolution
The limit of resolution is the smallest distance by
which two objects can be separated and still be
distinguishable as two separate objects.
The greatest resolution in light microscopy is
obtained with the shortest wavelength of visible light
and an object with the maximum NA.
The relationship between NA and resolution can be
expressed as follows:
d = /2NA
Where, d=resolution, and = wavelength of light.
23
24. Highest magnification in Bright-field
Microscopes
To achieve high magnification (100X) with good resolution, the
lens must be small.
To preserve the direction of light rays at the highest magnification,
immersion oil is placed between the glass slide and the oil
immersion objective lens.
The immersion oil has the same refractive index as glass, so oil
becomes part of optics of the glass of the microscope.
Unless immersion oil is used, light rays are refracted as they enter
the air from the slide, and the objective lens would have to be
increased in diameter to capture them.
Immersion oil improves the resolving power of the lenses.
If oil is not used with oil immersion objective lens, the image
becomes fuzzy, with poor resolution.
25. Refraction in the compound microscope
using an oil immersion objective lens
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. 25
