Microbiology
Definition:
Microbiology is the science that deals with microorganisms that can not be seen
by the
naked eye
.
The Importance
of
Microbiology
:
1
-
The Environment
:
Microbes
are responsible for the cycling of carbon, nitrogen and
phosphorus
.
2
-
Medicine:
Production
of antibiotics.
3
-
Food:
Wine
and bread making, cheese and milk products production
.
4
-
Biotechnology:
Some
microorganisms have been used to synthesize many chemicals
such
as acetone and acetic acid.
5
-
Research:
Microbes
have been used as
model
organisms for the biochemical and
medical
investigations
.
What
is a Microbe?
A microbe or microorganism is:
A
-
A living creature which can not be seen except by microscope.
B
-
It is simple in structure.
C
-
Usually unicellular.
D
-
It can be either prokaryotic cell or a eukaryotic cell.
Microbiology
Nature
Range of size
Microbe
Cellular
20
-
300 nm
Viruses
Prokaryote
0.1
-
10
µm
Bacteria
Eukaryote
2 µm
-
1 meter
Fungi
Eukaryote
2
-
1000
µm
Protozoa
Prokaryotes and
Eukaryotes:
M
icrobes
Prokaryotes
Eukaryotes
Cell Structure
Simple structure
Complex structure
Nucleus
Primitive nucleus
True nucleus
Nuclear Membrane
Absent
Present
Nucleoli
Absent
Present
Mitochondria
Absent
Present
Enzyme System
Simple
Complex
Multiplication
Binary Fission
Mitosis
Examples
Bacteria, Rickettsia
Fungi, Protozoa
Microscopy:
It is the technical field of using a
microscope
to view
samples and objects
that can
not
be seen by unaided eye ( objects that aren't within the resolution range of normal
eye).
A
microscope
is the most
important
piece of
equipment
used
in clinical
microbiology.
Microscopy
forms 70
-
90% of
the
work
.
Microscopy
Working principle of a microscope:
A microscope is a magnifying instrument.
The magnified image of the object (specimen) is first produced by a lens close to
the object called
the
objective lens
.
Objective lens magnification = 4 X to 100 X
A
second lens near the eye called the
eyepiece
enlarges the primary image,
converting it into one that can enter the pupil of the eye
.
Eyepiece
magnification =
8 X to 12 X typically.
(but 10 X is most common)
Total
magnification = objective magnification* eyepiece magnification
(4, 10, 40, 100 X) * ( 10 X
)
So, final magnification ranges from 40 X up to 1000 X
Microscopy
Resolution of Microscope:
It is the ability to distinguish two
very small
and
very closed
objects as
separate
entities.
Resolution is
best
when the distance separating the two tiny objects is
small.
So, Resolution can be defined in a different way as:
It is the
smallest
distance that separate two sources of light points reflected from two
particles close together on object.
Resolution is determined by certain physical parameters that include the
wavelength
of light
, and the
light
-
gathering power
of the objective & condenser lenses.
Resolution
= light wavelength / 2
Example:
yellow light of a wavelength of 0.4
μm
give a resolution of
0.2
μm
.
Microscopy
Types
of microscopy:
1
-
The light microscope: (simple and compound).
A
-
The
student
(
Brightfield
) microscope
B
-
Phase contrast microscope.
C
-
Darkfield
illumination microscope.
2
-
The Fluorescence microscope
.
3
-
The Electron microscope
.
1
-
The Light Microscope:
-
Brieghtfield
microscope
is most likely found in classrooms or labs.
-
Better equipped classrooms and labs have
darkfield
&/or
phase contrast microscope
.
Microscopy
A
-
The
student
microscope:
Image quality is based largely on your ability to use the microscope properly.
Uses:
Brightfield
illumination microscope is mainly used to examine
stained
smear
Or
naturally
pigmented specimens.
Microscopy
Light path consists of:
Transillumination
light source
, commonly a halogen lamp in the
microscope
stand.
Condenser lens
which focuses light from the light source onto the sample.
Objective lens
which collects the light from the sample and magnifies the image.
Oculars
and /or a camera to view the sample image.
Objects seen in the light path because:
-
Natural pigmentations or stains absorb light differentially.
Or
-
They are thick enough to absorb a significant amount of light despite
being colorless.
Microscopy
B
-
Phase contrast microscope
:
This is needed to visualize
transparent
microorganisms
suspended
in a fluid.
(does
not
require staining to view the slide)
The phase microscope takes advantage of the fact that light waves passing
through
transparent slide
or objects (cells) emerge in different phases (different times).
A special optical system
converts
difference in phase into difference
in intensity. So that some
structure appear darker
than others
.
This microscope made it possible to
study
cell cycle.
Microscopy
C
-
Dark field illumination microscope
:
This method is used for visualizing organisms
suspended in fluid
and can
not
be
stained by Gram’s stain.
Both the
structure
and the
motility
of the organisms can be seen
.
Used for
: Unstained biological samples such as a smear from a tissue culture.
In this
method:
the light enters the special
condenser which
has a central
blacked
-
out
area
, so that light can not
pass
directly through it to
enter
the objective
.
Instead,
the light is reflected to pass
through the outer rim of the
condenser at a wide angle which
illuminates the microorganism
by a ring of light surrounding them.
Microscopy
Micrcoccus
species
examined by
phase contrast microscope
(
left
) and
Leptospira
examined by
Darkfield
microscope
(
Right
):
Microscopy
2
-
The
Fluorescence Microscope:
In this method, microorganisms must be stained with a
fluorescent dye
such
as
rhodamine
.
A fluorescent lamp emits visible light which is filtered off
using
optical
filters
.
(Ultraviolet (UV) wavelengths will not be filtered off).
A fluorescent dye will absorb this UV
-
light and change it to visible (yellow or
orange)
light
.
Microscopy
3
-
The
Electron Microscope:
-
Electron
microscope is an instrument that
magnify very small
objects which can
not be seen by light microscopy.
-
Resolution
of this microscope is higher than the light one.
-
The
energy source of this kind is the
electron beam
while light waves are used in
the light microscope.
-
Electromagnetic
lenses
are used instead of the glass lenses of light microscope
.
-
Visible
light of 500 nm wavelength give a resolution of 250 nm.
Electron
beam of 0.001 nm wavelength give a resolution of 0.0005 nm.
-
Viruses
with a diameter of 0.01
-
0.2 micrometer can be easily seen
.
-
Magnification of electron microscope is up to about 10.000.000 X .
Microscopy
Microscopy
Microscopy
Light waves and Electron beam:
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