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Microbiology:
It is the science that deals with
the study of micro-organisms (very
small organisms) that are invisible
to the naked eye
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Experiments and procedures involving
handling of living pathogenic micro-
organisms.
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Handling and examining cultures of
microorganisms.
→ good aseptic technique
(not contaminated/ not inadvertently dispersed)
even if the microorganisms being studied are
not considered to be pathogenic.
Any culture of any organism should be handled
with respect for its potential pathogenicity. 4
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Sterilization:
Killing or removal of all living microorganisms (from a particular location or
material).
Sterile article: completely free of all
living micro-organisms
Disinfection:
Destruction of vegetative conspiring
micro-organisms.
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Disinfectants:
Chemicals which cause disinfection
Bacterial spores, mycobacteria, some
viruses → considerable resistance
Antiseptics:
Disinfectants which can be safely applied to
skin & mucous membranes.
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Contamination:
Introduction of undesirable m.o.
Asepsis:
Processes designed to prevent m.o. from
reaching a protected environment.
Aseptic technique: Practices used by
microbiologists to exclude all organisms from
contaminating media or contacting living
tissues.
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Discard cultures and other infectious
materials:
 Petri dishes→ Plastic bag → Autoclave.
 Test tube cultures → wire basket → Autoclave.
 Used pipettes → Jar containing a disinfectant →
Plastic bag → Autoclave
 Used slides, covers → Jar containing a
disinfectant
 Broken glass → swept in a dustpan → container
for broken glass.
NEVER place contaminated material in waste basket.
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Broken or spilled living cultures:
 Clothing → Autoclave plastic bag →
Autoclave.
 Flood the area with
a disinfectant ( or
paper towels are placed over the spills).
 After
20- 30min→ wipe up & discard the
waste in autoclavable dustpan→ Autoclave.
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☠ Lab coat & marker.
☠ No eating, drinking, or
mouth pipetting.
☠ Benches disinfection.
☠ Inoculating loop
sterilization.
☠ Aseptic technique.
☠ Discarded cultures &
infectious materials.
☠ Broken or spilled
living cultures.
☠ Microscope.
☠ Laboratory notes.
☠ At the end of each lab
check:
Gas tap is turned off.
Water tap is closed
properly.
Microscope lamp
is turned off.
☠ Finally wash your
hands thoroughly.
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A Culture medium:❊ An artificial preparation which contains
the essential elements and nutrients
needed by the m.o
to grow. (most bacteria &fungi)
❊ It may be:
• Liquid (broth)
• Solid (containing agar)
• Semisolid (containing low conc of agar)
Strict intracellular organisms (e.g., some
bacteria & all viruses)→ only cultures of
living eukaryotic cells.
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❊
Inoculation:
Culturing of sterile media with m.o
[Inoculation loop].
Incubation:
Placing the culture into the incubator at
optimum temperature for growth.
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Growth:
Multiplication (↑number) to quantities
sufficient to be seen by naked eye..
Bacterial growth in the lab has 2 main
forms:
1- Development of Colonies ( the
macroscopic products of 20-30 cell divisions of
a single bacterium on solid media)
2- Turbidity (macroscopic clumps) of a clear
fluid medium.
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Bacterial Growth
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1- Macroscopical Examination
(colony morphology):
• Characters of colonies.
• Hemolysis on blood agar.
• Pigment production.
2- Microscopical Examination:
• Examination of wet mount preparation.
• Examination of stained preparation.
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3-Biochemical Tests:
(The ability to attack various substances
e.g., carbohydrate breakdown;
or to produce particular metabolic products
e.g., enzymes.
4-Additional Tests:
such as seriological tests
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Colony vs. Cell
Colonies morphology
(Macroscopical examination)
Cells
(Microcopical
examination)
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Colony vs. Cell
Colonies morphology
(Macroscopical
examination)
Cells
(Microcopical
examination)
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The microscope
It is the most important
tool used for examination
and identification of
microorganisms.
It produces magnified
images of microorganisms.
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Types of microscopes
1- Light microscope:


Use light beams and lenses
The most common one used in the lab

Max. magnification is about 900-1000
times.
2- Electron microscope:

Use electron beams and magnetic fields

Magnification power is 100.000
times or
more.

Used for examination of viruses and sections
of bacteria
The microscope
The light microscope
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The electron microscopy
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Light microscope vs. Electronic
microscope
light microscope
Electron microscope
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Light microscope vs. Electronic
microscope
Light microscope
Electron microscope
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Light microscope vs. Electronic
microscope
Light microscope
Electron microscope
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Electron microscope
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The Light Microscope
Types:
a- Simple microscope: single system of
lenses
b- Compound microscope: has two lens
system, the ocular lens and the objective
lens
The two lenses system give greater
magnification
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Components of the compound
microscope
Ocular lens
Objective lens
Stage
Iris diaphram
Lamp box
Slide movement knob
Fine adjustment
Coarse
adjustment
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Theoretical principles of the light
microscope
Magnification
Resolution
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Theoretical principles of the light
microscope
Magnification:
It means enlargement of the linear
diameter of an object.
It is the function of two lens system
[the ocular and the objective lens].
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Total magnification =
mag. power of ocular lens
× mag. power of objective lens used
The magnification power of ocular lens (eye
piece) is 10×
1.
2.
3.
4.
The magnification power of objective lens
Objective lens
mag. Power
Scanning lens
4×
Low power objective lens
10×
High power objective lens
40×
Oil immersion lens
100×
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The total magnification will be:
40 times → when using the scanning lens
100 times → when using the low power lens
400 times → when using the high power lens
1000 times → when using the oil immersion lens
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Working distance:
It is the distance between the objective
lens and the slide.
General rule:
As the magnification of the lens increase
the working distance decrease.
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Resolution:
It is the ability of a lens to reveal
two closely adjacent points as
separate and distinct points.
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Resolution
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Resolving power =
0.61× wavelength of light used
--------------------------------------------numerical aperture.
Numerical aperture = n sin θ
n = refractive index of the medium through
which light passes before entering the
objective lens.
θ = an angle equal to half the angle of the
maximum cone of light that may enter the
objective.
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The max. resolution power of the
compound light microscope with the oil
immersion lens is 0.2
µm
(blue filter, the condenser at the highest
position, oil immersion lens).
(0.001
µm
→ for the electron microscope)
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The resolving power of the oil immersion lens
depends on the addition of special oil (Ceder
wood oil) between the specimen slide and the
objective lens.
Saved light
Objective
lens
Lost light
oil
specimen
Light source
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Uses of the light microscope
1. Identification of microbial groups.
(Bacterial, Fungi, Protozoa)
2. Morphological studies of m.o
(size, shape, arrangement…..)
3. Physiological studies
(motility and reproduction….)
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Care of the microscope
Transport: Use both hands.
Uncluttered workstation.
Avoid Electrical cord dangling → foot
entanglement.
Dust cover.
Lens care: Cleaning lens tissue
moistened with Xylene,
 Occasional wiping off the top surface of
the condenser with lens tissue.
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Examination of living bacteria for motility
(Hanging – drop technique)
For non-pathogenic bacteria:
Direct observation of a drop from a liquid
containing bacteria → an excellent method of
studying motility
e.g. Hanging
– drop technique
In stained slide preparation the cells are heatkilled prior to staining→ the motility in not
observable.
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True motility:- it is the active directional
movement of the organism from place to
place.
(several times the long dimension of the
organism
& In different directions)
Brownian movement:- is a vibratory
nondirectional movement of the cells due to
their bombardment by water molecules in the
suspension
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Materials:
Culture of Proteus vulgaris
 Slide, coverslip.
 Plasticine.
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