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MICROSCOPES
Microscopes
Microscope : an instruments used to
examine very small objects
(Specimens) in microbiology.
Functions :
To allow us to study the morphology of
bacteria (size, shape, arrangement,
motility and staining reaction )
Types of microscopes
1-light microscope
2-Dark field microscope
3-fluorescent microscope
4-Phase contrast microscope
5-Electron microscope
• Light Microscope
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Eye piece
Objective lenses
Stage
Diaphragm
Condenser
Light source
Adjustment knobs
STAINING METHODS
Types of stain
1-Simple stain (one dye is used )
Methylene blue, safranin, crystal violet.
2-differential stains( two dyes are used)
Gram stain ,Ziehl-Neelsen stain.
3-special stains
fontana stain.
4- Fluorescent stained antibody
Antibodies for surface antigens of bacteria carrying
a fluorescent dye
Simple stains
1- preparation of smear:
• Take a loopfull of bacterial
suspension and spread it onto an
area of about 1 cm square
• Leave to dry in air or at the
warmth of a Bunzen flame
• Fix the smear by:
a- passing it into a flame three times
b- dipping into ethyl alcohol
Fixation leads to coagulation of proteins
and fixation on the surface of slide.
Simple stains
2-staining of the smear:
a- Flood the smear with
several drops of a stain like
methylene blue and leave for 2
min.
b- wash the stain gently with
water
c- Dry the smear by leaving it
in air for a few minutes or by
blotting with a filter paper.
Simple stains
3- Examination under
the microscope
• Put a drop of oil onto
the stained smear
• Examine the smear
under the oil immersion
lens
• Describe what you can
see under the
microscope
Chains of bacillui
Chains of beads
Bunches of cocci
Tetrads of cocci
Gram-stain
• Principles:
• Bacteria differ from one another chemically and
physically and may react differently to stains.
• Bacteria are divided into
1- Gram-positive bacteria (violet in colour)
2- And Gram-negative bacteria (red in colour)
Gram-stain:Steps of staining:
• 1- Prepare a bacterial smear and fix it
• 2- Flood with crystal violet (primary
stain) and leave 60 sec. Wash gently with
tap water.
• 3- flood with iodine solution (mordant)
and leave for 2 min and then wash gently
with tap water.
Gram-stain:Steps of staining:
4-Declourize with drop by drop 95% ethanol alcohol
(decolourization of Gram-negative bacteria), till the
washing becomes faint purple (this takes about 20
sec). Wash gently with water.
5- Flood the smear with safranin (counter stain) and
leave for 2 min.
6- Dry the smear by blotting, add a drop of oil and
examine under oil immersion lens
Technique of isolation of
separate colonies of
bacteria
Technique for isolates
• Sterilize the loop by heating to redness
on the top of a flame.
• Take one loop-full of bacteria and
streak the surface of nutrient agar
plate to spread bacteria in area [1] as
indicated in the photo.
• Sterlilize the loop and make other
streaks starting from the side of area
[1] as shown in the photo to spread
bacteria in area [2].
• Repeat the same procedure to spread
bacteria in area [3].
Colonies isolated
Types of media :
Types of media :
1-basal media
2-Enriched media
3-Selective media
4- Enrichment media
5-Differential media
6- selective and indicator media
1-Basal media
1- Nutrient broth
2-Peptone water
(Water soluble
products obtained
from protein materials
digested by
proteolytic enzyme as
trypsin and pepsin).
3- Nutrient agar
4-Nutrient gelatin
nutrient agar
2-Enriched media
Useful for fastidious
organisms
Prepared by Addition of
blood, yeast extract, brain
heart infusion
Examples:
1-Blood agar
2-Chocolate agar
3-Selective media
Contain substances that inhibit the growth
of some organism but have no effect on
the organism of test .these inhibitors may
be dyes ,chemicals or antibiotics.
An example is Mannitol salt agar used to
separate staphylococcus aureus
Mannitol salt agar
Staphylococcus aureus produce yellow colouration
4- Enrichment media
These are liquid media with
selective properties enhance the
multiplication of organisms.
As:
1-Selenite broth.
2-Alkaline peptone water
3-Tetrathionate broth medium
5-Differential media
Contain some substances that are
changed visibly as a result of
metabolic activity of organism as:
1-Sugar media
2-Triple sugar iron agar (TSI)
6- Selective and indicator media
.
Composed of selective and indicator
supplements in basal media as:
1- MacConkey’s agar
2-Deoxycholate citrate agar (DCA).
3-TCBS(Thiosulphate citrate and bile
salt (selective) sucrose (test sugar)
and bromothymol blue (pH indicator)
MacConkey agar