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MICROSCOPES

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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 ... – PowerPoint PPT presentation

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Title: MICROSCOPES


1
MICROSCOPES
2
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 )

3
Types of microscopes
  • 1-light microscope
  • 2-Dark field microscope
  • 3-fluorescent microscope
  • 4-Phase contrast microscope
  • 5-Electron microscope

4
  • Light Microscope
  • Eye piece
  • Objective lenses
  • Stage
  • Diaphragm
  • Condenser
  • Light source
  • Adjustment knobs

5
STAINING METHODS
6
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

7
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.

8
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.

9
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

10
Bunches of cocci
Chains of bacillui
Tetrads of cocci
Chains of beads
11
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)

12
Gram-stainSteps 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.

13
Gram-stainSteps 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

14
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15
Technique of isolation of separate colonies of
bacteria
16
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.

17
Colonies isolated
18
Types of media
19
Types of media
  • 1-basal media
  • 2-Enriched media
  • 3-Selective media
  • 4- Enrichment media
  • 5-Differential media
  • 6- selective and indicator media

20
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
21
2-Enriched media
  • Useful for fastidious organisms
  • Prepared by Addition of blood, yeast extract,
    brain heart infusion
  • Examples
  • 1-Blood agar
  • 2-Chocolate agar

22
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

23
Mannitol salt agar Staphylococcus aureus produce
yellow colouration
24
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

25
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)

26
6- Selective and indicator media .
  • Composed of selective and indicator supplements
    in basal media as
  • 1- MacConkeys agar
  • 2-Deoxycholate citrate agar (DCA).
  • 3-TCBS(Thiosulphate citrate and bile salt
    (selective) sucrose (test sugar) and bromothymol
    blue (pH indicator)

27
MacConkey agar
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