????? ????? ??????? Streak Plate Method - PowerPoint PPT Presentation

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Title:

????? ????? ??????? Streak Plate Method

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Title: Practical Medical Microbiology PHT382 Author: User Last modified by: Toshiba Created Date: 2/11/2008 7:22:38 PM Document presentation format – PowerPoint PPT presentation

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Title: ????? ????? ??????? Streak Plate Method


1
????? ????? ??????? Streak Plate Method
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2
Streak Plate Method
3
Gram Stain
spread as a thin layer
One drop of water Loop of Bacteria
1-allowed to air dry
smear
2-fixed
clean glass slide
2. Saturate the smear with crystal violet for 1
minute. 3. Rinse the slide gently with water. 4.
Saturate the smear with iodine for 30 Second. 5.
Rinse the slide gently with water. 6. Decolorize
(alcohol) 7. Rinse the slide gently with
water. 8. Saturate the smear with safranin for 1
minute. 9. Rinse the slide gently with water. 10.
Carefully blot the slide dry with bibulous
paper. 11. Observe the slide under the microscope
4
Gram staining Results
Gram positive bacteria will stain purple. Gram
negative bacteria will stain red/pink. Gram
positive bacteria have a thick cell wall made of
peptidoglycan, whereas gram negative bacteria
have thin layer of peptidoglycan.
5
Cells Description under the microscope
1- Cell response GV or GV-
6
Cells Description under the microscope
2- Cell shape and arrangement
Cocci
single- Diplo Grape like-Strepto
7
Cells Description under the microscope
Road Short or long
Single or Chain
8
Classification of Bacteria
9
Gram-Positive Bacteria
10
Gram-Positive Cocci
11
Catalase test
  • The catalase test is distinguished streptococci
    from staphylococci
  • flood culture with drops of 3 H2O2
  • Catalase-positive cultures bubble at once
  • The test should not be done on blood agar because
    blood itself will produce bubbles

H2O2
Catalase
H2O O2 (gas, ?)
Staphylococci
12
Species of Satphylococci
  • Three species of staphyloccoci have medical
    importance
  • S. aureus Pathogenic commensally found in nose
    (nares)
  • S. epidermidis non pathogenic common
    commensals in nares skin
  • S. saprophyticus Cause UTI in female
    occasionally commensally found skin

13
Coagulase Test
  • Principle
  • This test used to differentiate between S. aureus
    (CPS) other Staphylococcus species (CNS)

Fibrinogen (Plasma)
Coagulase
Fibrin (Clot)
14
Staphylococci
  • General characters
  • Gram Positive Cocci
  • Grape-like
  • Non Motile
  • Non Spore Forming
  • Non Capsulated
  • Non Fastidious
  • Facultative Anaerobes
  • Fermentative
  • Catalase positive

15
Gram stain of Staphylococcus
16
Laboratory diagnosis of Staphylococcus
  • Gram Stain
  • Gram Positive Cocci, arranged in cluster
  • Culture 1- Blood agar , 2- MSA media
  • Blood agar (Non-Selective Media)
  • Coagulase Positive Staphylococci are Pigmented
    hemolytic
  • Coagulase Negative Staphylococci are
    non-pigmented non-hemolytic

17
  • MSA is selective differential medium for
    staphylococci
  • It contains NaCl (7.5), Mannitol, Phenol Red
  • The cause of selectivity due to presence of high
    salt concentration
  • The cause of differential because contains
    mannitol (sugar) and phenol red (pH indicators
    turns yellow in acidic pH and turns red in
    alkaline pH).

18
Coagulase Test
  • The tube coagulase test (Free)
  • Procedure
  • Mix 0.1 ml of culture 0.5 ml of plasma
  • Incubate at 37C for 4 h
  • Observing the tube for clot formation
  • Any degree of clotting constitutes a positive
    test
  • Advantage
  • More accurate
  • Disadvantage
  • Time consumed

S. aureus
S. epidermidis
19
Coagulase Test
  • Two Methods
  • The slide Method
  • Tube Method
  • The slide coagulase test
  • Used to detect bound coagulase or clumping factor
  • Add one drop heavy bacterial suspension and one
    drop of plasma on clean slide
  • Mixing well and observing for clumping within 10
    seconds
  • Advantage
  • Rapid diagnosis
  • Disadvantage
  • Less accurate

20
Deoxyribonuclease (DNAase) test
  • Principle
  • DNA is insoluble in acid
  • DNA is hydrolyzed into oligo nucleotides by the
    action of DNase
  • Nucleotides soluble in acid

21
DNase Test
  • Procedure result
  • Inoculate DNA agar with tested organism in
    circular motion
  • Incubate at 37C for 24-48h
  • Observe DNase activity by adding 1N HCl to the
    agar surface, a zone of clearing indicates a
    positive test
  • The zone represents the absence of DNA
  • The medium around colonies not producing DNase
    remains opaque, which is a reflection of the
    precipitation of DNA by the added acid.

22
Novobiocin Sensitivity
  • A simple disk diffusion test for estimating
    novobiocin susceptibility used to distinguish S.
    saprophyticus from other clinically species
  • Inoculated overnight culture on Mueller-Hinton
    agar
  • Add novobiocin disk on inoculated plate
  • Incubate at 370C overnight
  • Novobiocin resistance is intrinsic to S.
    saprophyticus but uncommon in other clinically
    important species.

23
Practical Work
  • Gram stain
  • Catalase test
  • Mannitol fermentation on MSA
  • Coagulase Test by Tube and Slide Method
  • DNAase Test
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