Lab 3: Visualizing and Identifying Microorganisms: Gram and Acid-Fast Staining - PowerPoint PPT Presentation

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Lab 3: Visualizing and Identifying Microorganisms: Gram and Acid-Fast Staining

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Lab 3: Visualizing and Identifying Microorganisms: Gram and Acid-Fast Staining Gram positive Gram negative Why do we need to stain microorganisms? – PowerPoint PPT presentation

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Title: Lab 3: Visualizing and Identifying Microorganisms: Gram and Acid-Fast Staining


1
Lab 3 Visualizing and Identifying
Microorganisms Gram and Acid-Fast Staining
Gram positive
Gram negative
2
Why do we need to stain microorganisms?
3
Bacterial Smears
  • Before bacteria can be stained a smear must be
    made and fixed.
  • A bacterial smear is made by spreading bacteria
    on a clean slide and allowing it to air dry or
    place on electric slide warmer.
  • The slide is passed over the incinerator to heat
    fix the bacteria.

4
Preparing a Bacterial Smear
  1. Get clean microscope slide (handle carefully, no
    fingerprints!)
  2. Vortex (mix) the broth culture until it is cloudy
    (or add 1-2 drops of water to the slide if the
    starter culture a solid)
  3. Using a loop, spread a thin film of bacteria
    (smear) on the slide and let it air dry
  4. Gently heat the slide by placing it near the
    front of the incinerator or on the electric slide
    warmer to fix the bacteria onto the slide

5
For Gram Stain Lab your slide should look like
this.
6
For Acid-Fast Stain Lab your slide should look
like this.
7
Part 1 The Gram Stain
  • The Gram Stain is a differential stain used to
    classify Gram positive and negative bacteria.
  • Developed by Hans Christian Gram in 1884 when he
    was studying bacteria from different respiratory
    diseases.
  • The single most important technique in
    microbiology.

8
Reagents for Gram Staining
  • Crystal violet (CV) primary stain all bacteria
    stain blue.
  • Grams Iodine (I) not a stain it forms a crystal
    violetiodine complex inside the cell wall.
  • Decolorizer ethyl alcohol, used to remove the
    primary stain. It removes the CV-I complex from
    cells without teichoic acid.
  • Safranin a counter stain it stains the
    decolorized cells a red color.
  • Whats the difference in a Gram positive vs. gram
    negative cell?
  • Gram positive bacteria have many layers of
    peptidoglycan in their cell wall making it easier
    to hold onto the CV-I complex.
  • Gram negative bacteria have one layer of
    peptidoglycan. Thus the CV-I complex is not
    retained in the cell.

9
The Gram Stain
  • The uptake and retaining of the dye depends on
    the cell wall.

10
(No Transcript)
11
Gram Staining
12
Gram Stain Lab Procedure
  • The detailed procedure can be found on pages 34-35

13
Part 2 Acid-Fast Staining
  • Acid fast is a differential stain that detects
    acid-fast and non-acid fast organisms.
  • Acid fast bacteria have a waxy cell wall (mycolic
    acid) that makes them difficult to stain with
    traditional methods.

Acid-fast positive (red) Acid-fast negative
(blue)
14
Part 2 Acid-Fast Staining
  • Reagents used in Acid-fast staining
  • Carbol fuchsin fuchsin is the red dye and carbol
    is the acid (carbolic acid)
  • Acid alcohol decolorizer
  • Methylene blue a blue counter stain

Acid-fast positive (red) Acid-fast negative
(blue)
15
Acid-Fast Staining Kinyoun Method
16
Acid-Fast Stain (Kinyoun) Procedure
  • The detailed procedure can be found on pages 41-42

17
Assignments for this week
  • Lab Reports
  • Gram stain all questions
  • AF stain drawings and questions 2-4

18
Lab 2 Examining Human Specimens
19
Lab 2 Examining Human Specimens
20
Lab 2 Examining Human Specimens
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