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Exercise 2

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Exercise (Env Iso A1, Env Iso B1, etc.) Incubate plates upside ... Touch to back of hand. Heat fixing. Kills bacterial cells. Adheres cells to the ... – PowerPoint PPT presentation

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Title: Exercise 2


1
Exercise 2
  • Pre-lab for Exercise 2 due
  • Quiz 1
  • Continue Purification of Environmental Isolate
  • Begin Staining

2
Environmental Isolate
  • Last week
  • Started purification of microorganisms from the
    environment
  • Throughout the semester
  • Isolate, characterize, identify one species
  • Continue purification

3
Environmental Isolate
  • For the two colonies that you chose…
  • Record colony color, morphology, size
  • Record source
  • Please remember…
  • You are responsible for your environmental
    isolate culture for the REMAINDER of the semester

4
Environmental Isolate
5
Environmental Isolate Isolation Streak Pattern
6
Environmental Isolate
  • Label plates
  • Name
  • Lab Section
  • Date
  • Exercise (Env Iso A1, Env Iso B1, etc.)
  • Incubate plates upside down at room temp
  • Check every 24 hours until growth appears
  • Record hours (ideally 24-48 hours)

7
Microscopy and Staining
  • Phase contrast microscopy
  • Used to view live cells
  • Bright field microscopy
  • Used to view stained

8
Staining techniques may be
  • SIMPLE
  • Simple stains use a single dye
  • OR
  • DIFFERENTIAL
  • Differential stains use more than one dye

9
SIMPLE STAIN
  • Uses a single dye
  • Used to determine the basic cell morphology and
    arrangement
  • Examples include Methylene Blue, Crystal Violet,
    or Safranin

10
DIFFERENTIAL STAIN
  • Uses two or more dyes
  • Used to distinguish between two different
    organisms or between two different parts of a
    single organism
  • Examples include Gram stains, capsule stains,
    spore stains, and acid-fast stains

11
Stains are either
  • CATIONIC (positively charged)
  • Bind to negatively charged cell walls
  • Most microorganisms have negatively charged cell
    walls
  • OR
  • ANIONIC (negatively charged)
  • Bind to positively charged cell walls
  • Often used in negative staining techniques

12
NEGATIVE STAIN
  • Cells are not stained
  • Background is stained
  • Typically uses anionic stains
  • Negative charge on the surface of the cell repels
    the stain
  • Used for bacteria that are too delicate to
    withstand heat fixing

13
Staining Techniques
  • Wet Mount Slides used to view live cells
  • Dry Mount Slides used for staining cells
  • Heat Fix
  • Methylene Blue Stain (simple stain)
  • Gram Stain (differential stain)
  • Capsule Stain (differential and negative stain)

14
Dry Mount Slides
  • Microorganisms from a broth culture (or from a
    plate culture mixed with a drop of water) are
    spread on a slide
  • Stain cells
  • View with bright field microscopy
  • No cover slip

15
Dry Mount Preparation
  • Clean the slide
  • Rinse slide with 70 ethanol and wipe dry
  • Spread cells on the slide
  • A loop of broth culture -OR-
  • Emulsify (mix thoroughly) a small amount of
    bacteria from a plate culture into a drop of
    water on the slide
  • Allow the slide to air dry
  • Spread thin for faster drying

16
Dry Mount Preparation Heat Fixing
  • Gently heat the slide by passing it through the
    flame of a Bunsen burner 3 or 4 times
  • Touch slide to back of hand
  • Heat fixing
  • Kills bacterial cells
  • Adheres cells to the slide
  • Alters the cell membrane to make it permeable to
    most dyes
  • Not required for all dry mount preparations

17
Methylene Blue Stain (a simple stain)
  • Uses one dye Methylene Blue
  • Used to determine the basic cell morphology and
    arrangement
  • Organism Bacillus megaterium

18
Methylene Blue Stain
  • Simple stain of
    a Bacillus spp.

19
Methylene Blue Procedure
  • Prepare a dry mount
  • Heat fix the cells
  • Flood slide with Methylene Blue
  • Gently rinse slide with distilled water
  • Blot dry with Bibulous paper
  • View slide under 100x oil immersion
  • (Focus at 10x, proceed to 40x, then to 100x)

20
Gram Stain (a differential stain)
  • Uses two dyes to differentiate between two major
    cell wall types
  • Gram-positive cells appear purple in color
  • Gram-negative cells appear pink in color
  • Widely used to aid in identification of bacteria
  • Originally devised by Hans Christian Joachim
    Gram, a Danish doctor

21
Gram Stain
  • Distinguishes between Gram-positive and
    Gram-negative microorganisms based upon amount of
    peptidoglycan in the cell wall
  • Gram-positive
  • Cell walls contain large amounts of peptidoglycan
    and no lipopolysaccharides
  • Gram-negative
  • Cell walls contain small amounts of peptidoglycan
    and lipopolysaccharides

22
Gram -
Gram
23
Gram Stain
  • Organisms
  • Escherichia coli Gram-negative rods
  • Bacillus megaterium Gram-positive rods
  • Staphylococcus epidermidis Gram-positive cocci
  • Prepare two cultures on the same slide
  • Use one Gram-positive organism and one
    Gram-negative organism
  • Mix one loop-ful of each culture on the slide
  • Remember to flame sterilize the loop between
    cultures!

24
Gram Stain
Escherichia coli Gram-negative rods Opportunistic
Streptococcus pneumoniae Gram-positive cocci
Pathogenic
Staphylococcus epidermidis Gram-positive cocci
Opportunistic
25
Gram Stain Procedure
  • Flood slide with Crystal Violet
  • Gently rinse with distilled water
  • Flood slide with Grams Iodine
  • Decolorize with ethanol, then rinse with
    distilled water
  • Flood slide with Safranin, then rinse with
    distilled water
  • Blot dry and view slide

26
Capsule Stain (a differential stain and a
negative stain)
  • Used to identify bacteria that have capsules
  • Capsule
  • Protective layer around a bacterium
  • Made of glycoprotein or polypeptides
  • Impervious to stains
  • Many pathogens have a capsule

27
Capsule Stain Procedure
  • Emulsify Klebsiella pneumoniae into 1 drop of
    Congo Red
  • Spread the emulsion over the entire slide using
    another slide (similar to blood smear)
  • Allow to air dry, but do not heat fix
  • Flood the slide with Maneval's Stain
  • Gently rinse with distilled water
  • Blot dry and view slide

28
Next Week
  • Mini Writing Assignment 3 DUE
  • Pre-lab for Exercise 3 DUE
  • Quiz 2
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