Exercise 2

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Exercise 2

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

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Environmental Isolate

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

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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

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Environmental Isolate
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Environmental IsolateIsolation Streak Pattern
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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)

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Microscopy and Staining

• Phase contrast microscopy
• Used to view live cells
• Bright field microscopy
• Used to view stained

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Staining techniques may be

• SIMPLE
• Simple stains use a single dye
• OR
• DIFFERENTIAL
• Differential stains use more than one dye

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SIMPLE STAIN

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

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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

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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

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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

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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)

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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

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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

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Dry Mount PreparationHeat 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

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Methylene Blue Stain(a simple stain)

• Uses one dye Methylene Blue
• Used to determine the basic cell morphology and
  arrangement
• Organism Bacillus megaterium

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Methylene Blue Stain

• Simple stain of
  a Bacillus spp.

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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)

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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

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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

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Gram -
Gram
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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!

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Gram Stain
Escherichia coli Gram-negative rods Opportunistic
Streptococcus pneumoniae Gram-positive cocci
Pathogenic
Staphylococcus epidermidis Gram-positive cocci
Opportunistic
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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

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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

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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

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Next Week

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