Studying Microbes

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Studying Microbes

• Seeing the Invisible

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WBC with Engulfed Yeast
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RBC and Bacteria
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Lymphocyte (WBC) and HIV
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Inoculate and Incubate
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Isolate, Inspect, and Identify
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Why might there be more than one microbe in the
sample?
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Streak Plate
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Appearance of Streak Plate After Culture

• How many different kinds of bacteria are on this
  plate?
• How could you test your answer?

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Loop Dilution
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Spread Plate
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Physical State of Media
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Chemical Content

• Synthetic all components known and quantified
• Complex some components not known or quantified
• may contain mixture of partially digested
  proteins, serum, or RBC

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Functional Types of Media

• Enriched
• Selective
• Differential

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Microbial Growth

• Incubate at correct temperature and atmosphere
• Inspect to verify pure culture
• Identify - macroscopic and microscopic
  appearance, biochemical tests, genetics
• Maintenance and disposal

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Parts of the Light Microscope
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Resolution

• The ability of the lens to distinguish two points
  a specified distance apart.
• The shorter the wavelength, the greater the
  resolution.
• Best resolution of light microscopes is 0.2 mm
  (2,000X)

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Refractive Index

• Measure of light-bending ability of the medium.
• Changing the refractive index of the specimen
  relative to the medium increases the contrast
  between the two.
• Staining is used to change the refractive index.

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Immersion Oil

• Air has a different refractive index than glass.
• Most light would be lost if it had to travel
  through air between the specimen and the
  objective lens.
• Immersion oil has about the same refractive index
  as glass and increases the resolving power of the
  lens.

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Brightfield Illumination

• Usual configuration of the light microscope
• Light passes through the specimen and is seen in
  the eyepiece
• Staining gives increased contrast so cells can be
  seen.

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Light Microscopy Cyanobacteria
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Light Microscopy Fungus
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Infected Macrophage
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Light Microscopy
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Darkfield Illumination

• An opaque disk is put between the light and the
  specimen
• Only light refracted by the specimen reaches the
  eyepiece.
• Emphasizes edges of structures against a dark
  background unstained, live specimens

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Darkfield Treponema
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Phase-Contrast

• Light comes through an annular diaphragm (donut)
• Direct rays and refracted rays are combined at
  the eyepiece
• Where rays are in phase, see light where out of
  phase, see dark.

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Phase-Contrast

• Used for detailed examination of living cells.
• View internal details

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Phase Contrast Macrophage
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Phase Contrast
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Phase Contrast Bacteria
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Fluorescence Microscopy

• Fluorescence molecules absorb UV light, emit
  visible light
• Fluorochromes are attached to antibodies that
  bind specifically to organisms or cells

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Fluorescence Microscopy Fig 3.6
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Immunofluorescence
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Fluorescence Bacteria in Paramecium
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GFP E. coli
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Confocal Microscopy

• Laser beam scans various depths of the specimen

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Electron Microscopy

• Uses electron beam instead of light (105 shorter
  wave length) 2-20nm
• Focused with magnets
• Transmission (TEM)thin section
• Scanning (SEM) 3D
• Specimen dried, stained with metals

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Vibrio cholerae in intestine(TEM)
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Bacterial Plasmid (TEM)
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TEM Bacterial Flagella
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TEM Shadowing
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Diphtheria phage(TEM)
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TEM Viruses
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TEM Viruses
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SEM Parasite Head
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SEM Macrophage
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SEM Biofilm on Contact Lens Case
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SEM Mars Rock
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Staining for Light Microscopy

• Simple stains increase contrast
• Differential stains differentiate between
  organisms or structures
• Gram stain differentiates between two kinds of
  cell wall structures

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Gram and Cell Walls
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Gram Stain
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Gram Positive Bacteria
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Gram Negative Bacteria
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Acid Fast StainMycobacteria
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Spore Stain
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Capsule Stain
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Microbe of the Day

• Escherichia coli
• Common intestinal bacterium
• Most strains non-pathogenic

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Escherichia coli
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Escherichia coli

• Gram negative rod
• Divides in 20 minutes under optimal conditions
• Widely used in molecular biology

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Escherichia coli

• Ferments lactose to produce acid
• Indicator organism for fecal contamination of
  water or food

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Escherichia coli

• Some strains produce exotoxins that cause
  intestinal damage
• Example E. coli 0157

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What You Should Know

• Approximate size of eukaryotic cell, bacterium,
  and virus
• What limits ability of microscope to magnify
  specimen.
• Advantages of brightfield, darkfield, phase
  contrast, and fluorescence microscopy

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What You Should Know

• Difference between electron microscopy and light
  microscopy
• Purpose and types of information you get from
  Gram stain, spore stain, and capsule stain