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Microbiology Chapter 3
Culturing MicrobesThe Five Is
Innoculation: Producing a pure culture
Isolation: Colony on media, one kind of microbe,pure culture
Incubation: growing microbes under properconditions
Inspection: Observation of characteristics (data)Identification: use of data, correaltion, to IDorganism to exact species
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Microbiology Chapter 3
Culturing MicrobesThe Five Is
Innoculation: Producing a pure cultureIntroduce bacteria into a growth medium using aseptic technique to
prevent contamination. Tools: Bunsen burner, loop. Needle, etc.
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Microbiology Chapter 3
Innoculation: Producing a pure cultureIntroduce bacteria into a growth medium using aseptic technique toprevent contamination. Tools: Bunsen burner, loop. Needle, etc.
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Microbiology Chapter 3
Isolation: Colony on media, one kind ofmicrobe, pure culture: isolation on generaland special differential media
General growth media: NA, TSADifferential: Mac, EMB, SS
These have dyes, salts, inhibiting
agents : see differences onplates
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Microbiology Chapter 3
Isolation: Colony on media, one kind ofmicrobe, pure culture
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Microbiology Chapter 3
Isolation: Colony on media, one kind ofmicrobe, pure culture Streak Plates
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Microbiology Chapter 3
Isolation: Colony on media, one kind ofmicrobe, pure culture. Many colonies? Usea needle, pick one, and redo streak plate
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Microbiology Chapter 3
Differential: Mac, EMB, SSThese have dyes, salts, inhibitingagents : see differences on plates
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Microbiology Chapter 3
Blood agar : rich with nutrients, can see adifference, thus differential; much morelater
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Microbiology Chapter 3 Incubation: Allow organisms to grow under the optimal
conditions
Temperature, with or without oxygen etc
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Microbiology Chapter 3 Incubation: Allow organisms to grow under the optimal conditions
Temperature, with or without oxygen etc
Candle jar reduces oxygen
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Microbiology Chapter 3
Inspection: Observation, description Colony Morphology, Microscopic examination (gramsstain)
Systematic recording of DATA
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Microbiology Chapter 3
Microscopic study: Gram + bacilli, Gram -bacilli
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Microbiology Chapter 3
Microscopic study: Acid fast, and capsule
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Microbiology Chapter 3 Identification: Correlating data from all observations to
ID organism to species
Resources: flow charts, Bergeys manual etc.
Ex. Gram bacilli, ferments lactose, green sheen onEMB: E.coli
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Microbiology Chapter 3 Identification: Correlating data from all observations to
ID organism to species Gram + cocci, grape like clusters, golden yellow colonies, catalase
+, coagulase +, resistant to Methicillin (MRSA)
Staphylococcus aureus
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Microbiology Chapter 3, part 2
Light can be described as a form of energy that moves in waves .Wavelengths of light in the visible spectrum are used in mostmicroscopes. Remember the prism? Light is composed of differentcolors of light. Each color has different wavelength. Longerwavelengths have less energy (red end). Shorter; more energy (violet toUV).
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Microbiology Chapter 3, part 2
When light strikes an object the light can be:
Reflected Bounces off (Mirror)
Transmitted Passes through (GLASS)
Absorbed Soaked (black colored paper)
Diffracted Scattered as it passes through(bugs on a dirty windshield)
Refracted Bent as it passes (objects seen
under water) Glass lensesRefractive index: degree of bending,
based on lens material and shapeof lens
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Microbiology Chapter 3, part 2
So What? It is a big deal. When light in a scope strikes an
object (stained bacteria on a slide) some of the light is:Absorbed A pattern is collected by the lenses and our
Refracted eyes see a magnified object
Diffracted
ReflectedTransmitted
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Microbiology Chapter 3, part 2
Compound Light Microscope: Lens system with two
magnifying lenses, magnification is calculated by multiplyingthe power of the two lenses (10 X 10 = 100 power)
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Microbiology Chapter 3, part 2
Technicality
Contrast: Bacteria have little contrast unstained. Light isonly slightly refracted diffracted reflected etc. as itpasses through the cells. To see them we usually stainthem. Stains are colored dyes (chromophores) that increase
contrast. Without stains, special expensive microscopesare needed.
Resolution: aka resolving power The ability of a lens
system to allow an observer to see fine detail. Quality oflens systems (fine quality of glass and special lenscoatings). The best lens systems allow one to see twopoints as distinct points eve when they are tiny and veryclose together.
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Microbiology Chapter 3, part 2
The best light microscopes can resolve objects to only about
0.2 0.5 microns. It is a function of the energy of visiblelight and its wavelength (we make really good lenses). Toincrease resolving power we need and energy source withmore energy (shorter wavelength) thus the electron
microscope.
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Microbiology Chapter 3, part 2
The best magnification on our scopes is achieved with the
oil immersion objective. Oil is used with the lens becauseit has the same refractive index as glass. We can seeobjects with clarity at about 1000X magnification. Less lightis refracted away from the tiny lens and objects are clearer.No oil = fuzzy poor quality image.
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Microbiology Chapter 3, part 2
Types of Light Microscopes
Brightfield most common, objects are darkagainst a bright background
Darkfield - special condenser, objects are
light against a dark background used to seelive microbes unstained (spirochetes in fluid)
Phase contrast expensive condenser andinternal lens components, change phase of
light, so live specimens appear with moreinternal contrast
Fluorescence fluorescent dyes and UV light
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Microbiology Chapter 3, part 2
Brightfiled
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Microbiology Chapter 3, part 2
Darkfield
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Microbiology Chapter 3, part 2
Phase contrast
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Microbiology Chapter 3, part 2
Fluorescence Microscope
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Microbiology Chapter 3, part 2
Electron Microscope: energy source for magnification is a beam of
electrons (negative charged subatomic particles
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Microbiology Chapter 3, part 2
Transmission electron microscope veryhigh magnification (100,000 X)
Scanning: tremendous surface detail
Transmission Scanning
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Microbiology Chapter 3, part 2
Tunneling scanning electron microscope
Molecular and atomic level? Research
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Microbiology Chapter 3, part 2
Compare and contrast Light and Electron Microscope
Light Electron
Energy light Energy electron beam
Cost - $1200 Cost $120,000
Simple to use Complex processes.
trained technician
Magnification 1200X Magnification 100,000X
Viewed by eye, camera Viewed with CRT, photos
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Microbiology Chapter 3, part 2
Compare and contrast Light and Electron Microscope
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Microbiology Chapter 3, part 2
Preparation of samples for light microscope
Wet mounts (ex. Hanging drop) for live observation
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Microbiology Chapter 3, part 2
Simple stain one dye
Differential stain complex procedure, seedifference between cells
Grams + and (-)
Acid fast + and (-)
Negative acid dye stains background andcells are white (cell wall repels stain)
Capsule modified negative stain to showcapsule layer
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Microbiology Chapter 3, part 2
Grams
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Microbiology Chapter 3, part 2
Acid fast (for tb)
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Microbiology Chapter 3, part 2
Capsule
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