Chp 3 Ppt Bakt

8/8/2019 Chp 3 Ppt Bakt

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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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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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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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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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Isolation: Colony on media, one kind ofmicrobe, pure culture


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Isolation: Colony on media, one kind ofmicrobe, pure culture Streak Plates


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Isolation: Colony on media, one kind ofmicrobe, pure culture. Many colonies? Usea needle, pick one, and redo streak plate


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Differential: Mac, EMB, SSThese have dyes, salts, inhibitingagents : see differences on plates


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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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Inspection: Observation, description Colony Morphology, Microscopic examination (gramsstain)

Systematic recording of DATA


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Microscopic study: Gram + bacilli, Gram -bacilli


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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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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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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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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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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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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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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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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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Brightfiled


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Darkfield


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Phase contrast


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Fluorescence Microscope


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Electron Microscope: energy source for magnification is a beam of

electrons (negative charged subatomic particles


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Transmission electron microscope veryhigh magnification (100,000 X)

Scanning: tremendous surface detail

Transmission Scanning


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Tunneling scanning electron microscope

Molecular and atomic level? Research


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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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Compare and contrast Light and Electron Microscope


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Preparation of samples for light microscope

Wet mounts (ex. Hanging drop) for live observation


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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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Grams


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Acid fast (for tb)


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Capsule


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