02 BIO+210+FQ+2014+Ch+4+Microscopy+and+staining

8/10/2019 02 BIO+210+FQ+2014+Ch+4+Microscopy+and+staining

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Ch. 4. Microscopy and Staining

Learning Objectives:

Measurements

Magnification

ResolutionRefraction

Parts of the compound microscope

Different microscopes

Different Stains and Specimen Preparation


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Chicken

egg

Human red

blood cell

Large

protozoan

(Euglena)Chloroplasts

Flea

Typical bacteria

and archaeaDiameter

of DNA

VirusesProteins

Ribosomes

Amino

acids

Atoms

Transmission electron microscope (TEM)

Compound light microscope (LM)

Mitochondrion


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*textbook p.100


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Two key characteristics of a microscope:

Magnificationability to enlarge objects

Resolving power ability to show detail


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Ocular lens: the lens closest to the eye

Objective lens: the lens closest to the

specimen

Total power of magnification of the final image:

Power of objective X Power of Ocular = Total Magnification

10 x low power 10 x = 100 x

40 x high dry 10 x = 400 x

100 x oil immersion 10 x = 1,000 x


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Refractionbending or change in the angle of light as

it passes through a medium (e.g. a lens)

When using an oil immersion lens, need to use oil.

The oil has same optical qualities as glass and prevents

refractive loss and increases the numerical aperture.


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Resolutioncapacity of an optical system to distinguish or

separate two adjacent objects or points from one another.

Shorter visible wavelengths of light will provide better

resolution.

Blue filters may be placed over a light source to limit

longer wavelengths from entering the specimen.


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Bright-field microscopy: image is

formed when light is transmitted

through the specimen

Phase-contrast microscopy: changes

in light waves passing through

specimen are transformed intodifferences in light intensity.


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https://www.youtube.com/watch?v=hC0jeNU

yQ9s


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Dark-Field Microscopy: stop

disc added to condenser

block all but peripheral light from entering objective

lens

Reverse image

Specimen appears

bright on a dark

background


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Nomarski microscopy:

-uses two prisms and two beams

of light

-depends on differences in

refractive index

-provides a 3-D image


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Fluorescence

Microscopy:

Uses UV radiation source

and dyes that showfluorescence.


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Confocal Scanning Laser Microscopy:

-used to construct three-dimensional image of thicker

structures

-provides detailed sectional views of internal structures of anintact organism


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Confocal microscopy used to look at

Staphylococcusaureusbiofilms

http://www.vcu.edu/micro/lab_web/jefferson/research/index.html


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Electron Microscopy: Resolution is approx. 0.3 nm and

magnification 5,000 x to 1,000,000 x for biological specimens.

Uses a series of electromagnetic lenses, electrons, and fluorescent screen to

produce images


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Transmission Electron Microscopy (TEM):

Used to observe fine detail inside the cell

Directs beam of electrons at specimen

Electrons pass through or scatter at

surface

Specimen preparation through

Thin sectioning


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Scanning Electron

Microscopy (SEM):

Used to observe detail

on the surface of the

specimen

Specimen is coated with

metal (e.g. gold)

Beam of electrons scan

surface of specimen


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Microscope Techniques, Dyes, and Staining

Stains are made of organic salts

Basic dyes bind to cell structures thatcarry negative charge

Commonly stain the cell

Acidic dyes are repelled by cell structuresthat carry negative charge

Commonly stain the background


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Spread culture inthin film over slide

Pass slide throughflame to fix it

Air dry


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Negative Staining:

dye settles around specimen

e.g. capsule stain


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Differential Staining:

Differentialprimary dye and counterstain

e.g. Gram Stain

permits differentiation of major categories of

bacteria

Gram positive bacteria:

cells stain purple

Gram negative bacteria:

cells stain pink


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Gram Stain Video

https://www.youtube.com/watch?v=tg5P6M

M7AAs


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Ziehl-Neelsen acid-fast stain:

Can be used for presumptive identification in diagnosis ofclinical specimens

- Primary dye

Carbol fuchsin

Colors acid-fast bacteria red

- Decolorizer

Generally acid alcohol

Removes stains from non acid-fast bacteria

- Counter stain

Methylene blue

Colors non acid-fast bacteria blue

Color of acid-fast bacteria red


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Schaeffer-Fulton endospore stain (spore stain):

- Dye is forced by heat into resistant bodies called spores

- Vegetative cells stain pink; spores stain green


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Endospore stain video

https://www.youtube.com/watch?v=8tqNzsS

YQYA


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Flagella stain:

-Staining increases

the diameter of the

flagella and

increases the

visibility of the

flagella

02 BIO+210+FQ+2014+Ch+4+Microscopy+and+staining

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