ch02 microscopy

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Copyright 2004 Pearson Education, Inc., publishing as Benjamin Cummings


PowerPoint Lecture Slide Presentation prepared by Christine L. Case Modified by Nick Kapp

MicrobiologyB.E Pruitt & Jane J. Stein

AN INTRODUCTION9th EDITION

TORTORA FUNKE CASE

Chapter 3

Observing Microorganisms Through a

Microscope


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Units of Measurement Table 3.1

1 m micrometer = 10-6 m = 10-3 mm 1 nm nanometer = 10-9 m = 10-6 mm

1000 nm = 1 m

0.001 m = 1 nm


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Microscopy: The Instruments

A simplemicroscope has onlyone lens.

Figure 1.2b


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In a compoundmicroscope the

image from theobjective lens ismagnified again bythe ocular lens.

Total magnification

=

objective lens v

ocular lens Figure 3.1b


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Resolution is the ability of the lenses todistinguish two points.

A microscope with a resolving power of 0.4 nm

can distinguish between two points 0.4 nm.

Shorter wavelengths of light provide greater

resolution

Resolving power=Wavelength of light used/2x

numerical aperture(a property of the lens).


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Refractive index isthe light-bending

ability of a medium.

The light may bendin air so much that it

misses the small

high-magnificationlens.

Immersion oil isused to keep light

from bending. Figure 3.3


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Brightfield Illumination

Dark objects arevisible against a

bright background.

Light reflected offthe specimen does

not enter theobjective lens.

Figure 3.4a, b


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Darkfield Illumination

Light objects are

visible against adark background.

Light reflected

off the specimenenters theobjective lens.

Figure 3.4a, b


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Phase-Contrast Microscopy

Accentuates diffractionof the light that passesthrough a specimen.

Direct and reflectedlight rays are combined

at the eye. Increasingcontrast

Figure 3.4c


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eren a n er erence on ras

Microscopy

Accentuatesdiffraction of

the light that

passes through

a specimen;uses twobeams of light.

Adding colorFigure 3.5


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Fluorescence Microscopy

Uses UV light.

Fluorescent

substances absorb

UV light and emitvisible light.

Cells may be stainedwith fluorescentdyes

(fluorochromes).Figure 3.6b


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Confocal Microscopy

Uses fluorochromes

and a laser light. The laser

illuminates each

plane in a specimento produce a 3-Dimage.

Figure 3.7


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Electron Microscopy

Uses electrons instead of light.

The shorter wavelength of electrons gives greaterresolution. Why?


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ransm ss on ec ron croscopy

(TEM) Ultrathin sections of specimens.

Light passes through specimen, then anelectromagnetic lens, to a screen or film.

Specimens may be stained with heavy metal salts.

Figure 3.8a


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ransm ss on ec ron croscopy

(TEM)

10,000-100,000v; resolution 2.5 nm

Figure 3.9


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Scanning Electron Microscopy (SEM)

An electron gun produces a beam of electrons thatscans the surface of a whole specimen.

Secondary electrons emitted from the specimenproduce the image.

Figure 3.9b


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Scanning Electron Microscopy (SEM)

1000-10,000v; resolution 20 nm

Figure 3.8b


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Scanning-Probe Microscopy

Scanning tunneling

microscopy uses ametal probe to scan

a specimen.

Resolution 1/100 ofan atom.

Figure 3.9a


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Scanning-Probe Microscopy

Atomic forcemicroscopy uses a

metal and diamond

probe inserted intothe specimen.

Produces 3-Dimages.

Figure 3.9b


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Preparation of Specimens forLight Microscopy

A thin film of a solution of microbes on aslide is a smear.

A smear is usually fixed to attach themicrobes to the slide and to kill themicrobes.


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Preparing Smears for Staining

Live orunstained cells

have little

contrast with thesurrounding

medium.However,researchers do

makediscoveries

about cell


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Preparing Smears for Staining

Stains consist of a positive and negative ion.

In a basic dye, the chromophore is a cation.

In an acidic dye, the chromophore is an anion.

Staining the background instead of the cell is

called negative staining.


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Simple Stains

Use of a single basic dye is called a simple stain.

A mordant may be used to hold the stain or coatthe specimen to enlarge it.


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Differential Stains: Gram Stain

The Gram stain classifies bacteria into gram-

positive and gram-negative. Gram-positivebacteria tend to be killed by

penicillin and detergents.

Gram-negativebacteria are more resistant toantibiotics.


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Color of

Gram + cells

Color of

Gram cells

Primary stain:

Crystal violet

Purple Purple

Mordant:

Iodine

Purple Purple

Decolorizing agent:

Alcohol-acetone

Purple Colorless

Counterstain:

Safranin

Purple Red


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Figure 3.11b


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Differential Stains: Acid-Fast Stain

Cells that retain a basic stain in the presence ofacid-alcohol are called acid-fast.

Nonacid-fast cells lose the basic stain when

rinsed with acid-alcohol, and are usually

counterstained (with a different color basic stain)to see them.

Figure 3.12


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Special Stains

Negative staining isuseful for capsules.

Heat is required todrive a stain intoendospores.

Flagella stainingrequires a mordant

to make the flagellawide enough to see.

Figure 3.13a-c


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Know the parts of the microscope

Know the types of light and electronic microscopes Power

What they are good for observing

What are stains used for?How do you do a gram stain

ch02 microscopy

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