microscope 1

8/12/2019 Microscope 1

1/50


2/50

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display .

2

Scale


3/50


3

Discovery of

Microorganisms Antony van

Leeuwenhoek

(1632-1723) first person to

observe and

describe

micro-organisms

accuratelyFigure 1.1b


4/50


4

Lenses and the Bending of

Light light is refracted (bent) when passing

from one medium to another

refractive index a measure of how greatly a substance slows

the velocity of light

direction and magnitude of bending isdetermined by the refractive indexes of

the two media forming the interface


5/50


5

Lenses

focus light rays at a specific placecalled the focal point

distance between center of lens andfocal point is the focal length

strength of lens related to focal

length short focal length more

magnification


6/50


6

Figure 2.2


7/50


7

The Light Microscope

many types

bright-field microscope

dark-field microscope

phase-contrast microscope

fluorescence microscopes

are compound microscopes

image formed by action of 2 lenses


8/50


8

The Bright-Field

Microscope produces a dark image against a

brighter background

has several objective lensesparfocalmicroscopes remain in focus

when objectives are changed

total magnification product of the magnifications of the

ocular lens and the objective lens


9/50


9

Figure 2.3


10/50


11/50


11

Microscope Resolution

ability of a lens to separate or

distinguish small objects that are

close together wavelength of light used is major

factor in resolution

shorter wavelength greater resolution


12/50


12

working distancedistance between the front surface of lens and surface of

cover glass or specimen


13/50


13

Figure 2.5


14/50


14

Figure 2.6


15/50


15

The Dark-Field Microscope

produces a bright image of the

object against a dark background

used to observe living, unstainedpreparations


16/50


16

Figure 2.7b


17/50


17

The Phase-Contrast

Microscope enhances the contrast between

intracellular structures having slight

differences in refractive index

excellent way to observe living cells


18/50


18

Figure 2.9


19/50


19

Figure 2.10


20/50


20

The Differential Interference

Contrast Microscope creates image by detecting

differences in refractive indices andthickness of different parts of

specimen

excellent way to observe living cells


21/50


21

The Fluorescence

Microscope exposes specimen to ultraviolet,

violet, or blue light

specimens usually stained withfluorochromes

shows a bright image of the object

resulting from the fluorescent light

emitted by the specimen


22/50


22

Figure 2.12


23/50


23

Figure 2.13c and d


24/50


25/50


25

Fixation process by which internal and external

structures are preserved and fixed inposition

process by which organism is killed andfirmly attached to microscope slide

heat fixing

preserves overall morphology but not internal

structures chemical fixing

protects fine cellular substructure andmorphology of larger, more delicate organisms


26/50


26

Dyes and Simple Staining

dyes

make internal and external structuresof cell more visible by increasing

contrast with backgroundhave two common features

chromophore groups

chemical groups with conjugated doublebonds

give dye its color

ability to bind cells


27/50


27

Dyes and Simple Staining

simple staining

a single staining agent is used

basic dyesare frequently used dyes with positive charges

e.g., crystal violet


28/50


28

Differential Staining

divides microorganisms into groups

based on their staining properties

e.g., Gram stain e.g., acid-fast stain

C G C f


29/50


29

Gram staining

most widely used differential

staining procedure

divides Bacteria into two groupsbased on differences in cell wall

structure

C i ht Th M G Hill C i I P i i i d f d ti di l


30/50


30

Figure 2.14

primarystain

mordant

counterstain

decolorization

positive

negative


31/50

Copyright The McGraw Hill Companies Inc Permission required for reproduction or display


32/50


32

Acid-fast staining

particularly useful for stainingmembers of the genusMycobacter ium

e.g., Mycobacter ium tuberculosiscausestuberculosis

e.g., Mycobacter ium lepraecauses leprosy

high lipid content in cell walls isresponsible for their stainingcharacteristics

Copyright The McGraw Hill Companies Inc Permission required for reproduction or display


33/50


33

Staining Specific

Structures Negative staining

often used to visualize capsules

surrounding bacteria

capsules are colorless against a stained

background


34/50

Copyright The McGraw-Hill Companies Inc Permission required for reproduction or display


35/50


35

Electron Microscopy

beams of electronsare used toproduce images

wavelength ofelectron beam ismuch shorter than

light, resulting inmuch higherresolution

Figure 2.20



36/50

Copyright The McGraw Hill Companies, Inc. Permission required for reproduction or display .

36

The Transmission Electron

Microscope electrons scatter when they pass

through thin sections of a specimen

transmitted electrons (those that do

not scatter) are used to produce

image

denser regions in specimen, scatter

more electrons and appear darker



37/50


37

Figure 2.23

EM



38/50


38

Specimen Preparation

analogous to procedures used for

light microscopy

for transmission electronmicroscopy, specimens must be cut

very thin

specimens are chemically fixed andstained with electron dense material



39/50

py g p , q p p y

39

Other preparation methods

shadowing

coating specimen with a thin film of a

heavy metal freeze-etching

freeze specimen then fracture along

lines of greatest weakness (e.g.,membranes)



40/50

py g p , q p p y

40

Figure 2.25



41/50

py g p q p p y

41

Ebola



42/50

42

Fly head



43/50

43

The Scanning Electron

Microscope uses electrons reflected from the

surface of a specimen to create

image

produces a 3-dimensional image of

specimens surface features



44/50

44

Figure 2.27



45/50

45

Newer Techniques in

Microscopy confocalmicroscopyandscanning probe

microscopy have extremely

high resolution

can be used toobserve individualatoms

Figure 2.20



46/50

46

Confocal Microscopy

confocal scanning laser microscope

laser beam used to illuminate spots

on specimen computer compiles images created

from each point to generate a 3-

dimensional image



47/50

47

Figure 2.29



48/50

48

Figure 2.30



49/50

49

Scanning Probe

Microscopy scanning tunneling microscope

steady current (tunneling current)maintained between microscope probe

and specimen

up and down movement of probe as it

maintains current is detected and used

to create image of surface of specimen



50/50

Scanning Probe

Microscopy atomic force microscope

sharp probe moves over surface ofspecimen at constant distance

up and down movement of probe as it

maintains constant distance is detectedand used to create image

microscope 1

Documents