MICROSCOPY

“WINDOW INTO AN INVISIBLE WORLD”

 Objectives
• Describe how lenses bend light rays to produce enlarged
images of small objects

• Describe the various parts of the light microscope and how

each part contributes to the functioning of the microscope
(theory of practical)

• Describe the preparation and simple staining of specimens

for observation with the light microscope (theory of
practical)

• Describe the Gram-staining procedure and how it is used

to categorize bacteria
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• Describe the basis for the various staining procedures
used to visualize specific structures associated with
microorganisms (theory of practical)

• Compare the operation of the transmission and scanning

electron microscopes with each other and with light
microscopes

• Describe confocal microscopy and scanning probe

microscopy

• Compare and contrast light microscopes, electron

microscopes, confocal microscopes, and scanning
probe microscopes in terms of their resolution, the types of
specimens that can be examined, and the images produced.
Animalcules – Leeuwenhoek –
magnifying glasses- simple microscope

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Visualizing Cells

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Unit of measurement in Microbiology: µm
= 10-6 of a meter

Most bacterial and archael cells between 1

– 5 µm

Viruses are in nanometer range (billionth

of a meter; 10-9) , e.g., poliovirus = 20 nm
(0.02 µm)

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 MAGNIFICATION
2 key characteristics of a microscope:

◼ Magnification – ability to enlarge objects

◼ Resolving power – ability to show detail and

clarity

◼ Complex interaction between visible light

waves and curvature of lens
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 Lenses and the Bending of Light
light refracted (bent) when passing from one
medium to another, e.g., air →glass or glass →air

refractive index
◼ measure of how greatly a substance slows the velocity
of light

direction and magnitude of bending determined

by refractive indexes of two media forming
interface

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 Lenses
focus light rays at a specific place - focal point
(F)

distance between center of lens and focal point -

focal length (f)

strength of lens related to focal length!

◼ short focal length  more magnification – more
powerful lens

◼ Objective lenses: 4 - 100 × magnifying power

◼ Ocular lenses:10 - 20 × magnifying power

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Figure 2.2
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 Light Microscopy

Many types
◼ bright-field
◼ dark-field
◼ phase-contrast
◼ fluorescence

Compound microscopes
◼ image formed by action of 2 lenses

◼ ocular and objective lenses

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 Bright-Field Microscopy
Light passes directly through the lens and
specimen
Produces dark image against brighter
background

3-5 objective lenses

◼ parfocal microscopes remain in focus when

objectives changed

total magnification
◼ product of the magnifications of ocular lens

and objective lens (e.g. 10X100 = 1000X)

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 Microscope Resolution

Capacity to make clear images of very small objects

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 (λ) and higher numerical aperture (n
sin Θ)
 greater resolution

Resolving power = wavelength of light (nm)

2 × numerical aperture of objective lens

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•Working distance
— distance between the front surface of lens and surface of
cover glass or specimen
 d

Minimum distance between 2 objects (d) = 0.5λ

n sin Θ17
Cone of light focused on specimen
◼ Narrow vs broad angle

Angle depends on refractive index of medium in

which lens works

Refractive index of air = 1

Refractive index of glass = 1.3-1.5
Refractive index of oil = 1.5

Immersion oil increases numerical aperture

(1.25)
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Fig. 5.1a

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 Dark-Field Microscopy
Living and unstained cells

Image formed by light reflected or refracted by

specimen

Bright image of object against dark background

Outline of specimen with reduced internal cellular

detail

Internal structures in large eukaryotes 21

Fig. 5.1b

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 Phase-Contrast Microscopy
Enhances contrast between intracellular
structures having slight differences in refractive
index

Converts slight differences in refractive index and

cell density into easily detected variations in light
intensity

Observe living cells in a gray background

Great internal cellular detail – endospores and

inclusion bodies, motility 23
Fig. 5.1c

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Differential Interference Contrast
Microscopy

Creates 3D image by detecting differences

in refractive indices and thickness of
different parts of specimen

Observe living cells

Highly contrasting, brightly coloured, 3D

images – cell wall, endospores, granules
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Fig. 5.1d

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 Fluorescence Microscopy

Exposes specimen to ultraviolet, violet, or

blue light

Specimens stained with fluorochromes/

fluorescent antibodies

Bright image of the object resulting from

the fluorescent light emitted by the
specimen
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Acridine orange – stains DNA, fluoresces
orange

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Live-dead staining: GFP-green = live
cells; rhodamine-red = dead cells

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 Preparation and Staining of
Specimens

Increases visibility of specimen

Accentuates specific morphological

features

Preserves specimens

Wet mounts – examination of live cells

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 Fixation
Internal and external structures are preserved and
fixed in position

Organisms killed and firmly attached to microscope

slide

◼ heat fixing
preserves overall morphology but not internal structures

◼ chemical fixing
protects fine cellular substructure and morphology of
larger, more delicate organisms
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 Dyes and Simple Staining

Dyes

◼ Make internal and external structures of cell more

visible by increasing contrast with background

◼ Have two common features

chromophore groups
◼ chemical groups with conjugated double bonds
◼ give dye its color

ability to bind cells

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 Dyes and Simple Staining

Simple staining

◼ Single staining agent used

◼ Highlights cell size, shape and cell arrangement

◼ Basic (cationic) dyes frequently used

dyes with positive charges
e.g., crystal violet

Microbe surfaces negatively charged so

attract positively charged basic dyes
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 Differential Staining

Divides microorganisms into groups based

on their staining properties

Requires primary dye and contrasting

counterstain

◼ e.g., Gram stain

◼ e.g., acid-fast stain

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 Gram staining

Most widely used differential staining

procedure

Divides Bacteria into two groups - based

on differences in cell wall structure

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 Acid-fast staining

Particularly useful for staining members of

genus Mycobacterium

e.g., Mycobacterium tuberculosis – causes

tuberculosis
e.g., Mycobacterium leprae – causes leprosy

◼ high lipid content in cell walls - responsible for

their staining characteristics

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 Staining Specific Structures

Negative staining
◼ visualize capsules surrounding bacteria

◼ capsules are colorless against a stained

background

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Staining Specific Structures

Spore staining
◼ double staining technique

◼ bacterial endospore is one color and

vegetative cell is a different color

Flagella staining
◼ mordant applied to increase thickness of
flagella
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 Electron Microscopy
Beams of electrons are
used to produce images

Magnets instead of
lenses pinpoint the flow
of electrons onto an
object

Wavelength of
electron beam is much
shorter (0.005 nm) than
light (550 nm), resulting
in much higher
resolution (2 nm) 47
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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

100 – 200 000× magnification range – 2 nm resolving

power

Denser regions in specimen, scatter more

electrons and appear darker

Fine, detailed cell structure

Viruses 49
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 Specimen Preparation

Analogous to procedures used for light

microscopy

TEM - specimens must be cut very thin

Specimens chemically fixed and stained with

electron dense material – osmium or lead

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Fig. 5.1g

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 Scanning Electron Microscope
Specimen is coated with gold

Uses electrons reflected from external

surface of a specimen to create image

Produces a 3-dimensional image of

specimen’s surface features

10 – 50 000× magnification range – 7 nm

resolving power 54
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Newer Techniques in Microscopy
confocal laser
scanning microscopy
(CSLM) and Scanning
probe microscope
(SPM)

have extremely high

resolution

can be used to
observe individual
atoms 58
Confocal scanning laser microscope

CSLM

Laser beam used to illuminate spots on

specimen stained with fluorescent dyes

Computer compiles multiple images created

from each point to generate a 3-dimensional
image

Used to observe biofilms

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Fig. 5.1f

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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

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Atomic force microscope
◼ Sharp probe moves over surface of specimen at constant
distance
◼ Up and down movement of probe as it maintains constant
distance is detected and used to create image
◼ Living cells in aqueous conditions but attached to a
surface

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 Self study questions
Describe two important characteristics of a
microscope (2)
What is a focal length? (1)
What is the relationship between focal
length and magnification? (2)
Describe the relationship between
resolution and
◼ (i) wavelength of light (1)
◼ (ii) Numerical aperture (1)
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Name and describe four types of light
microscopy (8)
What is simple staining? (1)
Why are cationic dyes used for simple
staining? (1)
Describe four different types of differential
staining (8)
Differentiate between scanning and
transmission electron microscopy (6)
Name two newer techniques of
microscopy (2)
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