Microbiology

An Evolving Science
Third Edition

Joan L. Slonczewski and John W. Foster

Observing the
Microbial Cell
PowerPoint Lecture Outlines
Prepared by Johnny El-Rady, University of South Florida

Copyright 2014 W. W. Norton & Company, Inc. Permission required for reproduction or display

Chapter Overview
How microorganisms are observed
The physics of light
The bright-field microscope
Staining bacterial cells
The fluorescence, dark-field, and phase-contrast
microscopes
The electron microscope

Cutting-edge microscopes
How molecules are visualized
2

Figure 1.13

Introduction
Since Leeuwenhoeks time, powerful microscopes
have been devised to search for microbes in
unexpected habitats
- e.g., the human stomach

Microscopy revealed
the presence of
Helicobacter pylori,
the cause of
stomach ulcers
Figure 2.1
4

Microbial Size
Microbes differ in size, over a range of a few orders
of magnitude, or powers of ten.
- Eukaryotic microbes
- Protozoa, algae, fungi
- 10100 mm

- Prokaryotes
- Bacteria, archaea
- 0.410 mm

Nevertheless, a few bacterial species are large

enough to be seen by the unaided eye
- Thiomargarita namibiensis
5

Figure 2.4

Microbial Shape
Prokaryotic cell structures are generally simpler
than those of eukaryotes
Certain shapes of bacteria are common to many
taxonomic groups
- Bacilli = rods
- Cocci = spheres
- Spiral forms
- Spirochetes

- Spirilla
7

Figure 2.6

2.1 Observing Microbes

The size at which objects become visible depends
on the resolution of the observers eye
Resolution is the smallest distance by which two
objects can be separated and still be distinguished
The resolution of the human retina is about
150 mm (1/7 mm)
Contrast is the ability to distinguish an object
from its surrounding (background)
9

 We define what is visible and what is microscopic

in terms of the human eye
Figure 2.2

10

Microscopy for Different-Sized Scales

Different microscopes are required to resolve
various cells and subcellular structures.

Figure 2.7

11

Resolution Is Different from Detection

Detection is the ability to determine the presence of
an object

Figure 2.3

Magnification means an increase in the apparent size

of an image to resolve smaller separations between
objects
12

Theoretical
resolution limit = /2

13

Light Interacts with an Object

Absorption means that the photons energy is
acquired by the absorbing object

Reflection means that the wavefront bounces off

the surface of an object
Refraction is the bending of light as it enters a
substance that slows its speed
Scattering occurs when the wavefront interacts
with an object smaller than the wavelength of
light
14

Figure 2.9

15

2.2 Optics and Properties of Light

Light is part of the spectrum of electromagnetic
radiation.
- Wavelength of visible light = 400750 nm

For electromagnetic radiation to resolve an object,

certain conditions must exist:
1. Contrast between object and its medium
2. Wavelength smaller than the object
3. Magnification
16

Magnification by a Lens
Magnification requires the bending of light rays,
as in refraction

Wavefronts of light
shift direction as they
enter a substance of
higher refractive index

Figure 2.10
17

 When light rays enter a parabolic lens, parallel rays

each bend at an angle such that all of the rays meet at
a certain point, called the focal point

Figure 2.11

18

Figure 2.12

19

Figure 2.14
Figure 2.15

R = /2*NA
20

Resolution of Detail
It is resolution, not magnification, that limits the
ability of what we can see with a microscope
- Indeed, magnification without increasing detail is
called empty magnification

The resolution of detail in microscopy is limited

by the wave nature of light
- Light rays actually form wavefronts, which undergo
interference
- Can be constructive or destructive
21

2.3 Bright-Field Microscopy

Generates a dark image of an object over a light
background
To increase resolution:
- Use shorter wavelength light
- Lessen contrast

- Use immersion oil

- Use wider lens closer to specimen
- Higher numerical aperture (NA)
22

The Compound Microscope

A compound microscope is a system of
multiple lenses designed to correct or
compensate for aberration
- Ocular lens

- Objective lens
Total magnification = magnification of ocular
multiplied by that of the objective

23

Figure 2.16

24
-Objectives may be parfocal

Preparing a Specimen for Microscopy

A simple way to observe microbes is to place
them in a drop of water on a slide with a
coverslip.
- This is called a wet mount preparation.
- Advantages:
- Observation of cells in natural state

- Disadvantages:
- Little contrast between cell and background
- Sample may dry out quickly
25

Figure 2.17

26

Fixation and Staining

The detection and resolution of cells under a
microscope are enhanced by:
- Fixation = cells are made to adhere to a slide in a
fixed position
- Staining = cells are given a distinct color
- Most stains have conjugated double bonds or
aromatic rings, and one or more positive charges

27

Different Kinds of Stains

A simple stain adds dark color specifically to
cells, but not to the external medium or
surrounding tissue
- Most commonly used stain is methylene blue

A differential stain stains one kind of cell but

not another
- The most famous differential stain is the Gram stain
28

Figure 2.20

29

 The Gram stain was devised in 1884 by the Dutch

physician Hans Christian Gram (18531938)
- It differentiates between two types of bacteria:
- Gram-positive bacteria retain the crystal violet
stain because of their thicker cell wall

- Gram-negative bacteria do not

Figure 2.21

30

Figure 2.22

31

Other Differential Stains

Acid-fast stain = carbolfuchsin used to stain
Mycobacterium species
Spore stain = malachite green used to detect spores of
Bacillus and Clostridium

Negative stain = colors the background, which makes

capsules more visible
Figure 2.24

32

2.4 Fluorescence Microscopy

In fluorescence microscopy, the specimen absorbs
light of a defined wavelength, and then emits light of
lower energy, thus longer wavelength; that is, the
specimen fluoresces
Used to view marine and pathogenic bacteria

Figure 2.25

33

Excitation and Emission

The specimen absorbs light of a specific wavelength
(the excitation wavelength), then emits light at a
longer wavelength (the emission wavelength)

Figure 2.26

34

 The optical system for fluorescence microscopy

uses color filters

- To limit incident
light to the
wavelength of
excitation and
emitted light to
the wavelength
of emission
Figure 2.27
35

Fluorophores for Labeling

A fluorophore is a fluorescent chemical compound

- Its cell specificity can be determined in three ways:

- Chemical affinity
- Labeled antibodies
- DNA hybridization

Figure 2.29

36

2.5 Dark-Field Microscopy

Dark-field optics enables microbes to be visualized
as halos of bright light against darkness
Light shines at oblique angle
- Only light scattered by sample reaches objective
- Makes visible objects below resolution limit
- Flagella
- Very thin bacteria

37

Figure 2.31

Figure 2.32

Figure 2.33
38

2.5 Phase-Contrast Microscopy

Superimposes refracted light and transmitted light
shifted out of phase
- Reveals differences in refractive index as patterns of
light and dark
- Can be used to view live cells and cellular organelles

Figure 2.34

39

Figure 2.35

40

2.6 Electron Microscopy

Electrons behave like light waves
- Very high frequency
- Allow very great resolution

- A few nanometers
Sample must absorb electrons
- Coated with heavy metal

Electron beam and sample are in a vacuum

- Lenses are magnetic fields
41

Figure 2.36

42

2.6 Electron Microscopy

Two major types
- Transmission electron microscopy (TEM)
- Electrons pass through the specimen
- Reveals internal structures

- Scanning electron microscopy (SEM)

- Electrons scan the specimen surface
- Reveals external features in 3D

43

Figure 2.37

The TEM
closely parallels
the design of the
bright-field
microscope

44

Figure 2.38

The SEM is
arranged
somewhat
differently from
the TEM

45

Sample Preparation
The specimens for electron microscopy can be
prepared in several ways
- Embedded in a polymer for thin sections
- Microtome is used to cut slices

- Sprayed onto a copper grid

The specimen is then treated with a heavy-metal salt

such as uranyl acetate
Note: For SEM, specimen is coated with heavy
metal and it is not sliced
46

Figure 2.39

Figure 2.40

47

2.6 Tomography
In cryo-EM, or electron cryomicroscopy, the
specimen is flash-frozen
- Suspended in water and frozen rapidly in a refrigerant

Cryo-electron tomography, or electron

cryotomography, avoids the need to physically slice
the sample for thin-section TEM
- The images are combined digitally to visualize the
entire object in 3D
- Generates high-resolution models of virus particles
48

Figure 2.42

49

Figure 2.43

50

Figure 2.44

51

Emerging Methods of Microscopy

Scanning probe microscopy (SPM) enables
nanoscale observation of cell surfaces
The atomic force microscope (AFM) is an
example of an SPM
It measures the van der Waals forces between
electron shells of adjacent atoms of the cell surface
and the sharp tip
It can be used to observe live bacteria in water or
exposed to air (unlike electron microscopy)
52

Figure 2.45

53

2.7 Visualizing Molecules

X-ray diffraction analysis
- For samples that can be crystallized, X-ray diffraction
makes it possible to fix the position of individual
atoms in a molecule
- A beam of X-rays is shot at a crystallized sample
- Many molecules in identical conformation
- X-rays diffract according to position of atoms
- Compute position of atoms from pattern of scattered Xrays
54

Figure 2.46

55

 Today, X-ray data undergo digital analysis to

generate sophisticated molecular models
- Example: the anthrax lethal factor
- A toxin produced
by Bacillus anthracis
- Note: The model was
encoded in a protein
data bank (PDB)
text file
Figure 2.48
56

Chapter Summary
When observing microbes, resolution and
magnification are paramount
Different kinds of microscopes are required to
resolve cells and subcellular structures:
- Bright-field: employs various stains

- Fluorescence: employs fluorophores for labeling

- Dark-field: detects unresolved objects

- Phase-contrast: exploits differences in refractive

indices
57

Chapter Summary
Electron microscopes use beam of electrons instead
of light rays
- TEM: provides internal details in 2D

- SEM: provides external details in 3D

Scanning probe microscopes (SPMs) include the

atomic force microscope (AFM)
- Allow observation of living cells in water or in air

Molecules can be visualized by X-ray

crystallography
58

Concept CheckSection 2.1

Which of the following statements about the
size of microbes is FALSE?
a)

Eukaryotic microbes tend to have a size of

10100 mm.

b) Prokaryotic microbes tend to have a size that

is less than 10 mm.
c)

A few bacterial species are large enough to be

seen by the unaided eye.

d) Choose this answer if all the above are true.

59

Concept CheckSection 2.2

Which of the following properties is most
important for a lens to magnify an image?
a)

Absorption

b) Reflection
c)

Scattering

d) Refraction

60

Concept CheckSection 2.3

You are observing a bacterium using a 10
ocular lens and a 45 objective lens. What
would the total magnification be?
a)

10

b) 45
c)

145

d) 450
e)

Need more information

61

Concept CheckSection 2.3

What is the correct order of reagents in the Gram
stain?
a) Iodine, crystal violet, ethanol, safranin
b) Crystal violet, iodine, ethanol, safranin
c)

Crystal violet, ethanol, iodine, safranin

d) Iodine, ethanol, safranin, crystal violet

e)

Safranin, ethanol, iodine, crystal violet

62

Concept CheckSection 2.4

In fluorescence microscopy, the specimen
absorbs incident light and then re-emits it at a
_______ energy and thus, a _______
wavelength.
a)

lower; shorter

b) lower; longer
c)

higher; shorter

d) higher; longer
63

Concept CheckSection 2.5

Which of the following microscopes allows the
best view of bacterial flagella during motility?
a)

Bright-field microscope

b) Dark-field microscope

c)

Fluorescence microscope

d) Transmission electron microscope

e)

Scanning electron microscope

64

Concept CheckSection 2.5

Which of the following statements about
phase-contrast microscopy is true?
a)

It exploits differences in refractive indices

between cell parts and surrounding media.

b) It can be used to view live cells.

c)

Both A and B

d) Neither A nor B

65

Concept CheckSection 2.6

All of the following statements apply to
scanning electron microscopy EXCEPT
a)

The specimen is usually fixed and embedded

b) The embedded specimen is cut into thin

sections with a microtome
c)

It cannot be used to view live specimens

d) It provides 3D images of the specimen

66

Concept CheckSection 2.6

Which of the following statements about the
atomic force microscope is true?
a)

It is an example of a scanning-probe microscope

b) It measures van der Waals forces

c)

Both A and B

d) Neither A nor B

67

Concept CheckSection 2.7

What is the best technique for examining the
presence of a chemical structure with a diameter
of 3 nm?
a)

Light microscopy

b) Electron microscopy
c)

Ultracentrifugation

d) Tomography
e)

X-ray crystallography
68