Chapter 3

Observing Microorganisms
Through A Microscope
Q&A
Acid-fast staining of a
patients sputum is a
rapid, reliable, and
inexpensive method to
diagnose tuberculosis.
What color would bacterial
cells appear if the patient
has tuberculosis?

A Glimpse of History
Hans Christian Joachim Gram (18531938)
Danish physician working at morgue in Berlin
Worked for Dr. Carl Friedlander
Attempting to identify cause of pneumonia
Gram was developing methods to stain bacteria for easier
viewing under the microscope
With one method, bacteria stained unequally
Some retained dye, others did not
Revealed two different kinds of bacteria
Basis for modern Gram stain
Identifies two major groups of bacteria
Gram-positive and Gram-negative
Microscopy Reveals Two Cell Types
Microscopy reveals two fundamental cell types
Prokaryotic cells (Bacteria, Archaea)
Smaller size gives high surface area to low volume
Facilitates rapid uptake of nutrients, excretion of wastes
Allows rapid growth
Disadvantages include vulnerability to threats including
predators, parasites, and competitors
Eukaryotic cells (Eukarya)
Larger, more complex, many cellular processes take
place in compartments
1.5. Size in the Microbial World
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1 micron () = 10
6
meter
micrometer (m) = 10
6
meter = .000001 meter
Nucleus
Mitochondria Proteins Small
molecules
Atoms Lipids Ribosomes Smallest
bacteria
Most
bacteria
Most eukaryotic cells Adult roundworm
Human height
10 m 1 m 0.1 m 1 cm 1 mm 100 m 10 m 0.1 nm 1 nm 10 nm 100 nm 1 m
The basic unit of length is the meter (m), and all
other units are fractions of a meter.
These units of measurement correspond to units
in an older but still widely used convention.
1 angstrom () = 10
10
meter
nanometer (nm) = 10
9
meter = .000000001 meter
1 meter = 39.4 inches
millimeter (mm) = 10
3
meter = .001 meter
Prion fibril
Viruses
Electron microscope
Light microscope
Unaided human eye
Observing Microorganisms
Figure 3.2
Figure 3.2
Units of Measurement
1 m = 106 m = 103 mm
1 nm = 109 m = 106 mm
1000 nm = 1 m
0.001 m = 1 nm

3.1. Microscopic Techniques: The
Instruments
Light microscope can magnify 1,000x
Common, important tool in microbiology
Electron microscope (1931) can magnify more than
100,000x
Atomic force microscope (1980s) can produce
images of individual atoms on a surface
Figure 1.2b
Microscopy: The Instruments
A simple microscope has only one lens
Light Microscopy
Use of any kind of microscope that uses visible light
to observe specimens
Types of light microscopy
Compound light microscopy
Darkfield microscopy
Phase-contrast microscopy
Differential interference contrast microscopy
Fluorescence microscopy
Confocal microscopy

Figure 3.1a
The Compound Light Microscope
Figure 3.1b
Compound Light Microscopy
In a compound
microscope, the image
from the objective lens is
magnified again by the
ocular lens
Total magnification =
objective lens ocular
lens
Principles of Light Microscopy
Light passes through specimen and then series of
magnifying lenses
Bright-field microscope is most common type
Three key concepts
Magnification: apparent increase in size
Modern compound microscopes have two lens types: objective
and ocular

Magnification is product of
objective (4x, 10x, 40x, and
100x) and ocular lens (10x)
Condenser lens (between
light source and specimen)
focuses light on specimen,
does not magnify
Ocular lens
(eyepiece)
Magnifies the
image, usually
10-fold(10).
Specimen
stage
Condenser
Focuses
the light.
Iris diaphragm
Controls the
amount of light
that enters the
objective lens.
Objective lens
A selection of lens
options provide
different
magnifications. The
total magnification is
the product of the
magnifying power of
the ocular lens and
the objective lens.
Light source
Rheostat
Controls the
brightness of the
light.
Courtesy of Leica, Inc., Deerfield, FL
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Compound Light Microscopy
Resolution is the ability of the lenses to
distinguish 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
Maximum resolving power of light microscope is
0.2 m
Compound Light Microscopy
The refractive index is
a measure of the light-
bending ability of a
medium
The light may bend in
air so much that it
misses the small high-
magnification lens
Immersion oil is used to
keep light from bending
Contrast determines how easily cells can be seen
Transparent bacteria lack
contrast, difficult to see
against colorless background
Stains increase contrast but
kill microbes
Principles of Light Microscopy
(a) (b)
Light source Slide
Objective lens
Oil Air
(a): Richard Megna/Fundamental Photographs
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 3.4a
ANIMATION Light Microscopy
Brightfield Illumination
Dark objects are visible
against a bright background
Light reflected off the
specimen does not enter the
objective lens
Figure 3.4b
Darkfield Illumination
Light objects are visible
against a dark
background
Light reflected off the
specimen enters the
objective lens
Figure 3.4c
Phase-Contrast Microscopy
Accentuates diffraction
of the light that passes
through a specimen
Makes cells and other
dense material appear
darker

Figure 3.5
Differential Interference Contrast
Microscopy
Accentuates diffraction of the light that passes
through a specimen; uses two beams of light
Light waves are out of phase when recombined, yield
three-dimensional appearance of image

Figure 3.6b
Fluorescence Microscopy
Uses UV light
Fluorescent substances
absorb UV light and
emit visible light
Cells may be stained
with fluorescent dyes
(fluorochromes)
Scanning Laser Microscopes (SLM)
Obtains detailed views of interior of intact cells
Specimens usually stained with fluorescent dye
Tags bind specifically to certain internal compounds
Marks their location

Other Light Microscopes
Figure 3.7
Confocal Microscopy
Cells stained with
fluorochrome dyes
Short wavelength
(blue) light used to
excite the dyes
The light illuminates
each plane in a
specimen to produce a
three-dimensional
image
Up to 100 m deep
Figure 3.8
Two-Photon (Multiphoton) Microscopy
Cells stained with
fluorochrome dyes
Two photons of long-
wavelength (red) light
used to excite the dyes
Used to study cells
attached to a surface
Light also penetrates
deeper to give interior
views of relatively thick
structures
Up to 1 mm deep
Electron microscopy
(continued)
Can magnify images 100,000x
One drawback is that
lenses and specimen
must be in vacuum
Air molecules would interfere with
electrons
Results in large, expensive unit
and complex specimen
preparation
Two major types
Electron Microscopes
Electron beams
Electromagnet
Electromagnet
Electromagnet
Viewing screen Eye
Glass
Glass
Light
rays
Glass
Lamp
Transmission Electron
Microscope
Light Microscope
Ocular
lens
Image
Objective
lens
Specimen
Condenser
lens
Electron
gun
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Electron Microscopy
Uses electrons instead of light
The shorter wavelength of electrons gives greater
resolution
Figure 3.10a
Transmission Electron Microscopy (TEM)
Ultrathin sections of
specimens
Light passes through
specimen, then an
electromagnetic lens,
to a screen or film
Specimens may be
stained with heavy
metal salts
Figure 3.10a
Transmission Electron Microscopy (TEM)
10,000100,000; resolution 2.5 nm
Figure 3.10b
Scanning Electron Microscopy (SEM)
An electron gun
produces a beam of
electrons that scans
the surface of a whole
specimen
Secondary electrons
emitted from the
specimen produce the
image
Used to observe
surface details
Surface coated with
thin film of metal
Yields 3-D effect


Figure 3.10b
Scanning Electron Microscopy (SEM)
1,00010,000; resolution 20 nm
Figure 3.11b
Scanned-Probe Microscopy
Atomic force microscopy (AFM) uses a metal- and-
diamond probe inserted into the specimen.
Produces three-dimensional images.
Resolving power much greater than that of EM
Avoid special preparation required for EM
Sharp probe moves across samples surface
Feels bumps, valleys of atoms
Laser measures motion, computer produces surface map

Copyright 2010 Pearson Education, Inc.
Check Your Understanding
A.) Identify the type of
microscopy used.
B.) Explain the utility of each type
of microscopy.
Preparing Smears for Staining
Staining: Coloring the microbe with a dye that
emphasizes certain structures
Smear: A thin film of a solution of microbes on a
slide
A smear is usually fixed to attach the microbes to
the slide and to kill the microbes
Preparing Smears for Staining
Samples can be immobilized, stained to visualize
Live or unstained cells have little contrast with the
surrounding medium. Researchers do make
discoveries about cell behavior by observing live
specimens.
Figures B and C
Preparing Smears for Staining
Stains consist of a positive and negative ion
In a basic dye, the chromophore is a cation
Basic dyes (positive charge)
Attracted to negatively charged cellular components

In an acidic dye, the chromophore is an anion
Acidic dyes (negative charge)
Negative staining: cells repel, so colors background
Can be done as wet mount

Staining the background instead of the cell is called
negative staining
Negative Staining
an acidic (-) stain is utilized.
results in only the background being stained.
aids in observing the shape and size of a cell
www. smccd.net/.../case/ biol240/images/negstain.jpeg
Useful for cells that are unable
to be stained due to composition
of the cell wall
Simple Stains
Simple stain: Use of a single basic dye
A mordant may be used to hold the stain or coat the
specimen to enlarge it
Differential Stains
Used to distinguish between bacteria
Gram stain
Acid-fast stain
Gram Stain
Classifies bacteria into gram-positive
or gram-negative
Gram-positive bacteria tend to be killed by penicillin and
detergents
Gram-negative bacteria are more resistant to antibiotics
Gram stain most common for bacteria
Two groups: Gram-positive, Gram-negative
Reflects fundamental difference in cell wall structure
3.2. Microscopic Techniques: Dyes and
Staining
Crystal violet
(primary stain)
3
4
1
2
Cells stain purple.
Cells remain purple
Iodine
(mordant)
Safranin
(counterstain)
Gram-positive cells
remain purple;
gram-negative cells
appear pink.
Alcohol
(decolorizer)
Gram-positive cells
remain purple;
gram-negative cells
become colorless.
State of Bacteria Appearance Steps in Staining
(a) (b) 10 m
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
b: Leon J. Le Beau/Biological Photo Service
Color of
Gram-positive
cells
Color of
Gram-negative cells
Primary stain:
Crystal violet
Purple Purple

Mordant:
Iodine
Purple Purple
Decolorizing agent:
Alcohol-acetone
Purple Colorless
Counterstain:
Safranin
Purple Red
Gram Stain
Figure 3.12b
Micrograph of Gram-Stained Bacteria
Acid-fast staining used to detect Mycobacterium
Cell wall contains high concentrations of mycolic acid
Waxy fatty acid that prevents uptake of dyes
Harsher methods needed
Used to presumptively
identify clinical specimens
3.2. Microscopic Techniques: Dyes and
Staining
Differential Stains: Acid-Fast Stain
Cells that retain a basic stain in the presence of acid-
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 3.11
Color of
Acid-fast
Color of
NonAcid-fast
Primary stain:
Carbolfuchsin
Red Red
Decolorizing agent:
Acid-alcohol
Red Colorless
Counterstain:
Methylene blue
Red Blue
Acid-Fast Stain
Acid-Fast Stain
Stained waxy cell wall is not decolorized by acid-
alcohol
used to identify bacteria in the genus
Mycobacterium
Includes causative agents of tuberculosis and Hansens
disease (leprosy)


Mycobacterium
Nocardia
Q&A
Acid-fast staining of a
patients sputum is a
rapid, reliable, and
inexpensive method to
diagnose tuberculosis.
What color would
bacterial cells appear if
the patient has
tuberculosis?


Special Stains
Negative staining is
useful for capsules.


Heat is required to drive
a stain into endospores.

Flagella staining
requires a mordant to
make the flagella wide
enough to see.
Figure 3.12a-c
Special Stains

Capsule stain both the
cell and background are
stained. (Negative stain)
Endospore stain - heat
is required to drive a
malachite green stain
into endospores.
Flagella staining -
requires a mordant to
make the flagella wide
enough to see.
Figure 3.12a-c
Case Study
An outbreak of an infectious disease sent more than 25 children from a local day care
to the hospital. Five of the children were hospitalized with an upper respiratory tract
infection.
The initial onset of the clinical signs and symptoms of the disease included fever, sore
throat, and headache. Later, signs included creamy yellow pus produced on the tonsils
(exudative tonsillitis). Some also developed infections in deeper tissue, or a high fever and
bright-red rash.
Stainings revealed a purple-stained, round shaped organism arranged in chains (figure
1A). .
Questions:
1. Is this a gram-positive or
gram-negative bacterium?
Explain your answer.
2. What stain would help in
identification of the species?
3. Briefly outline the process of
this ___________-stain.
4. What bacterial genus does
this organism belong?
Figure 1a
A Bacterial Outbreak in a Prison Housing Inmates Infected with HIV
In early July 1999. a 34-year old HIV-infected man housed in a dormitory in
the prison was taken to the prison hospital with a 2-week history of fatigue, fever,
abdominal pain, and cough with blood. His chest radiograph was normal. The staff
neglected to obtain a sputum specimen for culture and the prisoner was returned to
his dormitory.

In mid-August 1999, the man was evaluated at a community hospital. A sputum
sample was obtained and an acid fast stained was performed. Figure 1.
Later that year, 31 inmates and a medical student who examined the patient
exhibited signs of this infection. Of the 31 inmates, all 31 resided in the same
dormitory as the original infected patient.
Case Study #2:
Questions:
1. What stain would help identify
this organism?
2. Describe the ____________
stain method.
3. What color is the positive cells?
4. What molecule is detected
using this stain?
5. What genus of bacteria is the
cause of this disease outbreak?
Simple staining involves one dye
Differential staining used to distinguish different types
of bacteria
3.2. Microscopic Techniques: Dyes and
Staining