Observing Microorganisms Through A Microscope: Lectures Prepared by Christine L. Case

Chapter 3
Observing
Microorganisms
Through A
Microscope
Copyright © 2010 Pearson Education, Inc.

Lectures prepared by Christine L. Case
Q&A
 Acid-fast staining of a
patient’s sputum is a
rapid, reliable, and
inexpensive method to
diagnose tuberculosis.
What color would
bacterial cells appear if
the patient has
tuberculosis?
 Look for the answer in the chapter .

Observing Microorganisms
Copyright © 2010 Pearson Education, Inc. Figure 3.2

Units of Measurement
Learning Objectives
3-1 List the metric units of measurement that are
used for microorganisms.

Units of Measurement
 1 µm = 10–6 m = 10–3 mm
 1 nm = 10–9 m = 10–6 mm
 1000 nm = 1 µm
 0.001 µm = 1 nm

Check Your Understanding
 If a microbe measures 10 μm in length, how long is
it in nanometers? 3-1

Microscopy: The Instruments
Learning Objectives
3-2 Diagram the path of light through a compound
microscope.
3-3 Define total magnification and resolution.

 A simple microscope has only one lens
Copyright © 2010 Pearson Education, Inc. Figure 1.2b

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

The Compound Light Microscope
Copyright © 2010 Pearson Education, Inc. Figure 3.1a

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

 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

 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

Refraction in the Compound Microscope

Figure 3.3
 Through what lenses does light pass in a compound
microscope? 3-2
 What does it mean when a microscope has a
resolution of 0.2 nm? 3-3

Learning Objectives
3-4 Identify a use for darkfield, phase-contrast,
differential interference contrast, fluorescence,
confocal, two-photon, and scanning acoustic
microscopy, and compare each with brightfield
illumination.
3-5 Explain how electron microscopy differs from light
microscopy.
3-6 Identify one use for the TEM, SEM, and scanned-
probe microscopes.

Brightfield Illumination
 Dark objects are visible

against a bright background
 Light reflected off the
specimen does not enter
the objective lens
ANIMATION Light Microscopy

Darkfield Illumination
 Light objects are visible

against a dark
background
 Light reflected off the
specimen enters the
objective lens

Phase-Contrast Microscopy
 Accentuates diffraction
of the light that passes
through a specimen
Copyright © 2010 Pearson Education, Inc. Figure 3.4c

Differential Interference Contrast
Microscopy
 Accentuates diffraction of the light that passes
through a specimen; uses two beams of light

Fluorescence Microscopy
 Uses UV light
 Fluorescent
substances absorb UV
light and emit visible
light
 Cells may be stained
with fluorescent dyes
(fluorochromes)

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 µm deep

Two-Photon 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
 Up to 1 mm deep

Scanning Acoustic Microscopy (SAM)
 Measures sound
waves that are
reflected back from
an object
 Used to study cells
attached to a
surface
 Resolution 1 µm

Electron Microscopy
 Uses electrons instead of light

 The shorter wavelength of electrons gives greater
resolution
ANIMATION Electron Microscopy

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

Transmission Electron Microscopy (TEM)
 10,000–100,000; resolution 2.5 nm

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

Scanning Electron Microscopy (SEM)
 1,000–10,000; resolution 20 nm

Scanned-Probe Microscopy
 Scanning tunneling microscopy (STM) uses a

metal probe to scan a specimen
 Resolution 1/100 of an atom

Scanned-Probe Microscopy
 Atomic force microscopy (AFM) uses a metal-

and-diamond probe inserted into the specimen.
 Produces three-dimensional images.

 How are brightfield, darkfield, phase-contrast, and
fluorescence microscopy similar? 3-4
 Why do electron microscopes have greater resolution than
light microscopes? 3-5
 For what is TEM used? SEM? Scanned-probe microscopy?
3-6

Preparation of Specimens for Light
Microscopy
Learning Objectives
3-7 Differentiate an acidic dye from a basic dye.
3-8 Explain the purpose of simple staining.
3-9 List the steps in preparing a Gram stain, and
describe the appearance of gram-positive and
gram-negative cells after each step.
3-10 Compare and contrast the Gram stain and the
acid-fast stain.
3-11 Explain why each of the following is used:
capsule stain, endospore stain, flagella stain.

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

 Live or unstained cells have little contrast with the

surrounding medium. Researchers do make
discoveries about cell behavior by observing live
specimens.
ANIMATION Microscopy and Staining: Overview
Copyright © 2010 Pearson Education, Inc. Figures B and C

 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

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
ANIMATION Staining

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
Color of Color of
Gram-positive cells Gram-negative cells
Primary stain: Purple Purple

Crystal violet
Mordant: Purple Purple

Iodine
Decolorizing agent: Purple Colorless

Alcohol-acetone
Counterstain: Purple Red

Safranin

Micrograph of Gram-Stained Bacteria

 Why doesn’t a negative stain color a cell? 3-7
 Why is fixing necessary for most staining
procedures? 3-8
 Why is the Gram stain so useful? 3-9

Acid-Fast Stain
 Stained waxy cell wall is not decolorized by acid-

alcohol
 Mycobacterium
 Nocardia

Acid-Fast Stain
Color of Color of
Acid-fast Non–Acid-fast
Primary stain: Red Red

Carbolfuchsin
Decolorizing agent: Red Colorless

Acid-alcohol
Counterstain: Red Blue

Methylene blue

Acid-Fast Bacteria

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

Special Stains
 Used to distinguish parts of cells

 Capsule stain
 Endospore stain
 Flagella stain

Negative Staining for Capsules
 Cells stained
 Negative stain

Endospore Staining
 Primary stain: Malachite green, usually with heat

 Decolorize cells: Water
 Counterstain: Safranin

Flagella Staining
 Mordant on flagella
 Carbolfuchsin simple stain
Copyright © 2010 Pearson Education, Inc. Figure 3.14c

 Which stain would be used to identify microbes in the genera
Mycobacterium and Nocardia?
3-10
 How do unstained endospores appear?
Stained endospores? 3-11

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Chapter 3

Copyright © 2010 Pearson Education, Inc.

 Look for the answer in the chapter .

Copyright © 2010 Pearson Education, Inc. Figure 3.2

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc. Figure 3.2

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

 A simple microscope has only one lens

Copyright © 2010 Pearson Education, Inc. Figure 1.2b

 Use of any kind of microscope that uses visible light

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc. Figure 3.1a

Copyright © 2010 Pearson Education, Inc. Figure 3.1b

 Resolution is the ability of the lenses to distinguish

Copyright © 2010 Pearson Education, Inc.

 The refractive index is a measure of the light-

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

 Dark objects are visible

ANIMATION Light Microscopy

Copyright © 2010 Pearson Education, Inc. Figure 3.4a

 Light objects are visible

Copyright © 2010 Pearson Education, Inc. Figure 3.4b

Copyright © 2010 Pearson Education, Inc. Figure 3.4c

Copyright © 2010 Pearson Education, Inc. Figure 3.5

Copyright © 2010 Pearson Education, Inc. Figure 3.6b

 Cells stained with

Copyright © 2010 Pearson Education, Inc. Figure 3.7

 Cells stained with

Copyright © 2010 Pearson Education, Inc. Figure 3.8

Copyright © 2010 Pearson Education, Inc. Figure 3.9

 Uses electrons instead of light

ANIMATION Electron Microscopy

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc. Figure 3.10a

 10,000–100,000; resolution 2.5 nm

Copyright © 2010 Pearson Education, Inc. Figure 3.10a

Copyright © 2010 Pearson Education, Inc. Figure 3.10b

Copyright © 2010 Pearson Education, Inc. Figure 3.10b

 Scanning tunneling microscopy (STM) uses a

Copyright © 2010 Pearson Education, Inc. Figure 3.11a

 Atomic force microscopy (AFM) uses a metal-

Copyright © 2010 Pearson Education, Inc. Figure 3.11b

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

 Staining: Coloring the microbe with a dye that

Copyright © 2010 Pearson Education, Inc.

 Live or unstained cells have little contrast with the

ANIMATION Microscopy and Staining: Overview

Copyright © 2010 Pearson Education, Inc. Figures B and C

 Stains consist of a positive and negative ion

Copyright © 2010 Pearson Education, Inc.

 Simple stain: Use of a single basic dye

Copyright © 2010 Pearson Education, Inc.

 Used to distinguish between bacteria

Copyright © 2010 Pearson Education, Inc.

 Classifies bacteria into gram-positive

Copyright © 2010 Pearson Education, Inc.

Primary stain: Purple Purple

Mordant: Purple Purple