Ch 3:
Observing
Microorganisms
Through a
Microscope
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?
Objectives
Review the metric units of measurement
Define total magnification and resolution
Explain how electron and light microscopy differ
Differentiate between acidic and basic dyes
Compare simple, differential, and special stains
List the steps in preparing a Gram stain. Describe the
appearance of Gram-positive and Gram-negative
cells after each step
Compare and contrast Gram stain and acid-fast stain
Explain why endospore and capsule stains are used
Units of Measurement
Review Table 3.1
• 1 µm = ______ m = ______ mm
• 1 nm = ______ m = ______ mm
• 1000 nm = ______ µm
• 0.001 µm = ______ nm
Sizes Among Microorganisms
• Protozoa: 100 µm
• Yeasts: 8 µm
Cells Alive –
How big is a . . .?
• Bacteria: 1 - 5 µm (some much longer than
wide)
• Rickettsia: 0.4 µm = _________ nm
• Chlamydia and Mycoplasma: 0.25 µm
• Viruses: 20 – 250 nm
Principles of the Compound Light
Microscope
Magnification: Ocular and
objective lenses of
compound microscope (total
mag.?)
Resolution: Ability of lens to . . .
Maximum resolving power ___ m
Contrast: Stains change refractive
index  contrast between
bacteria and surrounding medium
Fig 3.1
Refractive Index
• Measures light-bending
ability of a medium
• Light may bend in air so
much that it misses the
small high-magnification
lens.
• Immersion oil is used to
keep light from bending.
Fig 3.3
Microscopy: The Instruments
Brightfield Microscopy
• Simplest of all the
optical microscopy
illumination. techniques
• Dark objects are visible
against a bright
background.
Darkfield Microscopy
• Light objects visible
against dark background.
• used to enhance the
contrast in unstained
samples.
• Instrument of choice for
spirochetes
Spirochetes (Treponema pallidum) viewed with darkfield microscope
Fluorescence Microscopy
• Uses UV light.
• Fluorescent substances
absorb UV light and emit
visible light.
• Cells may be stained with
fluorescent chemicals
(fluorochromes).
• Immunofluorescence
Fig 3.6; T. pallidum
Figure 3.6a
Fig 3.6b
Principle of
Immunofluorescence
Electron Microscopy: Detailed Images of
Cell Parts
Uses electrons, electromagnetic lenses, and
fluorescent screens
Electron wavelength ~ 100,000 x smaller than
visible light wavelength
Specimens may be stained with heavy metal
salts
Two types of EMs:?
SEM or TEM?
Bacterial division
Leaf surface
?
10,000-100,000; resolution 2.5 nm.
Rod-shaped Mycobacterium avium
Preparation of Specimens for Light
Microscopy
• Staining Techniques Provide Contrast
• Smear  air-dry  heat-fix
• Basic dyes: cationic chromophore
• Acidic dyes: anionic chromophore 
negative staining (good for capsules)
• Three types of staining techniques:
Simple, differential, and special
Simple Stains
• Use a single basic
dye.
• A mordant may be
used to hold the
stain or coat the
specimen to
enlarge it.
Differential Stains
React differently with
different bacteria
• Gram stain
• Acid fast stain
Review of different staining techniques
Important Staining Reactions in Microbiology
For Gram stain
technique compare
to Fig 3-12
Gram Stain
crystal violet
safranin
Acid Fast Stain
• Cells that retain a basic stain in the presence of
acid-alcohol are called acid-fast.
• Non–acid-fast cells lose the primary stain when
rinsed with acid-alcohol, and are counterstained
with a different color basic stain
Special Stains
See Fig 3.14
• Endospore stain: Heat is
required to drive a stain into the
endospore.
• Flagella staining: requires a
mordant to make the flagella wide
enough to see.
• Capsule stain uses basic stain
and negative stain