Download microscopy and staining

MICROSCOPY AND
STAINING
CHAPTER 3
Metric Units
2
Light Properties

Wavelength
3
polarity
Light is a wave
Filters can block waves in off axis planes
4
Waves can be added
+
+
=
=
5
Light Properties

Resolution
6
Wavelength/Resolution Interaction
7
Light Properties

Reflection

Transmission
8
Light Properties

Absorption

Refraction
bending
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Light Microscopy Types

Compound
Bright Field
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Oil immersion
Oil with intermediate
refractive index
With oil
without
Some info lost
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Microscopy — Dark Field
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Microscopy — Phase Contrast

Dual Beam
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Phase contrast

Phase-contrast microscopy was invented in 1936 by Frits
Zernike, a Dutch mathematical physicist. It is based on the
principle that cells differ in refractive index (a factor by
which light is slowed as it passes through a material) from
their surroundings. Light passing through a cell thus
differs in phase from light passing through its
surroundings. This subtle difference is amplified by a
device in the objective lens of the phase-contrast
microscope called the phase ring, resulting in a dark image
on a light background (Figure 2.5b). The ring consists of a
phase plate—the key discovery of Zernike—that amplifies
the minute variation in phase. Zernike’s discovery of
differences in contrast between cells and their background
stimulated other innovations in microscopy, such as
fluorescence and confocal microscopy (discussed below).
For his invention of phase-contrast microscopy, Zernike
was awarded the 1953 Nobel Prize in Physics.
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Microscopy — DIC

Differential
Interference
Contrast
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DIC “differential interference
contrast”
Similar to phase contrast, but input light is polarized
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Microscopy — Fluorescence

Ultraviolet light
flourescein
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Advantages of fluorescence
Can use specialized chemical probes that target specific features
and then tag with fluorescent dyes
Downside: must use expensive filters and excitory frequencies
18
Microscopy — Confocal

Confocal
Allows 3 dimensional viewing
Allows multiple dyes to be overlaid
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Confocal microscopy
Allows 3 dimensions
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Combined confocal and
fluorescence
Antibody labeling
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Microscopy Imaging

Digital
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Fig. 2-15
23
Electron Microscopy

Transmission
(TEM)

Scanning (SEM)

Scanning
Tunneling (STM)
24
TEM
Most popular for
bacteria. Allows imaging
internal features, but
requires heavy metal
staining.
25
Electron Microscopy Images
26
Microscopy Techniques

Wet Mounts
 Smears
 Staining
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Fig. 2-3
Spread culture in thin
film over slide
Dry in air
I. Preparing a smear
Pass slide through
flame to heat fix
Flood slide with stain;
rinse and dry
II. Heat fixing and staining
100
Slide
III. Microscopy
Oil
Place drop of oil on
slide; examine with 100
objective lens
Staining cells - increases contrast
Simple stain - one dye - shows size, shape,
and arrangement
Methylene blue -
yeast
29
Cheek cell
Common stains
Safranin (*basic, + charge)
red
Crystal violet
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Differential stains
Use multiple dyes or dyes that interact with organisms
differently.
Primary stain / counterstain
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Gram Stain

Gram Stain The single
most important stain in
microbiology. Set the initial
taxonomy of bacteria.

Crystal violet *(basic stain)
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Gram Stain
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Acid Fast Stain
Carbol-fuchsin stains acid fast organisms
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Acid Fast

The Ziehl-Neelsen stain, also known as the acid-fast stain,
was first described by two German doctors; Franz Ziehl
(1859 to 1926), a bacteriologist and Friedrich Neelsen (1854
to 1894), a pathologist. It is a special bacteriological stain
used to identify acid-fast organisms, mainly Mycobacteria.
Mycobacterium tuberculosis is the most important of this
group, as it is responsible for the disease called
tuberculosis (TB). It is helpful in diagnosing Mycobacterium
tuberculosis since its lipid rich cell wall makes it resistant
to Gram stain. It can also be used to stain few other
bacteria like Nocardia. The reagents used are Ziehl-Neelsen
carbolfuchsin, acid alcohol and methylene blue.
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Acid Fast of Mycobacterium
tuberculosis
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Negative Stain
India ink or nigrosin
Sometimes referred to as capsular stain
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Flagellar Stain
Salmonella typhimurium
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Endospore Stain

Used on spore
forming bacteria
such as Bacillus
sp.
Malachite green
stains spores
39