Transcript A Brief History of Microbiology

Tools of the
Laboratory: The
Microscope
Chapter 3
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Learning Goals
• Name parts of the microscope and describe their function
• List and describe the three elements of good microscopy:
magnification, resolution, and contrast.
• Identify a use for darkfield, phase-contrast, differential interference
contrast (DIC), fluorescence, confocal, transmission and scanning
electron microscopy, and compare each with brightfield
illumination.
• Explain the purpose for simple staining. Differentiate between
positive and negative staining.
• List the steps in preparing a Gram stain, and describe the
appearance of G+ and G- cells after each step.
• Compare and contrast Gram stain and acid-fast stain
• Explain why each of the following is used: capsule stain,
endospore stain, flagella stain.
Viewing Microbial Cells
• Too small for the
human eye
• Need to magnify
the image
• Leeuwenhoek
used simple
microscopes
• Hooke used an
early compound
microscope
Compound Microscope
Microscope provides
• Magnification: creates an optical image of the
object which appears enlarged.
• Resolution: capacity to separate two adjacent
objects.
• Contrast.
Refraction
• Refraction:
bending or
changing an
angle of the
light ray as it
passes
through a
medium such
as lens
• Magnification
of an object by
a compound
microscope
occurs in two
phases
Resolution
• Resolution is the ability to distinguish two adjacent
objects or points from one another
• Also called resolving power (smaller is better)
Resolving power (RP) = Wavelength of light in nm
2 x Numerical aperture
• Shorter wavelengths provide a better resolution
• Numerical aperture is how well the lens gathers
light
Effect of Wavelength on Resolution
The Oil Immersion Lens
• Immersion oil reduces
refraction of light
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Objective lens
• More light is gathered
• Numerical aperture
increases
• Resolution improved
Air
Oil
Slide
Increasing Contrast
• Most microbes are…
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(a)
Simple Stains
• Colorless
• Very Small
• Difficult to see
• Staining increases
contrast by increasing
refractive index
• Increases size
sometimes
Crystal violet stain of
Escherichia coli
Methylene blue
stain of Corynebacterium
a1: © Kathy Park Talaro; a2: © Harold J. Benson
Conclusions:
• The most common microscope used in microbiology is
the compound light microscope. It uses visible light.
• The total magnification of an object is calculated by
multiplying the magnification of the objective lens by the
magnification of the ocular lens
• The maximum resolution the ability to distinguish between
two points) of a compound light microscope is 0.2m,
maximum magnification is 2000x.
• Immersion oil is used with the oil immersion lens to
reduce light loss between the slide and the lens
• Specimens are stained to increase the difference
between the refractive indexes of the specimen and the
medium
Light Microscopy
• Bright-field
• Dark-field
Light Microscopy
• Phase contrast
• DIC
Brightfield
Darkfield
Phase-contrast
Light Microscopy
• Fluorescence
• Confocal
Electron Microscopy
• Focused electron beam
• Wavelength = 6pm
• Magnification100,000 X
• Offers much better
resolution
Conclusions
• Brightfield illumination is used for stained smears.
• Unstained cells are more productively observed using dark
field, phase-contrast, or DIC microscopy.
• The darkfield microscope shows a light silhouette of an
organism against a dark background. It is most useful for
examining living organisms that are invisible in brightfield, do
not stain easily, or are distorted by staining.
• A phase contrast microscope brings direct and reflected light
rays together to form an image. It allows detailed observation
of living organisms.
• The DIC microscope provides a colored, three-dimensional
image of the object. It allows detailed observation of living
cells.
Conclusions:
• In fluorescence microscopy, specimens are first stained with
fluorochromes and then viewed through a compound
microscope by using an ultraviolet as a light source. The
organisms appear as bright objects against a dark background.
• In confocal microscopy, a specimen is stained with a fluorescent
dye and illuminated one plane at a time. Using computer, twoand three-dimensional images can be produced.
• A beam of electrons, instead of light, used in an electron
microscope. Electromagnets, instead of glass lenses, control
focus, illumination, and magnification.
• Thin sections of microorganisms can be observed using
transmission electron microscopy (TEM). Three-dimensional of
the surfaces of microorganisms can be obtained with scanning
electron microscope (SEM)
Microbial Stains
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• Simple staining:
Positive staining
Negative staining
• Differential staining:
Gram staining
Acid fast staining
Spore staining
• Special staining
Capsule staining
Flagella staining
Differential
(a) Simple Stains (b)
Stains
Crystal violet
Gram stain
stain of Escherichia Purple cells are G+
coli
Red cells are G -
(c) Special Stains
India ink capsule stain of
Cryptococcus neoformans
Acid-fast stain
Flagellar stain of Proteus
Red cells are acid-fast.
vulgaris
Methylene blue
Blue cells are nonA basic stain was used to
stain of
acid-fast.
build up the flagella.
Corynebacterium
Spore stain, showing spores (red)
and vegetative cells (blue)
Gram Staining
• The most universal
diagnostic staining
technique
• Differentiation of
microbes as gram
positive(purple) or
gram negative (red)
Gram stain
Purple cells are gram-positive.
Red cells are gram-negative.
Gram Staining Procedure
Over-decolorized
Positive control
Under-decolorized
Conclusions:
• Staining means coloring microorganisms with a dye to
make some structures more visible.
• Fixing uses heat or alcohol to kill and attach
microorganisms to a slide.
• Simple stains make cellular shapes and arrangements
visible. Bacteria are negatively charged, and the colored
positive ion of basic stain will stain bacterial cells. The
colored negative ion of an acidic dye will stain the
background of a bacterial smear producing a negative
stain.
• Differential stains differentiate bacteria according to their
reaction to the stains.
Conclusions
• The gram stain procedure uses a purple stain (crystal
violet), iodine as a mordant, an alcohol decolorizer, and a
red counterstain. G+ bacteria retain the purple stain after
the decolorization step; G- bacteria do not, and thus
appear pink from the counterstain.
• Acid-fast microbes, such as members of genera
Micobacterium and Nocardia, retain carbolfuchsin after
acid-alcohol decolorization and appear red; non-acid-fast
microbes take up the methylene blue counterstain and
appear blue.
• Negative staining is used to make microbial capsules
visible.
• The endospore stain and flagella stain are special stains
that only color certain parts of bacteria.