Microbiology and Parasitology

CHAPTER 1/ FEBRUARY/ PPT BASED/

MICROSCOPY AND STAINING

MICROSCOPY

is the technology of making very small thing

visible to the human eyes

METRIC UNITS

micrometer - 0.000001 m PARTS OF A TRANSVERSE WAVE

nanometer - 0.000000001 m Wavelength - the distance between two adjacent

crest of two adjacent troughs, designated by the
angstrom - 0.0000000001 m Greek letter lambda (λ)

WAVELENGTH AND RESOLUTION

NATURE OF LIGHT The wavelength used for observation is crucially

Light is a wave and a particle
Albert Einstein establishes that light consists of
particles or discrete quanta. These particles later
became known as photons.
PROPERTIES OF LIGHT
Light is a wave that travels in straight lines.
Light travels VERY FAST - around 300,000,000
meters per second.
Light travels much faster than sound.
We see things because they reflect light into our
eyes.
Shadows are formed when light is blocked by an
object.
related to the resolution that can be obtained
RESOLUTION refers to the ability to see two
items as separate and discrete units rather than as
fuzzy, overlapped single image
Microscopists use shorter and shorter wavelength
of electromagnetic radiation to improve resolution
RESOLVING POWER (RP) of a lens is a
numerical measure of the resolution that can be
obtained with that lens
PROPERTIES OF LIGHT
Reflection occurs if the light strikes an object and
bounces back
Transmission refers to the passage of light through
an object.

* In order to see objects through a microscope,

light must either be reflected from the objects or
transmitted through them.

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Absorption of light rays occurs when they neither
bounce off nor pass through an object but are
taken up by that object.
Luminescence is a phenomenon that occurs when
absorbed light rays are changed into longer
wavelengths and reemitted.
Fluoresce happens when reemission occurs during
irradiation (when light rays are striking an object)
Phosphorescence is when reemission continues
after irradiation
PROPERTIES OF LIGHT
Refraction is the bending of light as it passes from
one medium to another of different density.
The change in direction of a wave as is crosses the
boundary between two media in which the wave
travels at different speeds.
Refraction of light is responsible for the image
formation in our eyes and lenses
Light passing through a glass microscope slide,
through air, and then through a glass lens is
refracted each time it goes from one medium to
another.
Effect: loss of light and a blurred image
Solution: use immersion oil, which has the same
index of refraction as glass, to replace the air
Result: The slide and the lens are joined by a
layer of oil; there is no refraction to cause the
image to blur
INDEX OF REFRACTION
It is the ratio of speed of light in a vacuum to that
in the substance
It is a measure of the speed of light at which light
passes through the material.
When two substances have different indices of
refraction, light will bend as it passes from one
material into the other.
Diffraction is the neding of light waves as they
pass through a small opening, such as hole, a slit, a
space between two adjacent cellular structures.
Diffraction is a problem for microscopists
because the lens acts as a small aperture through
which the light must pass. A blurry image result.
The higher the magnifying power of a lens, the
smaller the lens must be, and therefore the greater
the diffraction and blurring it causes
It is this diffraction, or spreading of light, that
makes it possible to observe magnified images of
specimens in the microscope, however it is also
diffraction that limits the size of objects that can
be resolved.
THE MICROSCOPE
MAJOR PARTS OF A COMPOUND LIGHT
MICROSCOPE
BASE - supporting structure that generally
contains the light source
CONDENSER - converges light beam to pass
through the specimen
IRIS DIAPHRAGM - controls the amount of
light passing through the specimen
OBJECTIVE LENS - magnifies image
BODY TUBE - conveys light to the ocular lens
OCULAR LENS - magnifies the image from the
objective.
Monocular - one ocular lens
Binocular - two oculars
MECHANICAL STAGE - allows precise control
in moving the slide
COARSE ADJUSTMENT - knob used to locate
specimen
FINE ADJUSTMENT - knob used to bring
specimen into sharp focus
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DARK FIELD MICROSCOPY
Bright-field illumination is used in the ordinary
light microscope, with light passing directly
through the specimen
Dark-field illumination uses a special condenser
that causes light to reflect off the specimen at an
angle rather than pass directly through it
USES
Bright-field illumination
Vastly used in Biology, Cellular Biology, and
Microbiological Laboratory studies.
It can be used to identify basic bacteria cells and
parasitic protozoans such as Paramecium
Dark-field illumination
It is used to visualize the internal organs of larger
cells such as the eukaryotic cells
Identification of bacterial cells with distinctive
shapes such as Treponema pallidum, a causative
agent of syphilis.
PHASE CONTRAST MICROSCOPY
Phase Contrast Microscopy uses microscope
with special condensers that accentuate small
difference in the refractive index of structures
within the cell, allowing, live, unstained organisms
to be examined
USES
Determine morphologies of living cells such as
plant and animal cells
Studying microbial motility and structures of
locomotion
To detect certain microbial elements such as the
bacterial endospores
NOMARSKI (DIFFERENTIAL
INTERFERENCE CONTRAST)
MICROSCOPY
Nomarski microscopy uses microscope that
operates essentially like phase contrast
microscopes but with a much greater resolution
and a very short depth of field. They produce a
nearly 3D image
USES
Visualizes live and unstained biological samples,
such as a smear from a tissue culture or individual
water borne single- celled organisms.
FLUORESCENCE MICROSCOPY
Fluorescence microscopy uses ultraviolet light
instead of white light to excite molecules within
the specimen or dye molecules attached to the
specimen. These molecules emit different
wavelengths, often brilliant colors
USES
Visualization of bacterial agents such as
Mycobacterium tuberculosis.
Identify specific antibodies produced against
bacterial antigens/pathogens in
immunofluorescence techniques by labeling the
antibodies with fluorochromes.
Used in ecological studies to identify and observe
microorganisms labeled by the fluorochromes.
Differentiate between dead and live bacteria by the
color they emit when treated with special stains.
CONFORAL MICROSCOPY
Conforal microscopy uses laser light to obtain
thin, focal- level sections through a specimen, with
40 times greater resolution, and less out-of-focus
light
USES
Observing cellular morphology in multilayered
specimen.
Used in diagnosing cervical cancer
Evaluation and diagnosis of basal cell or
carcinoma of skin.
DIGITAL MICROSCOPY
Digital microscopy uses computer technology to
automatically focus, adjust light, and take
photographs of specimens. These can be directly
uploaded and viewed online
ELECTRON MICROSCOPY
Electron microscope (EM) uses a beam of
electrons instead of a beam of light and
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electromagnets instead of glass lenses for
focusing. They are much more expensive and
difficult to use but give magnification of up to
500,000X and a resolving power of less than 1 nm
ADVANCED TYPES ELECTRON
MICROSCOPY
Transmission Electron Microscope (TEM)
Uses a high voltage electron beam to illuminate
the specimen and create an image.
Transmits electron through the specimen.
The specimen must be sectioned into extremely
thin slices (20-100nm thick) and stained or coated
with metals to increase image contrast.
Creates a two-dimensional image
USES
TEMs are ideal for a number of different fields
such as life sciences, nanotechnology, medical,
biological and material research, forensic analysis,
gemology and metallurgy, and industry and
education.
TEMs provide topographical, morphological,
compositional and crystalline information.
TEMs can be used in semiconductor analysis and
production and the manufacturing of computer and
silicon chips
Scanning Electron Microscope (SEM)
SEM produces images by probing the specimen
with a focused electron beam that is scanned
across a rectangular area of the specimen (raster
scanning)
Produces an striking three-dimensional realistic
images.
Magnifies external surface of specimen
USES
SEMs can be used in a variety of industrial,
commercial, and research applications.
SEMs are used in materials science for research,
quality control and failure analysis.
Just about any material science industry, from
aerospace and chemistry to electronics and energy
usage, have only been made possible with the help
of SEMs.
Criminal and other forensic investigations utilize
SEMs to uncover evidence and gain further
insight.
In biological sciences, SEMs can be used on
anything from insects and animal tissue to bacteria
and viruses.
Geological sampling using a scanning electron
microscope can determine weathering processes
and morphology of the samples.
MAGNIFICATION
to determine the magnification, just multiply the
magnification of the ocular lens with the
magnification of the objective lens.
Example:
 Ocular lens 10X,
 Objective lens 40X
 Total Magnification is 400X, which
means that the object is 400X larger
PREPARATION OF SPECIMENS FOR THE
LIGHT MICROSCOPE
Wet Mounts are used to view living organisms.
Hanging Drop - special version of wet mount,
often used to determine whether organisms are
motile and is mainly used with dark-field
illumination
Smears, in which microorganisms from a loopful
of medium are spread onto the surface of a glass
slide, can be used to view killed organisms. After a
smear is made, it is allowed to air-dry completely.
Then it is quickly passed 3 to 4 times through an
open flame (heat fixation process)
PRINCIPLES OF STAINING
Stain, or dye, is a molecule that can bind to a
cellular structure and give it color.
SIMPLE STAINS
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use single dye; do not distinguish organisms or Schaeffer-Fulton spore stain
structures by different staining reactions
allows visualization of hard-to-stain bacterial
Examples: methylene blue, safranin, crystal violet endospores such as members of genera
Clostridium and Bacillus
Result: methylene blue - uniform blue stain,
safranin – uniform red stain, crystal violet -
uniform purple stain

Uses: shows sizes, shapes, and arrangements of

cells

DIFFERENTIAL STAINS

Use two or more dyes that react differently with

various kinds or parts of bacteria, allowing them to
be distinguished

Gram stain

devised by Hans Christian Gram

Result:

Gram + - purple with crystal violet

Gram - - red with safranin counterstain

Gram-variable - intermediate or mixed colors

Gram-nonreactive - stain poorly or not at all

Uses: distinguish gram+, gram-, gram-variable,

and gram nonreactive organisms

Ziehl-Neelsen acid-fast stain

Used to detect tuberculosis- and leprosy-causing

organisms of the genus Myobacterium

Negative stain

Allows visualization of organisms with structures

that will not accept most stains, such as capsules

Capsules appear clear against a dark background

SPECIAL STAINS

Identify various specialized structures

Flagellar stain

indicates presence of flagella by building up layers

of stain on their surface

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