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

related to the resolution that can be obtained
Light is a wave and a particle
RESOLUTION refers to the ability to see two
Albert Einstein establishes that light consists of items as separate and discrete units rather than as
particles or discrete quanta. These particles later fuzzy, overlapped single image
became known as photons.
Microscopists use shorter and shorter wavelength
PROPERTIES OF LIGHT of electromagnetic radiation to improve resolution
Light is a wave that travels in straight lines. RESOLVING POWER (RP) of a lens is a
numerical measure of the resolution that can be
Light travels VERY FAST - around 300,000,000
obtained with that lens
meters per second.
PROPERTIES OF LIGHT
Light travels much faster than sound.
Reflection occurs if the light strikes an object and
We see things because they reflect light into our
bounces back
eyes.
Transmission refers to the passage of light through
Shadows are formed when light is blocked by an
an object.
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 It is a measure of the speed of light at which light
bounce off nor pass through an object but are passes through the material.
taken up by that object.
When two substances have different indices of
Luminescence is a phenomenon that occurs when refraction, light will bend as it passes from one
absorbed light rays are changed into longer material into the other.
wavelengths and reemitted.
Diffraction is the neding of light waves as they
Fluoresce happens when reemission occurs during pass through a small opening, such as hole, a slit, a
irradiation (when light rays are striking an object) space between two adjacent cellular structures.

Phosphorescence is when reemission continues Diffraction is a problem for microscopists

after irradiation 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.
PROPERTIES OF LIGHT THE MICROSCOPE
Refraction is the bending of light as it passes from MAJOR PARTS OF A COMPOUND LIGHT
one medium to another of different density. MICROSCOPE
The change in direction of a wave as is crosses the BASE - supporting structure that generally
boundary between two media in which the wave contains the light source
travels at different speeds.
CONDENSER - converges light beam to pass
Refraction of light is responsible for the image through the specimen
formation in our eyes and lenses
IRIS DIAPHRAGM - controls the amount of
Light passing through a glass microscope slide, light passing through the specimen
through air, and then through a glass lens is
refracted each time it goes from one medium to OBJECTIVE LENS - magnifies image
another.
BODY TUBE - conveys light to the ocular lens
Effect: loss of light and a blurred image
OCULAR LENS - magnifies the image from the
Solution: use immersion oil, which has the same objective.
index of refraction as glass, to replace the air
Monocular - one ocular lens
Result: The slide and the lens are joined by a
Binocular - two oculars
layer of oil; there is no refraction to cause the
image to blur MECHANICAL STAGE - allows precise control
in moving the slide
INDEX OF REFRACTION
COARSE ADJUSTMENT - knob used to locate
It is the ratio of speed of light in a vacuum to that
specimen
in the substance
FINE ADJUSTMENT - knob used to bring
specimen into sharp focus

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DARK FIELD MICROSCOPY USES

Bright-field illumination is used in the ordinary Visualizes live and unstained biological samples,
light microscope, with light passing directly such as a smear from a tissue culture or individual
through the specimen water borne single- celled organisms.

Dark-field illumination uses a special condenser FLUORESCENCE MICROSCOPY

that causes light to reflect off the specimen at an
angle rather than pass directly through it Fluorescence microscopy uses ultraviolet light
instead of white light to excite molecules within
USES the specimen or dye molecules attached to the
specimen. These molecules emit different
Bright-field illumination wavelengths, often brilliant colors
Vastly used in Biology, Cellular Biology, and USES
Microbiological Laboratory studies.
Visualization of bacterial agents such as
It can be used to identify basic bacteria cells and Mycobacterium tuberculosis.
parasitic protozoans such as Paramecium
Identify specific antibodies produced against
Dark-field illumination bacterial antigens/pathogens in
It is used to visualize the internal organs of larger immunofluorescence techniques by labeling the
cells such as the eukaryotic cells antibodies with fluorochromes.

Identification of bacterial cells with distinctive Used in ecological studies to identify and observe
shapes such as Treponema pallidum, a causative microorganisms labeled by the fluorochromes.
agent of syphilis. Differentiate between dead and live bacteria by the
PHASE CONTRAST MICROSCOPY color they emit when treated with special stains.

Phase Contrast Microscopy uses microscope CONFORAL MICROSCOPY

with special condensers that accentuate small Conforal microscopy uses laser light to obtain
difference in the refractive index of structures thin, focal- level sections through a specimen, with
within the cell, allowing, live, unstained organisms 40 times greater resolution, and less out-of-focus
to be examined light
USES USES
Determine morphologies of living cells such as Observing cellular morphology in multilayered
plant and animal cells specimen.
Studying microbial motility and structures of Used in diagnosing cervical cancer
locomotion
Evaluation and diagnosis of basal cell or
To detect certain microbial elements such as the carcinoma of skin.
bacterial endospores
DIGITAL MICROSCOPY
NOMARSKI (DIFFERENTIAL
INTERFERENCE CONTRAST) Digital microscopy uses computer technology to
MICROSCOPY automatically focus, adjust light, and take
photographs of specimens. These can be directly
Nomarski microscopy uses microscope that uploaded and viewed online
operates essentially like phase contrast
microscopes but with a much greater resolution ELECTRON MICROSCOPY
and a very short depth of field. They produce a
nearly 3D image 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 usage, have only been made possible with the help
focusing. They are much more expensive and of SEMs.
difficult to use but give magnification of up to
500,000X and a resolving power of less than 1 nm Criminal and other forensic investigations utilize
SEMs to uncover evidence and gain further
ADVANCED TYPES ELECTRON insight.
MICROSCOPY
In biological sciences, SEMs can be used on
Transmission Electron Microscope (TEM) anything from insects and animal tissue to bacteria
and viruses.
Uses a high voltage electron beam to illuminate
the specimen and create an image. Geological sampling using a scanning electron
microscope can determine weathering processes
Transmits electron through the specimen. and morphology of the samples.
The specimen must be sectioned into extremely MAGNIFICATION
thin slices (20-100nm thick) and stained or coated
with metals to increase image contrast. to determine the magnification, just multiply the
magnification of the ocular lens with the
Creates a two-dimensional image magnification of the objective lens.
USES Example:
TEMs are ideal for a number of different fields
 Ocular lens 10X,
such as life sciences, nanotechnology, medical,
 Objective lens 40X
biological and material research, forensic analysis,
 Total Magnification is 400X, which
gemology and metallurgy, and industry and
means that the object is 400X larger
education.
PREPARATION OF SPECIMENS FOR THE
TEMs provide topographical, morphological,
LIGHT MICROSCOPE
compositional and crystalline information.
Wet Mounts are used to view living organisms.
TEMs can be used in semiconductor analysis and
production and the manufacturing of computer and Hanging Drop - special version of wet mount,
silicon chips often used to determine whether organisms are
motile and is mainly used with dark-field
Scanning Electron Microscope (SEM)
illumination
SEM produces images by probing the specimen
Smears, in which microorganisms from a loopful
with a focused electron beam that is scanned
of medium are spread onto the surface of a glass
across a rectangular area of the specimen (raster
slide, can be used to view killed organisms. After a
scanning)
smear is made, it is allowed to air-dry completely.
Produces an striking three-dimensional realistic Then it is quickly passed 3 to 4 times through an
images. open flame (heat fixation process)

Magnifies external surface of specimen PRINCIPLES OF STAINING

USES Stain, or dye, is a molecule that can bind to a

cellular structure and give it color.
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.
SIMPLE STAINS
Just about any material science industry, from
aerospace and chemistry to electronics and energy

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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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