Microbiology and Pasitology

CHAPTER 1/ FEBRUARY/ PPT BASED/

MICROSCOPY AND STAINING

MICROSCOPY

is the technology of making very small thing

visible to the human eyes

METRIC UNITS
PARTS OF A TRANSVERSE WAVE
micrometer - 0.000001 m
Wavelength - the distance between two
nanometer - 0.000000001 m
adjacent crest of two adjacent troughs,
angstrom - 0.0000000001 m designated by the Greek letter lambda (λ)

WAVELENGTH AND RESOLUTION

The wavelength used for observation is

NATURE OF LIGHT
crucially related to the resolution that can be
Light is a wave and a particle obtained

Albert Einstein establishes that light consists of RESOLUTION refers to the ability to see two
particles or discrete quanta. These particles items as separate and discrete units rather
later became known as photons. than as fuzzy, overlapped single image

PROPERTIES OF LIGHT Microscopists use shorter and shorter

wavelength of electromagnetic radiation to
Light is a wave that travels in straight lines. improve resolution
Light travels VERY FAST - around RESOLVING POWER (RP) of a lens is a
300,000,000 meters per second. numerical measure of the resolution that can
Light travels much faster than sound. be obtained with that lens

We see things because they reflect light into PROPERTIES OF LIGHT

our eyes. Reflection occurs if the light strikes an object
Shadows are formed when light is blocked by and bounces back
an object. Transmission refers to the passage of light
through an object.

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Microbiology and Pasitology
CHAPTER 1/ FEBRUARY/ PPT BASED/
Solution: use immersion oil, which has the
same index of refraction as glass, to replace
* In order to see objects through a the air
microscope, light must either be reflected from
the objects or transmitted through them. Result: The slide and the lens are joined by a
layer of oil; there is no refraction to cause the
Absorption of light rays occurs when they image to blur
neither bounce off nor pass through an object
but are taken up by that object. INDEX OF REFRACTION

Luminescence is a phenomenon that occurs It is the ratio of speed of light in a vacuum to

when absorbed light rays are changed into that in the substance
longer wavelengths and reemitted.
It is a measure of the speed of light at which
Fluoresce happens when reemission occurs light passes through the material.
during irradiation (when light rays are striking
When two substances have different indices of
an object)
refraction, light will bend as it passes from one
Phosphorescence is when reemission material into the other.
continues after irradiation
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

PROPERTIES OF LIGHT smaller the lens must be, and therefore the
greater the diffraction and blurring it causes
Refraction is the bending of light as it passes
from one medium to another of different It is this diffraction, or spreading of light, that
density. makes it possible to observe magnified images
of specimens in the microscope, however it is
The change in direction of a wave as is crosses also diffraction that limits the size of objects
the boundary between two media in which the that can be resolved.
wave travels at different speeds.
THE MICROSCOPE
Refraction of light is responsible for the image
formation in our eyes and lenses MAJOR PARTS OF A COMPOUND LIGHT
MICROSCOPE
Light passing through a glass microscope
slide, through air, and then through a glass BASE - supporting structure that generally
lens is refracted each time it goes from one contains the light source
medium to another. CONDENSER - converges light beam to pass
Effect: loss of light and a blurred image through the specimen

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Microbiology and Pasitology
CHAPTER 1/ FEBRUARY/ PPT BASED/
IRIS DIAPHRAGM - controls the amount of PHASE CONTRAST MICROSCOPY
light passing through the specimen
Phase Contrast Microscopy uses microscope
OBJECTIVE LENS - magnifies image with special condensers that accentuate small
difference in the refractive index of structures
BODY TUBE - conveys light to the ocular lens within the cell, allowing, live, unstained
OCULAR LENS - magnifies the image from the organisms to be examined
objective. USES
Monocular - one ocular lens
Determine morphologies of living cells such as
Binocular - two oculars plant and animal cells

MECHANICAL STAGE - allows precise control Studying microbial motility and structures of
in moving the slide locomotion

COARSE ADJUSTMENT - knob used to locate To detect certain microbial elements such as
specimen the bacterial endospores

FINE ADJUSTMENT - knob used to bring NOMARSKI (DIFFERENTIAL INTERFERENCE

specimen into sharp focus CONTRAST) MICROSCOPY

DARK FIELD MICROSCOPY Nomarski microscopy uses microscope that

operates essentially like phase contrast
Bright-field illumination is used in the ordinary microscopes but with a much greater
light microscope, with light passing directly resolution and a very short depth of field. They
through the specimen produce a nearly 3D image
Dark-field illumination uses a special USES
condenser that causes light to reflect off the
specimen at an angle rather than pass directly Visualizes live and unstained biological
through it samples, such as a smear from a tissue
culture or individual water borne single- celled
USES organisms.
Bright-field illumination FLUORESCENCE MICROSCOPY
Vastly used in Biology, Cellular Biology, and Fluorescence microscopy uses ultraviolet light
Microbiological Laboratory studies. instead of white light to excite molecules
within the specimen or dye molecules
It can be used to identify basic bacteria cells
attached to the specimen. These molecules
and parasitic protozoans such as Paramecium
emit different wavelengths, often brilliant
Dark-field illumination colors

It is used to visualize the internal organs of USES

larger cells such as the eukaryotic cells
Visualization of bacterial agents such as
Identification of bacterial cells with distinctive Mycobacterium tuberculosis.
shapes such as Treponema pallidum, a
Identify specific antibodies produced against
causative agent of syphilis.
bacterial antigens/pathogens in

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Microbiology and Pasitology
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immunofluorescence techniques by labeling Transmits electron through the specimen.
the antibodies with fluorochromes.
The specimen must be sectioned into
Used in ecological studies to identify and extremely thin slices (20-100nm thick) and
observe microorganisms labeled by the stained or coated with metals to increase
fluorochromes. image contrast.

Differentiate between dead and live bacteria Creates a two-dimensional image

by the color they emit when treated with
USES
special stains.
TEMs are ideal for a number of different fields
CONFORAL MICROSCOPY
such as life sciences, nanotechnology, medical,
Conforal microscopy uses laser light to obtain biological and material research, forensic
thin, focal- level sections through a specimen, analysis, gemology and metallurgy, and
with 40 times greater resolution, and less out- industry and education.
of-focus light
TEMs provide topographical, morphological,
USES compositional and crystalline information.

Observing cellular morphology in multilayered TEMs can be used in semiconductor analysis

specimen. and production and the manufacturing of
computer and silicon chips
Used in diagnosing cervical cancer
Scanning Electron Microscope (SEM)
Evaluation and diagnosis of basal cell or
carcinoma of skin. SEM produces images by probing the
specimen with a focused electron beam that is
DIGITAL MICROSCOPY scanned across a rectangular area of the
Digital microscopy uses computer technology specimen (raster scanning)
to automatically focus, adjust light, and take Produces an striking three-dimensional
photographs of specimens. These can be realistic images.
directly uploaded and viewed online
Magnifies external surface of specimen
ELECTRON MICROSCOPY
USES
Electron microscope (EM) uses a beam of
electrons instead of a beam of light and SEMs can be used in a variety of industrial,
electromagnets instead of glass lenses for commercial, and research applications.
focusing. They are much more expensive and
SEMs are used in materials science for
difficult to use but give magnification of up to
research, quality control and failure analysis.
500,000X and a resolving power of less than 1
nm Just about any material science industry, from
aerospace and chemistry to electronics and
ADVANCED TYPES ELECTRON MICROSCOPY
energy usage, have only been made possible
Transmission Electron Microscope (TEM) with the help of SEMs.

Uses a high voltage electron beam to

illuminate the specimen and create an image.

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Criminal and other forensic investigations SIMPLE STAINS
utilize SEMs to uncover evidence and gain
use single dye; do not distinguish organisms or
further insight.
structures by different staining reactions
In biological sciences, SEMs can be used on
Examples: methylene blue, safranin, crystal
anything from insects and animal tissue to
violet
bacteria and viruses.
Result: methylene blue - uniform blue stain,
Geological sampling using a scanning electron
safranin – uniform red stain, crystal violet -
microscope can determine weathering
uniform purple stain
processes and morphology of the samples.
Uses: shows sizes, shapes, and arrangements
MAGNIFICATION
of cells
to determine the magnification, just multiply
DIFFERENTIAL STAINS
the magnification of the ocular lens with the
magnification of the objective lens. Use two or more dyes that react differently
with various kinds or parts of bacteria,
Example:
allowing them to be distinguished
• Ocular lens 10X,
Gram stain
• Objective lens 40X
• Total Magnification is 400X, which devised by Hans Christian Gram
means that the object is 400X larger
Result:
PREPARATION OF SPECIMENS FOR THE
LIGHT MICROSCOPE Gram + - purple with crystal violet

Wet Mounts are used to view living organisms. Gram - - red with safranin counterstain

Hanging Drop - special version of wet mount, Gram-variable - intermediate or mixed colors
often used to determine whether organisms
Gram-nonreactive - stain poorly or not at all
are motile and is mainly used with dark-field
illumination Uses: distinguish gram+, gram-, gram-
variable, and gram nonreactive organisms
Smears, in which microorganisms from a
loopful of medium are spread onto the surface Ziehl-Neelsen acid-fast stain
of a glass slide, can be used to view killed
organisms. After a smear is made, it is allowed Used to detect tuberculosis- and leprosy-
to air-dry completely. Then it is quickly passed causing organisms of the genus Myobacterium
3 to 4 times through an open flame (heat Negative stain
fixation process)
Allows visualization of organisms with
PRINCIPLES OF STAINING structures that will not accept most stains,
Stain, or dye, is a molecule that can bind to a such as capsules
cellular structure and give it color. Capsules appear clear against a dark
background

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Microbiology and Pasitology
CHAPTER 1/ FEBRUARY/ PPT BASED/

SPECIAL STAINS

Identify various specialized structures

Flagellar stain

indicates presence of flagella by building up

layers of stain on their surface

Schaeffer-Fulton spore stain

allows visualization of hard-to-stain bacterial

endospores such as members of genera
Clostridium and Bacillus

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