Microscope

Microscopic Observations
of
Microorganisms
MUHAMMED MAHFUZUR RAHMAN
Assistant Professor
Department of PHARMACY

Introduction & Definition
 The word Microscope comes from Greek word “mikrós”
which means “small” & “skopeîn” which means “to look”
or “see”.
 Microscopes are instruments designed to produce
magnified visual or photographic images of small objects.
 A microscope is an instrument used to see objects that
are too small for the naked eye.

Introduction & Definition
 The science of investigating small objects using such an
instrument is called microscopy.
 Microscopic means invisible to the eye unless aided by a
microscope.
 Zacharias Janssen invented the microscope in 1590.
 The microscope must accomplish three tasks-
 Produce a magnified image of the specimen
 Separate the details in the image
 Render the details visible to the human eye or camera

Parts of Microscope and their functions
 Eyepiece
 Ocular lens
 Nosepiece
 Objective lens
 Stage
 Stage clip
 Light switch
 Light intensity knob
 Fine adjustment
 Coarse adjustment
 Stage manipulator knobs
 Condenser
 Light source
 Iris diaphragm knob
 Cord holder
 Microscope body

Arm
Light Source
Diaphragm
Stage
Stage Clips
Revolving
Nosepiece
Objective
Lenses
Ocular Lens
Fine adjustment knob
Coarse adjustment
knob

 Eyepiece and Body Tube
 The eyepiece contains the ocular lens which
magnifies objects a given amount that is
listed on the eyepiece.
 The body tube supports the eyepiece and
objectives
 Nosepiece, Objectives, and Stage Clips
 The nosepiece holds the 3 objectives.
 The objective lenses range in magnification
from 4X, 10X, and 40X.
 The stage clips holds the slide in place.

 Stage, Light & Diaphragm
 The stage supports the slide
being viewed.
 The light source projects
upward through the
diaphragm, the specimen
and the lenses.
 The diaphragm regulates the
amount of light on the
specimen.

 Arm and Base
 The arm is used to support
the microscope when it is
carried.
 The base supports the
microscope.

 Coarse Adjustment Knob
 Moves the stage up and down
for focusing.
 Fine Adjustment Knob
 Moves the stage slightly to
sharpen the image.
 Used with the 10X and 40X
objective to focus.

Types of Microscopes
 Optical Microscope
 This is an optical instrument using visible
light and a system of lenses to magnify
images of small samples.
 Typical magnification is up to 1250x with a
theoretical resolution limit of around
0.250 micrometres or 250 nanometres.
 It includes:
 Bright-field microscopy
 Dark-field microscopy
 Fluorescence microscopy
 Phase contrast microscopy

 Electron Microscope
 Passes electrons through the sample or looks at the surface of
bulk objects by scanning the surface with a fine electron beam.
 Types: Transmission electron microscope (TEM) & Scanning
electron microscope (SEM)
Objective
Lens

 Scanning probe Microscope
 Scanning probe microscopes also analyze a single
point in the sample and then scan the probe over a
rectangular sample region to build up an image.
 Many scanning probe microscopes can image several
interactions simultaneously. The manner of using
these interactions to obtain an image is generally
called a mode.
 Different types: AFM (atomic force microscopy) , BEEM
(ballistic electron emission microscopy), CFM (chemical
force microscopy) etc.

Preparations for Microscopic Examinations
 The Wet-Mount & Hanging drop
technique
 Wet preparations permit examination of
organisms in a normal living condition.
 A wet mount is made by placing a drop of
fluid containing the organisms onto a glass
slide & covering the drop with a cover slip.
 A special slide with a circular concave is
sometimes used.
 A suspension of microbial specimen is
placed on a cover slip, then inverted over
the concave depression to produce a
“hanging drop” of the specimen.

Preparations for Microscopic Examinations
 The Fixed, stained smears
 They are most frequently used for the
observations of the morphological
characteristics of microbes.
 The essential steps are:
 The preparations of the film or smear
 Fixation
 Application of one or more staining
solutions
 Advantages are:
 More clearly visible after staining
 Differentiation of cells of diff. species
or within the same species

Bright-Field Microscopy
 Dark sample on a bright background
 The Microscopic field is brightly lighted & the microbes appear
dark because they absorb some of the light
 Usually, microbes do not absorb much light, but staining them
with a dye greatly increases their light absorbing capacity
 Bright field illumination is useful for samples which have an
intrinsic colour, for example chloroplasts in plant cells.
 Some of the light is absorbed by stains, pigmentation, or dense
areas of the sample and this contrast allows us to see the
specimen.

Dark-Field Microscopy
 Dark background against which objects are brilliantly
illuminated.
 This is accomplished by equipping with a special
condenser that transmits a hollow cone of light.
 Most of the light directed through the condenser does
not enter the objective, the field is dark.
 However, some of the light rays will be scattered if the
medium contains objects.
 The diffracted light will enter the objective & reach
the eye, thus the object will appear bright in an dark
background.
 Best for observing pale objects, unstained microbes.

Fluorescence Microscopy
 After absorbing light of a particular wavelength & energy, some substances
will then emit light of a longer wavelength & a lesser energy content. The
phenomenon is termed fluorescence.
 Application of this phenomenon is the basis of fluorescence microscopy.
 In practice, microbes are stained with fluorescent dye & then illuminated
with blue light; the blue light is absorbed & green light is emitted by the
dye.
 Example of fluorescent dyes:
Fluorochrome Excitation Emission
Fluorescein Isothiocyanate (FITC) ~480 nm (Blue) ~520 nm (Green)
Rhodamine Isothiocyanate (RITC) ~540 nm (Green) ~590 nm (Red)

Phase-Contrast Microscopy
 The phase contrast microscopy made it possible to study living cells
and how they proliferate through cell division.
 Used to examine living organisms or specimens that would be
damaged/altered by attaching them to slides or staining.
 It uses a conventional light microscope fitted with a phase-contrast
objective & phase-contrast condenser.
 Light passing through one material & into another material of
slightly different refractive index or thickness will undergo a change
in phase. This change in are translated into variations in brightness
of the structures.

Transmission Electron Microscopy
 Electrons scatter when they pass through thin sections
of a specimen
 Transmitted electrons (those that do not scatter) are
used to produce image
 Denser regions in specimen, scatter more electrons and
appear darker
 Allows the observation of molecules within cells

Transmission Electron Microscopy
Chloroplast from a tobacco leaf

Scanning Electron Microscopy
 The specimen is subjected to a narrow electron beam which
rapidly move over the surface of the specimen.
 This causes the release of a shower of a secondary electrons &
other type of radiations.
 The intensity of these secondary electrons depends on the
shape & chemical composition of the irradiated objects.
 The secondary electrons are collected by a detector which
generates an electrical signal. These signals are then scanned
in the manner of a television system to produce an image.

Scanning Electron Microscopy Images

Optical Microscope vs Electron Microscope
FEATURE Optical Microscope Electron Microscope
Electromagnetic
spectrum used
Visible light
760nm (red) –
390nm
Electrons
app. 4nm
Maximum resolving
power
app. 200nm 0.2nm
Maximum
magnification x1000 – x1500 x500 000
Radiation source Tungsten or quartz
halogen lamp
High voltage (50kV)
tungsten lamp
Lenses Glass Magnets
Interior Air-filled Vacuum
Focussing screen Human eye (retina) fluorescent (TV)
screen

FEATURE Optical Microscope Electron Microscope
Preparation of
specimens
Temporary mounts
living or dead
Tissues must be
dehydrated (dead)
Fixation Alcohol OsO4 or KMnO4
Embedding Wax Resin
Sectioning Hand or microtome
slices  20 000nm
Microtome only.
Slices  50nm
Stains Water soluble dyes Heavy metals
Support Glass slide Copper grid

Image produced by an
optical microscope
Image produced by an
electron microscope

Some Important Terminals used in
Microscopy

Resolving power
 The ability to distinguish two adjacent points as distinct & separate is
known as resolving power.
 Mere increase in size without the ability to distinguish structural details is
not beneficial.
 In other words, the largest magnification produce by a microscope may not
be the most useful because the image obtained may be unclear or fuzzy.
 The more lines or dots per unit area that can be seen separately, the greater
is the resolving power.
 It is a function of the wavelengths of lights used & the numerical aperture of
the lens system.

Numerical Aperture
 In optics, the numerical aperture (NA) of an
optical system is a dimensionless number
that characterizes the range of angles over
which the system can accept or emit light.
 The sine value of half-aperture angle
multiplied by the refractive index n of the
medium gives the numerical aperture (NA)
Thus,
NA= n sin θ

The limit of Resolution
 The limit of resolution is the smallest distance by which two objects
can be separated & still be distinguished as two separate objects.
 The greatest resolution in optical microscopy can be obtained with
the shortest wavelength of visible light & an objective with maximum
NA.
 The relationship between NA & limit of resolution can be expressed
as follows:
d=
2NA
λ Here,
d= Resolution
λ= Wavelength of light

Magnification
 Magnification beyond the resolving power is of no value since the
larger image will be less distinct in detail & fuzzy in appearance.
 The situation is analogous to the of a movie screen: If we move
closer to the screen the image is larger but is also less sharp than
when viewed from distance.
 Most laboratory microscopes are equipped with three objectives,
each capable of a different degree of magnification.
 The total magnification of the system is determined by magnification
of the objective by that of eyepiece.

Lets play a GAME!!
Look at the following micrographs
(a picture made by a microscope)
and try to determine what the
object is!

EYE OF A NEEDLE WITH THREAD RUNNING THROUGH IT

A HUMAN CELL INFECTED WITH HIV VIRUS

Staining of Specimens
 Increases contrast & resolution by coloring specimens with stains/dyes.
 Smear of microorganisms (thin film) made prior to staining.
 Microbiological stains contain chromophore.
 Acidic dyes stain alkaline structures; more commonly, basic dyes stain
acidic structures.
 Types of staining:
 Simple staining
 Differential staining
o Gram stain
o Acid fast stain
o Endospore stain
 Special staining
o Negative (capsule) stain
o Flagellar stain

Fixation
 Process by which internal and external structures are preserved and
fixed in position
 Process by which organism is killed and firmly attached to microscope
slide
 Heat fixing
o preserves overall morphology but not internal structures
 Chemical fixing
o protects fine cellular substructure and morphology of larger,
more delicate organisms

Dyes and Simple Staining
 Dyes
 Make internal and external structures of cell more visible by increasing
contrast with background
 Have two common features:
o Chromophore groups
 Chemical groups with conjugated double bonds which give dye
its color
o Ability to bind cells
 Simple staining
 A single staining agent is used
 Basic dyes are frequently used
o Dyes with positive charges
o E.g. crystal violet

Differential Staining
 Use a primary stain & a counter stain to distinguish cell types or
parts.
 Divides microorganisms into groups based on their staining
properties
 E.g., Gram stain, acid-fast stain, Schaffer-Fulton endospore stain
etc.
Endospore Stain
Gram Stain
Acid-fast Stain

Gram Staining
 Most widely used differential
staining procedure.
 The name comes from its inventor,
Hans Christian Gram.
 Gram staining differentiates
bacteria by the chemical &
physical properties of their cell
walls by detecting peptidoglycan,
which is present in a thick layer in
gram-positive bacteria.
Primary stain
Positive
Negative
Mordant
Decolorization
Counter stain

Gram Staining
A Gram stain of mixed Staphylococcus aureus (gram-positive cocci, in
purple) and Escherichia coli (gram-negative bacilli, in red)

Acid-fast Staining
 Particularly useful for staining
members of the genus
Mycobacterium.
 E.g., Mycobacterium tuberculosis
– causes tuberculosis
 E.g., Mycobacterium leprae
– causes leprosy
 High lipid content in cell walls is
responsible for their staining
characteristics

Schaeffer-Fulton endospore Staining
 Double staining procedure.
 Bacterial endospore is one color & vegetative cell is a different
color.

Negative Staining
 Often used to visualize capsules surrounding bacteria
 Capsules are colorless against a stained background.
Bacteria
Capsule
Background
stain

Flagellar Staining
 Bacterial flagella are normally too thin to be seen under normal
microscopic conditions.
 The flagella stains employs a mordant to coat the flagella with stain
until they are thick enough to be seen.
Flagella

Microscope

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