The Microscope

A microscope is an instrument for producing enlarged images of objects too small to be seen
unaided.

Types of Microscopes
Microscopes are of two types:
Light (optical) and electrons depend on the principle on which magnification is done.

The Light Microscope

This is a type of microscope in which magnification is obtained by a system of optical lenses
using light waves. It includes:
1. Bright Field Microscope
2. Dark Field Microscope
3. Fluorescence Microscope
4. Phase Contract Microscope

Modern microscopes are compound microscopes. That is, the magnified image formed by the
objective lens is further enlarged by one or more
additional lenses.

Most undergraduate students of microbiology perform most of their examinations with the bright
field microscope which is the most widely used instrument for routine microscopic work. The
other types of microscope are used for special purposes or research investigation.

The Bright Field Microscope

The ordinary microscope is called a bright field microscope because it forms a dark image
against a brighter background.

The microscope consists of a sturdy metal body or stand made up of a base and an arm to which
the remaining parts are attached.

A light source, either a mirror or an electric illuminator, is located at the base.

Two focusing knobs, the fine and coarse adjustment knobs are located on the arm and can move
either the stage or the nose piece to focus the image.

The stage is positioned about halfway up the arm and holds microscope slides by slide clips or a
mechanical stage clip.
There is a substage condenser mounted within or beneath the stage which focuses a core of light
on the slide.

The upper part of arm of the microscope holds the body assembly to which a nose piece and
one or more eyepieces or ocular lenses are attached.

Most advanced microscopes have eyepieces for both eyes and are called binocular microscopes.

The nose piece holds three to five objective lenses of different magnifying powers and is easily
rotated to position any objective.

The image you see when viewing a specimen is focused on the objective and ocular lenses
working together.

Light from the specimen that has been illuminated is focused by the objective lens creating an
enlarged image within the microscope. The ocular lens further magnifies this primary image.

The total magnification is calculated by multiplying the objective and eye piece magnification
together; e.g. if a 45x objective is used with a 10x eyepiece, the overall magnification of the
specimen will be 450x.

The Dark-Field Microscope

The dark field microscope is used to observe living unstained cells and organisms as a result of
change in the way they are illuminated.

A hollow core of light is focused on the specimen in such a way that unreflected and unrefracted
rays do not enter the objective; only light that has been reflected or refracted by the image
forms an image.
The field surrounding the specimen appears black while the object itself is brightly illuminated.

The dark field microscope is useful in revealing many internal structures in larger eukaryotic
microorganisms. It is also used in the examination of unstained microorganisms suspended in
fluids, e.g. wet mount and hanging drop preparation.

The Phase-Contrast Microscope

This type of microscope converts slight differences in refractive index and cell density into easily
detected variations in light intensity and is used to view living cells. The background formed by
the undeviated light is bright while the unstained objects appear dark and well-defined.
This microscope is very useful for studying microbial motility, determining the shape of living
cells, and detecting some bacterial components such as endospores and inclusion bodies. It is
also used in studying eukaryotes.

The Fluorescent Microscope

This type of microscope exposes a specimen to ultraviolet, violet or blue light and forms an
image of the object with resulting fluorescent light.

The most commonly used fluorescence microscope light is the epifluorescence microscope
which is also called incident light or reflected light microscope. Epifluorescence microscope
employs an objective lens that also acts as a condenser. A mercury vapour arc lamp or other
source produces an intense beam of light that passes through an exciter filler. The exciter filler
transmits on the desired wavelength of excitation light.

The excitation light is directed down the microscope by a speed minor called the dichromatic
minor. This minor reflects light of shorter wavelength but allows light of longer wavelength to
pass through. The excitation light continues down through the objective lens to a specimen
stained with spaced dye molecules called fluorochromes.

Microscope Resolution
Resolution is the ability of a lens to separate or distinguish between small objects that are close
together, i.e. the microscope must produce a clear image and not just a magnified one. It is also
known as the resolving power.

Resolution is described mathematically by an equation in the 1870s by Ernest Abbe, a German

physicist. The Abbe equation states that the minimal distance (d) between two objects that reveal
them as separate entities depends on the wavelength of light () used to illuminate the specimen
and on the numerical aperture of the lens (nsin) which is the ability of the lens to gather light.
d = 0.5
NA=n×sin(θ)
as d becomes smaller, the resolution increases, and finer details can be discerned in a specimen;
d becomes smaller as the wavelength of light used decreases and as the numerical aperture (NA)
increases.

Hence, the greatest resolution is obtained using a lens with the largest NA and light with the
shortest wavelength.

Preparation for Light-Microscope Examination

There are two general methods used for preparing specimens for light-microscope examination.
i The organisms are suspended in a liquid (the wet-mount or the hanging drop technique), and
ii The organism is dried fixed, and stained before observing under the microscope.

The Wet Mount or Hanging Drop Technique

The technique permits the examination of organisms in a normal living condition. A wet mount
is made by placing a drop of fluid containing the organisms on a glass slide and covering the
drop with a cover slip.
Petroleum jelly may be used to provide a seal between the slide and cover slip after which the
slide is viewed under the microscope.
This method is desirable because,
● it prevents distortion of the morphology of spiral bacteria when they are stained and dried
● it reveals whether organisms are motile or not
● some cell inclusion bodies are easily observed
● spore formation and germination may also be observed in living cells.

Fixed, Stained Smears of Microorganisms

These are frequently used for the observation of the morphological characteristics of bacteria.
The procedure makes the cell more clearly
visible, and differences between cells of different species and within the same species can be
demonstrated. The essential steps in this procedure
are:
1. preparation of the film or smear
2. fixation and
3. application of one or more staining solutions.

Fixation
Fixation is the process by which the internal and external structures of cells and microorganisms
are preserved and fixed in position. It in-activates enzymes that might disrupt cell morphology
and tough cell structures so that they do not change during staining and observation. A
microorganism usually is killed and attached firmly to the microscope slide during fixation.

There are two fundamentally different types of fixation.

1. Heat Fixation: This is routinely used to observe prokaryotes. Typically, a film of cells (a
smear) is gently heated as a slide is passed through a flame. Heat fixation preserves overall
morphology but not structures within cells
.
2. Chemical Fixation: Is used to protect fine cellular sub-structure and the morphology of larger,
more delicate micro organisms.
Chemical fixatives penetrate cells and react with cellular components, usually proteins and
lipids, to render them inactive, insoluble, and immobile. Common fixative mixtures contain such
components as ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.

Staining of Specimens
Although living microorganisms can be directly examined with the light microscope, they often
must be fixed and stained to increase visibility,
accentuate specific morphological features, and preserve them for future study.

Types of Staining
Simple staining
This is a kind of staining in which a single stain or dye is used.
Basic dyes such as crystal violet, methylene blue, and carbol fuchsin are used in simple
staining to determine the size, shape, and arrangement of prokaryotic acids.

Differential staining
These are staining procedures that make visible the differences between bacterial cells or part of
a bacterial cell. It usually involves more than one dye used for staining.

Gram staining
The Gram stain was developed in 1884 by the Danish physician Christian Gram. It is the most
widely used differential staining procedure.
The steps involved are as follows:
i The smear is stained with crystal violet (which is the primary stain).
ii This is followed by treatment with iodine functioning as a mordant.
iii The smear is decolorised by washing with ethanol or acetone.
iv The smear is counterstained with a simple dye safranin.
Bacteria stained by the Gram stain method fell into two groups:
Gram-positive bacteria which retain the crystal violet and appear deep violet in colour and
Gram-negative bacteria which, lose the crystal violet and are counterstained with safranin appear
red in
colour.

Acid-fast staining
This is another differential staining procedure commonly used to identify Mycobacterium
tuberculosis and Mycobacterium leprae, the pathogens responsible for tuberculosis and leprosy
respectively.

These bacteria have cell walls with high lipid content, in particular, mycolic acid which prevents
dye from readily binding to the cells.
In the acid fast staining procedure, the red stain and carbol fuchsin is used as primary stain;
next acid-alcohol is used as a decolouriser. The acid-alcohol will remove the red stain form
bacteria such as Escherichia coli while the acid fast mycobacteria will remain red.

Electron Microscope
This type of microscope uses a beam of electrons in place of light waves to produce the image.
There are two types:
● scanning electron microscope
● transmission electron microscope.

The Transmission Electron Microscope

Electron microscopes use a beam of electrons to illuminate and create magnified images of
specimens. Electrons replace light as the illuminating beam. They can be focused, much as light
is in a light microscope, but their wavelength is around 0.005mm approximately 1000,000 times
shorter than that of visible light. Therefore, electron microscopes have a practical resolution
roughly 1,000 times better than the light microscope, with many electron microscopes point
closer than 0.5nm can be distinguished, and the useful magnification is well over 100,000x. In
the transmission electron microscope, the electron beam is transmitted through the specimen.

The Scanning Electron Microscope

The scanning electron microscope produces an image from electrons released from atoms on an
object‟ 's surface. It has been used to examine
the surfaces of microorganisms in great detail. Many SEM has a resolution of 7nm or less.