Roll Number 2k20/zoo/48

Name Japna kumri

D/o Abhimuni

Assignment Biological Techniques

Submitted to Nadir Ali Birmani

Department Zoology University of Sindh

 THE ELECTRON MICROSCOPES
The electron microscope is an instrument which utilizes the short wavelength of an
electron beam, rather than light waves, to obtain very high magnification and resolution
of minute structures for which a light microscope is inadequate.
It contains an electric gun whose beam is refracted and focused onto a specimen by an
electron lens system. The image of the specimen is magnified and projected onto a
stage or fluorescent screen.
On the basis of several earlier researches in Physics, Knoll and Ruska (1931) of Berlin
were the first to develop an electron microscope. With the help of their electron
microscope, they could magnify objects up to 12,000 times.
By an improved type of electron microscope developed by Borries and Ruska (1938),
they could obtain pictures of 20,000 times magnification. In this the magnification of
objective coil was 100 and the projector coil was 200. The resolution of this microscope
was 100Å. The electron microscopes with 4-10Å or even better resolution are now
available.

Construction:
An electron microscope consists of an electric gun, microscope column,
electromagnetic coils, a fluorescent screen and some other accessories
described below:
The Electron gun
(a) The electron gun is located at the top of the body of microscope. It is the source of
electrons. It is made up of a tungsten filament surrounded by a negatively biased shield
with an aperture. The electron beam is drawn off through this aperture.

The Microscope column

(b) The microscope column or central column is made up of an evacuated metal tube. It
protects the person operating the microscope from X-rays that are generated when the
electrons strike the surface of the metal tube.
The Electromagnetic coils
(c) The electromagnetic coils or lenses include projector coils, objective arid condenser.
In each coil, the coils of electric wire are wound on a hollow metallic cylinder. The
magnetic field, produced by passing the electric current through the magnetic coil,
functions as a magnifying lens.

The Fluorescent screen

(d) The fluorescent screen is used for observing the magnified image of the object. It
remains coated with a chemical which, on being excited, forms the image as on the
screen of television.

Other essential accessories

(e) Some other essential accessories of the electron microscope include high voltage
transformers (for developing high voltage current for the electron gun and
electromagnetic coils), vacuum pumps (for maintaining high vacuum inside the
microscope column), a water cooling system (for prevention from overheating of
various parts), a circulating pump, a refrigeration plant and also a filter system.
All these parts require elaborate arrangements and contribute to the massive size of the
electron microscope.

Image formation
The image formation in this microscope occurs by the scattering of electrons. The
electrons strike the atomic nuclei and get dispersed. These dispersed electrons form the
electron image. By projecting on a fluorescent screen or photographic film, this electron
image is converted into a visible image of the object.

The Electron beam

The electron beam in this microscope is made by accelerating electrons through a
potential difference of from 1-1500 kV in an electron gun.

Specimens
Only dried specimens are studied by electron microscope. Living cells cannot be studied
with this microscope because they possess water which causes large scale scattering of
electrons.

Ultrathin sections
Ultrathin sections (10-50 nm thickness), which are more than 200 times thinner than
those routinely used for light microscopy, are cut for electron microscopy. These are cut
with the help of diamond or glass knives of an ultra-microtome.

Working Principle:

An electron microscope uses an ‘electron beam’ to produce the image of the object and
magnification is obtained by ‘electromagnetic fields’; unlike light or optical microscopes,
in which ‘light waves’ are used to produce the image and magnification is obtained by a
system of ‘optical lenses’.

It has already been discussed that, the smaller is the wavelength of light, the greater is
its resolving power. The wavelength of green light (=0.55µ) is 1, 10,000 times longer
than that of electron beam (=0.000005µ or 0.05 Å; 1µ = 10,000 Å).
That is why, despite its smaller numerical aperture, an electron microscope can resolve
objects as small as 0.001µ (=10 Å), as compared to 0.2µ by a light microscope. Thus, the
resolving power of an electron microscope is 200 times greater than that of a light
microscope. It produces useful magnification up to X 400,000, as compared to X 2000 in
a light microscope. Thus, the useful magnification is 200 times greater in an electron
microscope than that in a light microscope.

There are three types of electron microscopes as described below:

(1) Transmission Electron Microscope (TEM):

In this microscope, an electron beam from an electron gun is transmitted through an

ultra-thin section of the microscopic object and the image is magnified by the
electromagnetic fields. It is used to observe finer details of internal structures of
microscopic objects like bacteria and other cells.

The specimen to be examined is prepared as an extremely thin dry film or as an ultra-

thin section on a small screen and is introduced into the microscope at a point between
the magnetic condenser and the magnetic objective

The point is comparable to the stage of a light microscope. The magnified image may be
viewed on a fluorescent screen through an airtight window or recorded on a
photographic plate by an in-built camera. Modern variants have facility to record the
photograph by digital camera.
 (2) Scanning Electron Microscope (SEM):

In a scanning electron microscope, the specimen is exposed to a narrow electron beam

from an electron gun, which rapidly moves over or scans the surface of the specimen
(Figure 4.13). This causes the release of a shower of secondary electrons and other
types of radiations from the specimen surface.

The intensity of these secondary electrons depends upon the shape and the chemical
composition of the irradiated object. These electrons are collected by a detector, which
generates electronic signals. These signals are scanned in the manner of a television
system to produce an image on a cathode ray tube (CRT).

The image is recorded by capturing it from the CRT. Modern variants have facility to
record the photograph by digital camera. This microscope is used to observe the surface
structure of microscopic objects.
 (3) Scanning and Transmission Electron Microscope (STEM):

It has both transmission and scanning electron microscope functions.

Applications
1) Electron microscopes are used to investigate the ultrastructure of a wide range of
biological and inorganic specimens including microorganisms, cells, large
molecules, biopsy samples, metals, and crystals.

2) Industrially, electron microscopes are often used for quality control and failure
analysis.

3) Modern electron microscopes produce electron micrographs using specialized

digital cameras and frame grabbers to capture the images.

4) Science of microbiology owes its development to the electron microscope. Study

of microorganisms like bacteria, virus and other pathogens have made the
treatment of diseases very effective.

Advantages

 Very high magnification

 Incredibly high resolution
 Material rarely distorted by preparation
 It is possible to investigate a greater depth of field
 Diverse applications

Limitations of Electron Microscopes:

The limitations of electron microscopes are as follows:

(a) Live specimen cannot be observed.

(b) As the penetration power of electron beam is very low, the object should be
ultra- thin. For this, the specimen is dried and cut into ultra-thin sections
before observation.
 Preparation of Permanent Slides
Introductions
Permanent slides are fundamental to preserve small specimens for scientific collections,
because temporary slide preparations can easily result in the loss of body parts in the
passage between slides and vial.
Permanent slides can be stored for a long time, provided that they were properly made.
The specimens have to be properly conserved and cut into thin sections before they are
mounted on the slide. Most permanent slides use a mounting medium which turns
solid. These are the most stable. Properly made permanent slides can remain useful for
a century. As a matter of fact, microscope slides from the 19th century are collector’s
items.
It is also possible to make permanent slides with a liquid mounting medium. In this case
the specimen is suspended in a small amount of liquid mounting medium and the cover
glass is fixed to the slide with nail polish. Permanent slides using a liquid mounting
medium are difficult to make and do have to be stored horizontally. I have also hear
them to be referred to as semi-permanent slides.

There are seven main processes involved in preparation of

permanent slides.

1. Killing
2. Fixing and Hardening
3. Staining
4. Dehydration
5. Clearing
6. Mounting
7. Labelling.

Process # 1. Killing:

It is the first step in the preparation of permanent mounts and is of

prime importance. By killing we mean the instantaneous stoppage of
all the activities of life in their respective original state without giving
the tissue enough opportunity to undergo any post-mortem changes.
This can only be achieved by not allowing any change in the form of
the tissue through use of reagents which are quick acting.

The best killing agents are:

1. 0.1% osmic acid and

2. Ether

Another good reagent which is most commonly used is absolute alcohol. But this
reagent has one major drawback, that it shrinks and contorts the tissue.

Process of Killing:
(1) The material to be killed is placed on a slide on which a very thin film of Mayer’s
albumen has been spread uniformly by rubbing a drop of albumen on surface of
slide vigorously with index finger.

(2) (a) The slide is either inverted for few seconds on the mouth of the bottle in which
osmic acid crystals have been put in distilled water.

Or

A few drops of ether or absolute alcohol are placed on the slide over the object
(material) and are allowed to evaporate.

Process # 2. Fixing and Hardening:

The fixing and hardening is the second major step in slide preparation. In case of minute
living organisms it is usually attained by the killing agents alone but in case of sections of
larger organisms or tissues it may be done through various fixing agents. The process of
fixing has multifarious importance i.e.

(1) Fixing stops any alteration in the form of tissues.

(2) It hardens the tissue and makes them fit for extensive subsequent treatment.

(3) It makes the tissue suitable and susceptive for the action of stains and various
other reagents.
 (4) Fixing agents make various constituents and components of a tissue optically
differentiated by changing their refractive indices.

The various important fixing agents used are:

(1) Bouin’s fluid,

(2) Mercuric chloride,
(3) 70% Alcohol,
(4) Acetic acid,
(5) Formaline (formaldehyde),
(6) Potassium dichromate and
(7) Osmic acid (osmium tetra-oxide).

After fixing in any of the aforesaid fixatives thorough washing of

material is very essential otherwise the tissue will be completely
spoiled, various mediums used for washing are:

(1) 70% Alcohol (for Bouin’s)

(2) Iodine+ 70% alcohol, (for mercuric chloride)
(3) 50% alcohol (for acetic acid)
(4) 70% alcohol (tor formaldehyde)
(5) Water and 0.12% chloral hydrate (for K2Cr2O7)
(6) Water (for osmic acid and K2C2O7)

Process # 3. Staining:

The process of colouring of various components and parts of a tissue for purpose of
clear and absolute differentiation through use of different dyes (strains) is called
staining. The nature of dyes may be acidic, basic or neutral.

Usually the acidic dyes stain (colour) the cytoplasmic part and basic dyes colour the
nuclear part. However, with a nuclear dye cytoplasm may also be stained. For the
purpose of undergraduates usually general staining (single staining) is used, which may
colour both nucleus and cytoplasm at the same time.
But, where specific stains are used, usually they are used in combinations of two
(double staining), three (triple staining), four or even more stains.

i. Single Staining:
For purpose of single staining the dyes used are picro-indigo-carmine or borax carmine
in 70% alcoholic solution or pircrocarmine in water.

Procedure:
The material after thorough washing is passed through the gradually increasing
percentage of the medium in which stain is made e.g., since the borax carmine or picro-
indigo-carmines are usually made in 70% alcohol the material should first be passed
through 30% alcohol, 50% alcohol and 70% alcohol for at least 3 minutes in each.

After the material has been saturated with the medium, it is put in the stain for about 3
minutes or till the material becomes dark red (Borax carmine) or dark green (Picro-
indigocarmine).

After staining, the material is washed with the medium or solvent (70% alcohol In case
of borax carmine and Picro-indigocarmine). If the stain is over it may easily be removed
from the tissue through use of acid alcohol in which material is put for few minutes (1-2
minutes) and after every one or two minutes it is washed in 70% alcohol and examined
under microscope, till desired results are achieved.

ii. Specific Staining:

For the purpose of double staining usually Delafleld’s Haematoxylin in distilled water
and Eosin in 70% alcohol are used.

Procedure:
The material, after thorough washing, is put in Haematoxylin for 5 minutes till it is dark
or blackish blue3. The material, after staining in haematoxylin, is passed through the
medium or solvent in which second stain is prepared e.g. since Eosin is prepared in 70%
alcohol the material is passed through 30% ale., 50% alc. and 70% alcohol and is kept in
Eosin for one minute. After staining it is just washed through a dip in 90% alcohol (never
use 70% alcohol).

Process # 4. Dehydration:
The process by which the traces of water present in the tissue are removed and
replaced by alcohol, in which clearing agent and solvent of mounting medium are
readily soluble, is called dehydration. It is done because water is un-miscible with
mounting medium, its solvent (usually Xylene) and the clearing agent (usually Xylene or
Benzene).

The dehydration is achieved by passing the tissue through gradually increasing

percentage of alcohol. Otherwise, if we directly put the material in absolute alcohol it
will shrink because of sudden loss of water.
The material is thus passed through 30%, 50%, 70%, 90% and 100% or absolute alcohol.
To achieve proper dehydration the material after absolute alcohol, should once again be
placed in fresh absolute alcohol for 3-5 minutes.

The alcohol may be better substituted by Cello solve (Ethylene glycol-mono-ethyl- ether)
because it freely mixes with alcohol, water, xylol and clove oil etc. and it doesn’t shrink
the tissue. As such it avoids repeated changes of alcohol in gradually increasing
strength.

1. It is always advisable to use regressive staining method i.e., the tissue should be first
over stained and then destained till desired depth of colour is achieved.
2. After single and double staining the excessive stair is removed by washing the tissue
in acid alcohol (or alcohol for prepared stains) or acid water (for water prepared stains)
for very short periods repeatedly till desired differentiation is achieved.

3. It is always advisable to use either staining tube or cavity blocks (covered) or any
thing else with a lid to avoid entry of atmospheric moisture, excessive
evaporation of alcohol and reagent and to safeguard against the loss of material.

Process # 5. Clearing (Dealcoholization):

The substitution of dehydrating agent (alcohol or cello solve) by the solvent of mounting
medium is called clearing. The term clearing is also used because of the fact that the
solvent or clearing agent imparts transparency to tissue.

The best clearing agents are Cedar wood oil and Clove oil but the most commonly used
reagent is Xylene. In its place Benzene may also be used Xylene makes the tissue hard
and brittle and also causes its shrinkage. As such it may be avoided if possible and
should be replaced by Cedar wood oil or Clove oil.

Procedure:
The material after absolute alcohol is placed in xylene or any other clearing agent. If the
clearing agent turns turbid or white, it shows that dehydration is not complete. Put the
material back in absolute alcohol for 5 minutes and then in clearing agent for 5 minutes
or till it becomes transparent. Still if turbidity comes give the material 2-3 changes in
clearing agent.

Process # 6. Mounting:

The material after it has been made transparent is transferred to a drop of mounting
medium which is placed in the centre of slide and is covered by a cover slip.

The mounting medium should be of the same refractive index as crown glass (R.I. 1.5).

The best mounting mediums are:

(1) Canada balsam dissolved in xylene (1.4 refractive index)

(2) Euparol (1.4 refractive index)

Procedure:
With a glass rod put a small drop of anada balsam in the centre of slide. Transfer the
material from Xylene to this drop with a brush. Take a cover slip and put its one edge on
the slide touching the balsam. The other end of cover slip should be held obliquely through a
needle as shown in fig. 41.

Now, bring down slowly the edge which is held with needle as shown in fig. 42. This will
prevent the air bubble from entering in between the balsam and coverslip. Clean the
oozing balsam with the help of a blotting paper.
 (1) There should be no air bubble in balsam.

(2) The material should be in the center of coverslip.

(3) The coverslip should be in the center of slide.

(4) The Canada balsam should not ooze out of coverslip.

Process # 7. Labelling:

Now, put down your name on one edge of the slide and identification of material on the
other edge of the slide and put it under microscope for examinatio

Reference
Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology (14 edition).

Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A.
(2018). Brock Biology of Microorganisms (15th Edition).

Diagram source: Google images

https://microbenotes.com/electron-microscope-principle-types-components-applications-advantages-
limitations/

https://www.yourarticlelibrary.com/microeconomics/working-principle-of-a-electron-microscopes-with-
diagram/26479

https://www.biologydiscussion.com/zoology/practicals/preparation-of-permanent-slides-
zoology/60158

http://www.microbehunter.com/what-are-the-different-kinds-of-microscope-slides/