BSCMRIT and 2nd Semester

General Microbiology (GEBT202)

2024-25

Microscopy
(General Microbiology and GEBT202)
__________________________________________________________________________________________

Table of Contents

S. No. CONTENT PAGE No.

2.1 Basic structure and Principles of 2

Microscope

Principle and working applications of

2.2 3
bright field microscopy

Principle and working applications of

2.3 3
dark field microscopy

Principle and working applications of

2.4 9
phase contrast microscopy

Principle and working applications of

2.5 12
electron microscopy

2.6 Gram staining 16

2.7 Model Questions 18

2.8 References 21

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2.1 BASIC STRUCTURE AND PRINCIPLES OF MICROSCOPE

History of Microscopes
In the late 17th century, Antoine van Leeuwenhoek of Holland created a simple single-lens microscope.
Similar to a modern magnifier in structure, this novel invention differed in that its magnification reached
more than 200 times. This new microscope allowed Leeuwenhoek to discover microorganisms and
spermatozoa.
Around the same time, Robert Hooke of England created a compound microscope that had two lenses,
objective and ocular lenses. Hooke observed cork tissue and called it cells because it looked like the small
cells of honeycomb. Thus he coined the biological term cell.
At that time, combining two lenses adversely affected the accuracy, partly due to the aberration of the
lenses, resulting in a lower resolution than a simple microscope.
In the 19th century, microscope resolution was improved dramatically through various measures.
Aberration correction was implemented by using better lenses or combination of lenses and was
supported by German companies such as Zeiss and Leitz who were the main contributors. Ernst Abbe of
Germany made theoretical and technical microscope innovations, and it can be said he established the
prototype of the modern optical microscope.
Various observation methods were invented in the 20th century. Both the phase contrast microscope
introduced in the 1930s and the differential interference contrast microscope invented in the 1950s,
contributed to high magnification of transparent samples such as cells. The confocal laser microscope, also
invented in the 1950s, marked the beginning of observation with clearer images. Fluorescence
microscopes have evolved together with the development of fluorescent dyes around the early 20th
century. Despite these significant improvements in microscopy, 19th century researcher George Airy
discovered a limit to resolution based on the nature of light. Soon after, Ernst Abbe introduced the concept
of numerical aperture and proved microscopic samples can be resolved only up to 200 nm under visible
light, regardless of the performance of the lens, which created a new challenge for magnified observation.
Principles of microscope

Figure: Principle that enables magnified observation with a biological microscope

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A general biological microscope mainly consists of an objective lens, ocular lens, lens tube, stage, and
reflector. An object placed on the stage is magnified through the objective lens. When the target is
focused, a magnified image can be observed through the ocular lens.
Telescopes also have a similar structure; however, they are used for observing distant objects. A telescope
receives light from a star or other distant object with the objective lens and adjusts the refracted light to
the focal point through the ocular lens. On the other hand, a microscope is designed to emit light onto or
through objects and magnify the transmitted or reflected light with the objective and ocular lenses.

2.2 PRINCIPLE AND WORKING APPLICATIONS OF BRIGHT FIELD MICROSCOPY

The most commonly used microscope for general purposes is the standard compound microscope. A
conventional light from an incandescent source is aimed toward a lens beneath the stage called the
condenser, through the specimen, through an objective lens, and to the eye through a second magnifying
lens, the ocular or eyepiece. Most microscopes will have a built-in illuminator. It magnifies the size of the
object by a complex system of lens arrangement. It has a series of two lenses; (i) the objective lens close
to the object to be observed and (ii) the ocular lens or eyepiece, through which the image is viewed by
eye. Light from a light source (mirror or electric lamp) passes through a thin transparent object. The
objective lens produces a magnified ‘real image’ first image) of the object. This image is again magnified
by the ocular lens (eyepiece) to obtain a magnified ‘virtual image’ (final image), which can be seen by eye
through the eyepiece. As light passes directly from the source to the eye through the two lenses, the field
of vision is brightly illuminated. That is why; it is a bright-field microscope.

Parts of a Compound Microscope:

(i) Mechanical Parts:

These are the parts, which support the optical parts and help in their adjustment for focusing the object

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1. Base or Metal Stand: The whole microscope rests on this base. Mirror, if present, is fitted to it.
2. Pillars: It is a pair of elevations on the base, by which the body of the microscope is held to the
base
3. Inclination joint: It is a movable joint, through which the body of the microscope is held to the
base by the pillars. The body can be bent at this joint into any inclined position, as desired by the
observer, for easier observation. In new models, the body is permanently fixed to the base in an
inclined position, thus needing no pillar or joint.
4. Curved Arm: It is a curved structure held by the pillars. It holds the stage, body tube, fine
adjustment and coarse adjustment.
5. Body Tube: It is usually a vertical tube holding the eyepiece at the top and the revolving nosepiece
with the objectives at the bottom. The length of the draw tube is called ‘mechanical tube length’
and is usually 140-180 mm (mostly 160 mm).
6. Draw Tube: It is the upper part of the body tube, slightly narrower, into which the eyepiece is
slipped during observation.
7. Coarse Adjustment: It is a knob with rack and pinion mechanism to move the body tube up and
down for focusing the object in the visible field. As rotation of the knob through a small angle
moves the body tube through a long distance relative to the object, it can perform coarse
adjustment. In modern microscopes, it moves the stage up and down and the body tube is fixed to
the arm.
8. Fine Adjustment: It is a relatively smaller knob. Its rotation through a large angle can move the
body tube only through a small vertical distance. It is used for fine adjustment to get the final clear
image. In modern microscopes, fine adjustment is done by moving the stage up and down by the
fine adjustment.
9. Stage: It is a horizontal platform projecting from the curved arm. It has a hole at the center, upon
which the object to be viewed is placed on a slide. Light from the light source below the stage
passes through the object into the objective.
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10. Mechanical Stage (Slide Mover): Mechanical stage consists of two knobs with rack and pinion
mechanism. The slide containing the object is clipped to it and moved on the stage in two
dimensions by rotating the knobs, so as to focus the required portion of the object.
11. Revolving Nosepiece: It is a rotatable disc at the bottom of the body tube with three or four
objectives screwed to it. The objectives have different magnifying powers. Based on the required
magnification, the nosepiece is rotated, so that only the objective specified for the required
magnification remains in line with the light path.

(ii) Optical Parts:

These parts are involved in passing the light through the object and magnifying its size.

The components of optical parts include the following:

1. Light Source: Modern microscopes have in-built electric light source in the base. The source is
connected to the mains through a regulator, which controls the brightness of the field. But in old
models, a mirror is used as the light source. It is fixed to the base by a binnacle, through which it
can be rotated, so as to converge light on the object. The mirror is plane on one side and concave
on the other.
2. Diaphragm: If light coming from the light source is brilliant and all the light is allowed to pass to
the object through the condenser, the object gets brilliantly illuminated and cannot be visualized
properly. Therefore, an iris diaphragm is fixed below the condenser to control the amount of light
entering into the condenser.
3. Condenser: The condenser or sub-stage condenser is located between the light source and the
stage. It has a series of lenses to converge on the object, light rays coming from the light source.
After passing through the object, the light rays enter into the objective.
The ‘light condensing’, ‘light converging’ or ‘light gathering’ capacity of a condenser is called
‘numerical aperture of the condenser’. Similarly, the ‘light gathering’ capacity of an objective is
called ‘numerical aperture of the objective’. If the condenser converges light in a wide angle, its
numerical aperture is greater and vice versa.
4. Objective: It is the most important lens in a microscope. Usually three objectives with different
magnifying powers are screwed to the revolving nosepiece.

The objectives are:

a) Low power objective (X 10): It produces ten times magnification of the object.
b) High dry objective (X 40): It gives a magnification of forty times.
c) Oil-immersion objective (X100): It gives a magnification of hundred times, when
immersion oil fills the space between the object and the objective The scanning objective
(X4) is optional. The primary magnification (X4, X10, X40 or X100) provided by each
objective is engraved on its barrel. The oil-immersion objective has a ring engraved on it
towards the tip of the barrel.

5. Resolving Power of Objective:

It is the ability of the objective to resolve each point on the minute object into widely spaced points,
so that the points in the image can be seen as distinct and separate from one another, so as to get a
clear un-blurred image. It may appear that very high magnification can be obtained by using more
number of high power lenses. Though possible, the highly magnified image obtained in this way is a
blurred, one. That means, each point in the object cannot be found as widely spaced distinct and
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separate point on the image. Mere increase in size (greater magnification) without the ability to
distinguish structural details (greater resolution) is of little value. Therefore, the basic limitation in light
microscopes is one not of magnification, but of resolving power, the ability to distinguish two adjacent
points as distinct and separate, i.e. to resolve small components in the object into finer details on the
image.

Wavelength of light (λ): The smaller is the wavelength of light (λ), the greater is its ability to resolve
the points on the object into distinctly visible finer details in the image. Thus, the smaller is the
wavelength of light, the greater is its resolving power.

Limit of resolution of objective (d): The limit of resolution of an objective (d) is the distance between
any two closest points on the microscopic object, which can be resolved into two separate and distinct
points on the enlarged image. Points with their in-between distance less than‘d’ or objects smaller
than ‘d’ cannot be resolved into separate points on the image. If the resolving power is high, points
very close to each other can be seen as clear and distinct. Thus, the limit of resolution (the distance
between the two resolvable points) is smaller. Therefore, smaller objects or finer details can be seen,
when’d’ is smaller. Smaller‘d’ is obtained by increasing the resolving power, which in turn is obtained
by using shorter wavelength of light (λ) and greater numerical aperture.
Limit of resolution = d = 0.61λ/NA

Where,

λ = Wave length of light and

NA = Numerical aperture of the objective.

If λ green = 0.55 p and NA = 1.30, then d = 0.61λ/NA = 0.61x0.55/1.30 = 0.26 µ. Therefore, the smallest
details that can be seen by a typical light microscope is having the dimension of approximately 0.2 µ.
Smaller objects or finer details than this cannot be resolved in a compound microscope.

5. Eyepiece: The eyepiece is a drum, which fits loosely into the draw tube. It magnifies the magnified
real image formed by the objective to a still greatly magnified virtual image to be seen by the eye.
Usually, each microscope is provided with two types of eyepieces with different magnifying powers
(X10 and X25). Depending upon the required magnification, one of the two eyepieces is inserted into
the draw tube before viewing. Three varieties of eyepieces are usually available.

They are the Huygenian, the hyper plane and the compensating. Among them, the Huygenian is very
widely used and efficient for low magnification. In this eyepiece, two simple Plano-convex lenses are
fixed, one above and the other below the image plane of the real image formed by the objective.

Bright-field microscopy is the simplest of all the optical microscopy illumination techniques. Sample
illumination is transmitted (i.e., illuminated from below and observed from above) white light, and
contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample.
Bright-field microscopy is the simplest of a range of techniques used for illumination of samples in light

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microscopes, and its simplicity makes it a popular technique. The typical appearance of a bright-field
microscopy image is a dark sample on a bright background, hence the name.

The light path of a bright-field microscope is extremely simple; no additional components are required
beyond the normal light-microscope setup. The light path therefore consists of:
 a trans illumination light source, commonly a halogen lamp in the microscope stand;
 a condenser lens, which focuses light from the light source onto the sample;
 an objective lens, which collects light from the sample and magnifies the image;
 Oculars and/or a camera to view the sample image.

Bright-field microscopy may use critical or Köhler illumination to illuminate the sample.
Bright-field microscopy typically has low contrast with most biological samples, as few absorb light to
a great extent. Staining is often required to increase contrast, which prevents use on live cells in many
situations. Bright-field illumination is useful for samples that have an intrinsic color, for
example chloroplasts in plant cells. Bright-field microscopy is a standard light-microscopy technique,
and therefore magnification is limited by the resolving power possible with the wavelength of visible
light.
Advantages:
 Simplicity of setup with only basic equipment required.
 Living cells can be seen with bright-field microscopes.

Limitations:
 Very low contrast of most biological samples.
 The practical limit to magnification with a light microscope is around 1300X. Although higher
magnifications are possible, it becomes increasingly difficult to maintain image clarity as the
magnification increases.
 Low apparent optical resolution due to the blur of out-of-focus material.
 Samples that are naturally colorless and transparent cannot be seen well, e.g. many types of
mammalian cells. These samples often have to be stained before viewing. Samples that do
have their own color can be seen without preparation, e.g. the observation of cytoplasmic
streaming in Chara cells.

Enhancements:
 Reducing or increasing the amount of the light source by the iris diaphragm.
 Use of an oil-immersion objective lens and a special immersion oil placed on a glass cover over
the specimen. Immersion oil has the same refraction as glass and improves the resolution of
the observed specimen.
 Use of sample-staining methods for use in microbiology, such as simple stains (methylene
blue, safranin, crystal violet) and differential stains (negative stains, flagellar stains, endospore
stains).
 Use of a colored (usually blue) or polarizing filter on the light source to highlight features not

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visible under white light. The use of filters is especially useful with mineral samples.

Fig: An example bright-field micrograph. This image shows a cross-section of the vascular tissue in a plant stem.

A. Binocular upright Bright Field Microscope B. Trinocular Upright Bright Field Microscope

2.3 PRINCIPLE AND WORKING APPLICATIONS OF DARK FIELD MICROSCOPY

Dark Field microscopy (also called dark-ground microscopy) describes microscopy methods, in
both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the
field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark.

The light's path

The steps are illustrated

1. Light enters the microscope for illumination of the sample.
2. A specially sized disc, the patch stop (see figure), blocks some light from the light source, leaving
an outer ring of illumination.

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3. The condenser lens focuses the light towards the sample.

4. The light enters the sample. Most is directly transmitted, while some is scattered from the sample.
5. The scattered light enters the objective lens, while the directly transmitted light simply misses the
lens and is not collected due to a direct-illumination block (see figure).
6. Only the scattered light goes on to produce the image, while the directly transmitted light is
omitted.

Advantages: Dark-field microscopy is a very simple yet effective technique and well suited for uses
involving live and unstained biological samples, such as a smear from a tissue culture or individual, water-
borne, single-celled organisms.

Dark-field microscopy techniques are almost entirely free of halo or relief-style artifacts typical of DIC and
phase-contrast imaging. This comes at the expense of sensitivity to phase information.

Considering the simplicity of the setup, the quality of images obtained from this technique is impressive.

Disadvantages: One limitation of dark-field microscopy is the low light levels seen in the final image. This
means that the sample must be very strongly illuminated, which can cause damage to the sample.
The interpretation of dark-field images must be done with great care, as common dark features of bright-
field microscopy images may be invisible, and vice versa. In general, the dark-field image lacks the low
spatial frequencies associated with the bright-field image, making the image a high-passed version of the
underlying structure.

2.4 PRINCIPLE AND WORKING APPLICATIONS OF PHASE CONTRAST MICROSCOPY

Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in light passing
through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible,
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but become visible when shown as brightness variations. It was first described in 1934 by Dutch physicist
Frits Zernike.
When light waves travel through a medium other than vacuum, interaction with the medium causes the
wave amplitude and phase to change in a manner dependent on properties of the medium. Changes in
amplitude (brightness) arise from the scattering and absorption of light, which is often wavelength-
dependent and may give rise to colors. Photographic equipment and the human eye are only sensitive to
amplitude variations. Without special arrangements, phase changes are therefore invisible. Yet, phase
changes often carry important information. Phase-contrast microscopy is particularly important in biology.
It reveals many cellular structures that are not visible with a simpler bright-field microscope, as
exemplified in the figure. These structures were made visible to earlier microscopists by staining, but this
required additional preparation and thus killing the cells. The phase-contrast microscope made it possible
for biologists to study living cells and how they proliferate through cell division. It is one of the few
methods available to quantify cellular structure and components that does not use fluorescence. After its
invention in the early 1930s, phase-contrast microscopy proved to be such an advancement in microscopy
that its inventor Frits Zernike was awarded the Nobel Prize in Physics in 1953. Phase-contrast microscopy
is basically a specially designed light microscope with all the basic parts in addition to which an annular
phase plate and annular diaphragm are fitted.

The basic principle to making phase changes visible in phase-contrast microscopy is to separate the
illuminating (background) light from the specimen-scattered light (which makes up the foreground details)
and to manipulate these differently.
In figure B, the ring-shaped illuminating light (green) that passes the condenser annulus is focused on the
specimen by the condenser. Some of the illuminating light is scattered by the specimen (yellow). The
remaining light is unaffected by the specimen and forms the background light (red). When observing an
unstained biological specimen, the scattered light is weak and typically phase-shifted by −90° (due to both
the typical thickness of specimens and the refractive index difference between biological tissue and the
surrounding medium) relative to the background light. This leads to the foreground and background
having nearly the same intensity, resulting in low image contrast.

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Working Principle
1. Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector
lens and focused on a specialized annulus (labeled condenser annulus) positioned in the sub stage
condenser front focal plane.
2. Wave fronts passing through the annulus illuminate the specimen and either pass through undeviated
or are diffracted and retarded in phase by structures and phase gradients present in the specimen.
3. Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a phase
plate and focused at the intermediate image plane to form the final phase-contrast image observed in the
eyepieces.

Components of a Phase Contrast Microscope

The annular diaphragm
● It is situated below the condenser.
● It is made up of a circular disc having a circular annular groove.
● The light rays are allowed to pass through the annular groove.
● Through the annular groove of the annular diaphragm, the light rays fall on the specimen or object
to be studied.
● At the back focal plane of the objective develops an image.
● The annular phase plate is placed at this back focal plane.

The phase plate

● It is either a negative phase plate having a thick circular area or a positive phase plate having a thin
circular groove.
● This thick or thin area in the phase plate is called the conjugate area.
● The phase plate is a transparent disc.
● With the help of the annular diaphragm and the phase plate, the phase contrast is obtained in this
microscope.
● This is obtained by separating the direct rays from the diffracted rays.
● The direct light rays pass through the annular groove whereas the diffracted light rays pass through
the region outside the groove.
● Depending upon the different refractive indices of different cell components, the object to be
studied shows a different degree of contrast in this microscope.

Advantages of Phase Contrast Microscopy

1. Living cells can be observed in their natural state without previous fixation or labeling.
2. It makes a highly transparent object more visible.
3. No special preparation of fixation or staining etc. is needed to study an object under a phase-
contrast microscope which saves a lot of time.
4. Examining intracellular components of living cells at relatively high resolution. E.g: The dynamic
motility of mitochondria, mitotic chromosomes & vacuoles.
5. It made it possible for biologists to study living cells and how they proliferate through cell division.
6. Phase-contrast optical components can be added to virtually any bright field microscope, provided
the specialized phase objectives conform to the tube length parameters, and the condenser will
accept an annular phase ring of the correct size.

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Limitations of Phase Contrast Microscopy

1. Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so
phase contrast is often not used in teaching labs except perhaps in classes in the health
professions.
2. To use phase-contrast the light path must be aligned.
3. Generally, more light is needed for phase contrast than for corresponding bright-field viewing,
since the technique is based on the diminishment of the brightness of most objects.

2.5 PRINCIPLE AND WORKING APPLICATIONS OF ELECTRON MICROSCOPY

An electron microscope is a microscope that uses a beam of accelerated electrons as a source of
illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible
light photons, electron microscopes have a higher resolving power than light microscopes and can reveal
the structure of smaller objects. A scanning transmission electron microscope has achieved better than
50 pm resolution in annular dark-field imaging mode and magnifications of up to about 10,000,000×
whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful
magnifications below 2000×.
Electron microscopes use shaped magnetic fields to form electron optical lens systems that are analogous
to the glass lenses of an optical light microscope.
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.
Industrially, electron microscopes are often used for quality control and failure analysis. Modern electron
microscopes produce electron micrographs using specialized digital cameras and frame grabbers to
capture the images.

Parts of Electron Microscope

Electron Microscope is in the form of a tall vacuum column that is vertically mounted. It has the following
components:
1. Electron gun
● The electron gun is a heated tungsten filament, which generates electrons.
2. Electromagnetic lenses
● The condenser lens focuses the electron beam on the specimen. A second condenser lens forms
the electrons into a thin tight beam.
● The electron beam coming out of the specimen passes down the second of magnetic coils called
the objective lens, which has high power and forms the intermediate magnified image.
● The third set of magnetic lenses called projector (ocular) lenses produce the final further
magnified image.
● Each of these lenses acts as an image magnifier all the while maintaining an incredible level of
detail and resolution.
3. Specimen Holder
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● The specimen holder is an extremely thin film of carbon or collodion held by a metal grid.
4. Image viewing and Recording System
● The final image is projected on a fluorescent screen.
● Below the fluorescent screen is a camera for recording the image.

Types of electron microscope:

Transmission electron microscope (TEM): The original form of the electron microscope, the transmission
electron microscope (TEM), uses a high voltage electron beam to illuminate the specimen and create an
image. The electron beam is produced by an electron gun, commonly fitted with
a tungsten filament cathode as the electron source. The electron beam is accelerated by
an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused
by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part
transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen,
the electron beam carries information about the structure of the specimen that is magnified by
the objective lens system of the microscope. The spatial variation in this information (the "image") may
be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with
a phosphor or scintillator material such as zinc sulfide. One major disadvantage of the transmission
electron microscope is the need for extremely thin sections of the specimens, typically about 100
nanometers. Creating these thin sections for biological and materials specimens is technically very
challenging. Biological tissue specimens are chemically fixed, dehydrated and embedded in a polymer
resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic
polymers, and similar materials may require staining with heavy atom labels in order to achieve the
required image contrast.
Electron microscopes use signals arising from the interaction of an electron beam with the sample to
obtain information about structure, morphology, and composition.
1. An electron gun generates electrons.
2. Two sets of condenser lenses focus the electron beam on the specimen and then into a thin tight
beam.
3. To move electrons down the column, an accelerating voltage (mostly between 100 kV-1000 kV) is
applied between the tungsten filament and anode.
4. The specimen to be examined is made extremely thin, at least 200 times thinner (20-100 nm) than
those used in the optical microscope.
5. The electronic beam passes through the specimen and electrons are scattered depending upon the
thickness or refractive index of different parts of the specimen.
6. The denser regions in the specimen scatter more electrons and therefore appear darker in the
image since fewer electrons strike that area of the screen. In contrast, transparent regions are
brighter.
7. The electron beam coming out of the specimen passes to the objective lens, which has high power
and forms the intermediate magnified image.

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8. The ocular lenses then produce the final further magnified image.
For TEM, samples must be cut into very thin cross-sections. This is to allow electrons to pass right through
the sample. After being fixed and dehydrated, samples are embedded in hard resin to make them easier
to cut. Then, an instrument called an ultramicrotome cuts the samples into ultra-thin slices (100 nm or
thinner).

Scanning electron microscope (SEM)

The SEM produces images by probing the specimen with a focused electron beam that is scanned across
a rectangular area of the specimen (raster scanning). When the electron beam interacts with the
specimen, it loses energy by a variety of mechanisms. The lost energy is converted into alternative forms
such as heat, emission of low-energy secondary electrons and high-energy backscattered electrons, light
emission (cathodoluminescence) or X-ray emission, all of which provide signals carrying information about
the properties of the specimen surface, such as its topography and composition. The image displayed by
an SEM maps the varying intensity of any of these signals into the image in a position corresponding to
the position of the beam on the specimen when the signal was generated.
Generally, the image resolution of an SEM is lower than that of a TEM. However, because the SEM images
the surface of a sample rather than its interior, the electrons do not have to travel through the sample.
This reduces the need for extensive sample preparation to thin the specimen to electron transparency.
The SEM is able to image bulk samples that can fit on its stage and still be maneuvered, including a height
less than the working distance being used, often 4 millimeters for high-resolution images. The SEM also
has a great depth of field, and so can produce images that are good representations of the three-
dimensional surface shape of the sample.
Conventional scanning electron microscopy depends on the emission of secondary electrons from the
surface of a specimen.
● Because of its great depth of focus, a scanning electron microscope is the EM analog of a stereo
light microscope.

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● It provides detailed images of the surfaces of cells and whole organisms that are not possible by
TEM. It can also be used for particle counting and size determination, and for process control.
● It is termed a scanning electron microscope because the image is formed by scanning a focused
electron beam onto the surface of the specimen in a raster pattern(the beam sweeps horizontally
left-to-right at a steady rate).
Samples destined for the SEM aren’t cut into thin sections, because the SEM visualises the surface of
three-dimensional objects. Instead, SEM samples are coated with a thin layer of metal (usually gold or
gold-palladium). The metal coating makes samples conductive.

2.6 STAINING
Types of Stain

Simple stains

A simple stain is a stain using only a single dye that does not differentiate between different types of
organisms. There is a single staining step and everything that stains is stained the same color.

Simple staining:

● A simple stain is an aqueous or alcohol solution of a single basic dye

● Simple stains usually highlight the entire microorganism so that cellular shapes and structures
are visible.
● Stain is applied to the fixed smear for a certain length of time and then washed off, and the slide
is dried and examined.
● Basic dyes like crystal violet, methylene blue and carbolfuchsin are frequently used to determine
the size, shape and arrangement of bacteria

Differential stain
A differential stain uses more than one dye and stains different kinds of organisms with different colors.
The differential stains are Gram stain and Ziehl - Neelsen acid fast stains. Differential staining requires the
use of chemical reagents that are applied sequentially to a heat-fixed smear. The first reagent is called
the primary stain. Its function is to impart color to all cells. In order to establish a color contrast, the
second reagent used is the decolorizing agent. Based on the chemical composition of the cellular
components, the decolorizing agent may or may not remove the primary stain from the entire cell or only
from certain cell structures. The final reagent, counter stain, has a contrasting color to that of the primary
stain.

Gram Staining: Principles and Method

Introduction:
Danish physician Hans Christian Gram developed the Gram staining method in 1884. Gram staining
procedure uses four chemicals; crystal violet, iodine, alcohol, and safranin, to stain bacteria. Gram staining
involves primary staining, decolorizing and counterstaining. Gram devised his technique not for the
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purpose of distinguishing one type of bacterium from another but to make bacteria more visible in stained
sections of lung tissue. He published his method in 1884, and included in his short report the observation
that the typhus bacillus did not retain the stain. Gram staining is still the cornerstone of bacterial
identification and taxonomic division. This differential staining technique separates most bacteria into two
groups based on cell wall composition.

1. Gram-positive bacteria- stains purple

2. Gram-negative bacteria-stains red/pink

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram-
positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet.
Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out on
addition of ethanol. They are stained pink or red by the counterstain, commonly safranin or fuchsine.
Lugol's iodine solution is always added after addition of crystal violet to strengthen the bonds of the stain
with the cell membrane. Gram staining is almost always the first step in the preliminary identification of a
bacterial organism.

Steps in Gram Staining

1. Fixation of clinical materials to the surface of the microscope slide either by heating or by using
methanol. (Methanol fixation is recommended rather than heat fixation. Methanol fixation
preserves the morphology of host cells and bacteria. Heating the slide causes cell distortion, could
increase cell debris, and may cause erroneous Gram stain results.).
2. Application of the primary stain (crystal violet). Crystal violet is a dark blue to purple dye. It stains
all cells blue/purple.
3. Application of mordant: The iodine solution (mordant) is added to form a crystal violet-iodine (CV-
I) complex; all cells continue to appear blue.
4. Decolorization step: The decolorization step distinguishes gram-positive from gram-negative cells.
The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-rich,
thin-walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-walled,
gram-positive bacteria. The gram-negative bacteria appear colorless, and gram-positive bacteria
remain blue.
5. Application of counterstain (safranin): The red dye safranin stains the decolorized gram-negative

Principle behind Gram Staining:

The differences in Gram-positive and Gram-negative bacteria cell wall composition account for the Gram
staining differences. Gram-positive cell wall contains a thick layer of peptidoglycan with numerous teichoic
acid cross-linking, which resists decolorization. In aqueous solutions, crystal violet dissociates into CV+ and
Cl – ions that penetrate through Gram-positive and Gram-negative cell walls. The CV+ interacts with
negatively charged components of bacterial cells, staining the cells purple. When added, iodine (I- or I3-)
interacts with CV+ to form large crystal violet-iodine (CV-I) complexes within the cytoplasm and outer
layers of the cell. The decolorizing agent (ethanol or an ethanol and acetone solution) interacts with the
lipids of both gram-positive and gram-negative bacteria membranes.
● The outer membrane of the Gram-negative cell is lost from the cell, leaving the thin peptidoglycan
layer exposed. With ethanol treatment, gram-negative cell walls become leaky and allow the large
CV-I complexes to be washed from the cell.
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● The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by
the addition of ethanol. Thus ethanol treatment traps the large CV-I complexes within the cell.

After decolorization, the gram-positive cell remains purple. In contrast, the gram-negative cell loses the
purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.

Illustrative figure showing major differences between Gram Positive and Gram Negative

Gram positive Gram Negative

Cell Wall
A single-layered, smooth cell wall A double-layered, wavy cell-wall
Cell Wall Thickness
The thickness of the cell wall is 20 to 80 The thickness of the cell wall is 8 to 10
nanometres nanometres
Peptidoglycan Layer
It is a thick layer/ also can be multilayered It is a thin layer/ often single-layered.
Teichoic acids
Presence of teichoic acids Absence of teichoic acids
Outer membrane
The outer membrane is absent The outer membrane is present (mostly)
Lipid content
Very low 20 to 30%
Toxin Produced
Exotoxins Endotoxins or Exotoxins
Gram Staining
These bacteria retain the crystal violet colour These bacteria do not retain the stain colour
even after they are washed with acetone or even after they are washed with acetone or
alcohol and appear as purple-coloured when alcohol and appear as pink-coloured when
examined under the microscope after gram examined under the microscope after gram
staining. staining.
Examples
Staphylococcus, Streptococcus, etc. Escherichia, Salmonella, etc.
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Limitations The sensitivity of the Gram stain procedure is low. Sometimes, you may fail to see the organism
in Gram Stain smear, but the same clinical specimen may yield organisms when cultured. To be visible on
a slide, organisms that stain by the Gram method must be present in about 104 to 105 organisms per
milliliter of centrifuged fluid. Gram staining technique is not recommended for spirochetes and
mycobacteria. Mycobacteria stain weakly with gram stain, and bacteria such
as Mycoplasma, Rickettsiae, Chlamydiae do not take up the dyes used in Gram stain or are too small to be
seen with light microscopy.

2.7 MODEL QUESTIONS

MCQ type Questions

1.In bright field microscopy, the specimen appears:

a. Dark against a bright background

b. Bright against a dark background

c. Colorful against a neutral background

d. Transparent against a colorful background

2.The objective lens in bright field microscopy is designed to:

a. Absorb light

b. Transmit all light

c. Reflect light

d. Scatter light

3.Dark field microscopy is used to observe specimens that are:

a. Opaque

b. Transparent

c. Stained with specific dyes

d. Fluorescent

4.In dark field microscopy, the specimen appears:

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a. Dark against a bright background

b. Bright against a dark background

c. Red against a green background

d. Yellow against a blue background

5.Phase contrast microscopy is particularly useful for visualizing:

a. Unstained living cells

b. Fixed and stained cells

c. Metal-coated specimens

d. Fluorescent specimens

6.In phase contrast microscopy, differences in refractive index are converted into differences in:

a. Absorption

b. Color

c. Intensity

d. Polarization

7.Which type of microscopy uses electrons instead of light for imaging?

a. Bright field microscopy

b. Dark field microscopy

c. Phase contrast microscopy

d. Electron microscopy

8. In electron microscopy, the wavelength of electrons is much ____________ compared to the

wavelength of visible light.

a. Shorter

b. Longer

c. Equal to
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d. Unrelated to

9. Which microscopy technique provides the highest resolution among the options mentioned?

a. Bright field microscopy

b. Dark field microscopy

c. Phase contrast microscopy

d. Electron microscopy

10. Electron microscopy is capable of achieving much higher magnifications than light microscopy
because:

a. Electrons have a shorter wavelength than light

b. Light microscopes are not capable of magnification

c. Electrons are less energetic than photons

d. Electron microscopes use visible light for imaging

Answers:

b. Bright against a dark background

b. Transmit all light
b. Transparent
b. Bright against a dark background
a. Unstained living cells
c. Intensity
d. Electron microscopy
a. Shorter
d. Electron microscopy
a. Electrons have a shorter wavelength than light

Short answer type Questions

1. What is the basic principle of bright field microscopy?
2. How does the specimen appear in bright field microscopy?
3. What is the primary purpose of dark field microscopy?
4. How does dark field microscopy enhance the visibility of specimens?
5. What type of specimens is phase contrast microscopy particularly useful for?
6. In phase contrast microscopy, what property of light is used to create contrast in the image?
7. What is the major difference between electron microscopy and light microscopy in terms of the
imaging source?
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8. Why can electron microscopy achieve much higher magnifications than light microscopy?
9. Which microscopy technique provides detailed images of internal cellular structures with the
highest resolution?
10. What are the primary advantages of electron microscopy over light microscopy?
11. Given that the limit of resolution (d) is determined by Abbe’s equation, d=λ/2NA, where λ is the
wavelength of the illuminating source and NA is the numerical aperture of the objective lens.
Calculate the resolution limits of a microscope A (λ ≈ 500 nm, NA = 1.4) and microscope B (λ ≈
0.005 nm, NA = 0.9). How do these values quantitatively explain the superior resolving power
among these 2 microscopes, interpret your answer?

Long answer type Questions

1. Describe the basic principles of bright field microscopy and how it produces an image. Include
information about the role of lenses and specimen contrast.
2. Explain the principle of dark field microscopy and how it differs from bright field microscopy.
Provide examples of situations where dark field microscopy is particularly useful.
3. Discuss the principles of phase contrast microscopy and its applications in biological research.
How does phase contrast microscopy overcome the limitations of traditional bright field
microscopy?
4. Examine the fundamental principles of electron microscopy and its advantages over light
microscopy. Discuss the types of electron microscopy and their specific applications.

2.7 REFERENCES
 Prescott’s and Dunn’s Microbiology. Prescott. Willey et al, 10th Ed. Tata McGraw-Hill.
 Brock: Biology of Microorganisms. Madigan et al, 15th Ed. Pearson.

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