Microbiology and Bioprocess Technology

(BTC 302)

Microbiology 2 – Microscopy, Specimen Preparation, Staining Methods

Most of the texts and images of this presentation are from Prescott, Harley, and Klein’s Microbiology book (7th Ed). Some are from
Brock Biology of Microorganisms (14th Ed)

The study materials/presentations are solely meant for academic purposes and they can be reused, reproduced, modified, and
distributed by others for academic purposes only with proper acknowledgements

Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 1

 HOW TO OBSERVE MICROORGANISMS
Types of Microscopes : Light Microscope, Electron Microscope
Light microscope - Bright-Field Microscope
Dark-Field Microscope
Phase Contrast Microscope
Differential Interference Contrast Microscope
Fluorescence Microscope
Electron Microscope – Transmission Electron Microscope (TEM)
Scanning Electron Microscope (SEM)
Some Advanced Microscope – Confocal Scanning Laser Microscope (CSLM),
Atomic Force Microscope (AFM)
Scanning Tunneling Microscope (STM)

Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 2

Resolution is the
ability of a lens to
separate or
distinguish between
small, tiny
objects/points that
are closed together.

Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 3

 from Brock Biology of Microorganisms (14th Ed)

Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 4

Different Types of Light Microscopes
The Bright-Field Microscope
The ordinary microscope is called a bright-field microscope - because it forms a dark image against a
brighter background. Un-pigmented (unstained) living cells are not clearly visible in the bright field
microscope because there is little difference in contrast between the cells and water.
A light source, either a mirror or an electric illuminator, is located in the base. Two focusing knobs, the
fine and coarse adjustment knobs, are located on the arm. The stage or the nose piece can be moved to
focus the object. More advanced microscopes have eyepieces for both the eyes and are called binocular
microscopes. The body assembly itself contains a series of mirrors and prisms. The nosepiece holds three
to five objective lenses of differing magnifying power and can be rotated to position any objective over
the sample.
The image is created by the objective and ocular lenses working together. Light from the illuminated
specimen 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 eyepiece magnifications together.
For example, if a 40X objective is used with a 10X eyepiece, the overall magnification of the specimen will
be 400X.
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 5
 Images from Brock Biology of Microorganisms (14th Ed)

Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 6

The Dark-Field Microscope
The dark-field microscope is used to observe living, unstained cells and organisms. A hollow cone 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 specimen forms an image. The field surrounding a
specimen appears black, while the object itself is brightly illuminated. The dark-field microscope can reveal
considerable internal structure in larger eukaryotic microorganisms.

Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 7

The Phase-Contrast Microscope
A phase-contrast microscope is an excellent way to observe living cells.
The condenser of a phase-contrast microscope has an annular stop, an opaque
disk with a thin transparent ring, which produces a hollow cone of light. As this
cone passes through a cell, some light rays are deviated/bent due to variations
in density and refractive index of cell components within the specimen and
these light rays are retarded. The deviated light is focused to form an image of
the object.

Un-deviated light rays pass through a phase ring in the phase plate, a special
optical disk located in the objective, while the deviated rays miss the ring and
pass through the rest of the plate. The phase ring is constructed in such a way
that the deviated and un-deviated waves will be out of phase and will come
together to form an image. The background, formed by un-deviated light, is
bright, while the image of the unstained specimen formed by deviated light
appears dark and well-defined. This type of microscopy is also called dark-phase-
contrast microscopy. Color filters are used to improve the image.

Phase-contrast microscopy is especially useful for studying motility of living

microorganisms, determining the shape of living cells, and detecting bacterial
intracellular components such as endospores and inclusion bodies, etc. Phase
contrast microscopes also are widely used in studying eukaryotic cells also.
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 8
 9
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur
The Differential Interference Contrast
Microscope
The differential interference contrast (DIC) microscope is
similar to the phase-contrast microscope in that it creates an
image by detecting differences in refractive indices and
thickness of cellular components.

Two beams of plane-polarized light at right angles to each

other are generated by prisms. In one design, the object
beam passes through the specimen, while the reference
beam passes through the clear area of the slide. After
passing through the specimen, the two light beams are
combined and interfere with each other to form an image.

A live, unstained specimen appears brightly colored and

three-dimensional. Cell walls, endospores, granules, vacuoles,
and eucaryotic nuclei are clearly visible under this
microscope. Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 10
PREPARATION AND STAINING (COLORING) OF SPECIMENS
Although living microorganisms can be directly examined with the light microscope, they often must be
fixed and stained to increase visibility, highlight specific morphological features, and preserve them for
future study.

Fixation:
Fixation is the process by which the internal and external structures of cells/microorganisms are fixed and
preserved in position. It inactivates enzymes that might disrupt cell morphology and toughens 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.
Heat fixation - is routinely used to observe microorganisms. Typically, a film of cells (a smear), made on a
slide, is gently heated as the slide is passed through a flame. Heat fixation preserves overall morphology but
not structures within cells.
Chemical fixation - is used to protect fine cellular internal structure as well as the morphology of
microorganisms. Chemical fixatives penetrate cells and react with cellular components, usually proteins and
lipids, to make them inactive, insoluble, and immobile. Common fixative mixtures contain components like
ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.
11
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur
Different types of dyes (coloring agents) for staining microorganisms
Dyes have two common features: (a) they have chromophore groups that give the dye its
color, (b) they can bind with cells by ionic, covalent, or hydrophobic bonding.

Dyes that bind cells by ionic interactions are probably the most commonly used dyes.
These ionizable dyes may be divided into two general classes -

1. Basic dyes —methylene blue, basic fuchsin, crystal violet, safranin, malachite green—
have positively charged groups in their molecular structure and therefore basic dyes can
bind to negatively charged molecules of cells like nucleic acids, many proteins, and the
surfaces of cells. Basic dyes are most effective at higher pHs (alkaline).

2. Acidic dyes —eosin, rose bengal, acid fuchsin, etc. — possess negatively charged groups
such as carboxyl (—COOH) and phenolic hydroxyl (—OH). Acidic dyes, therefore, can bind
to positively charged cell structures like some proteins, amino acids, etc. Acidic dyes stain
best under acidic conditions when proteins and many other molecules carry a positive
charge
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 12
Dyes that bind through covalent bonds – DNA can be stained by the
Feulgen procedure in which the staining compound (Schiff’s
reagent) is covalently attached to its deoxyribose sugars.
Hydrophobic dyes - Sudan III (Sudan Black) selectively stains lipids
because it is lipid soluble but will not dissolve in aqueous portions of
the cell.

Staining Procedures
Most dyes (like safranin, crystal violet, etc.) are used to directly stain
the cell or object of interest, but some dyes (e.g., India ink, nigrosine
dye) are used in negative staining, where the background is stained,
but not the cell. The unstained cells appear as bright objects against
a dark background.
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 13
Staining Procedures
Simple Staining

In simple staining, a single dye is used. Simple

staining procedure is very easy and simple.

Cover the fixed smear of microorganisms with

stain for a short period of time - then wash the
excess stain off with water - blot the slide dry -
observe under microscope.

Basic dyes like crystal violet, methylene blue,

and carbolfuchsin are frequently used in simple
staining to determine the size, shape, and
arrangements of cells.
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 14
Differential Staining
Gram staining
The Gram stain, developed in 1884 by the Danish physician Christian Gram, is the most widely employed
staining method in bacteriology. It is an example of differential staining procedures that are used to
distinguish organisms based on their staining properties. Use of the Gram stain divides bacteria into two
classes — gram-negative bacteria and gram-positive bacteria.

In the first step, the microbial culture smear is heat fixed on a glass slide and is stained with the basic dye
crystal violet, the primary stain. This is followed by treatment with iodine solution functioning as a
mordant. The iodine increases the interaction between the cell and the dye so that the cell is stained
more strongly.

Next, the smear is decolorized by washing with ethanol or acetone. This step generates the differential
aspect of the Gram stain; gram-positive bacteria retain the crystal violet color, whereas gram-negative
bacteria lose their crystal violet and become colorless.

Finally, the smear is counterstained with a simple, basic dye different in color from crystal violet. Safranin,
the most common counterstain, colors gram-negative bacteria pink to red. Gram-positive bacteria remain
dark purple.
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 15
 Gram Staining

Simple Staining
16
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur from Brock Biology of Microorganisms (14th Ed)
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 17
Acid-fast staining is another important
differential staining procedure
It is most commonly used to identify Mycobacterium tuberculosis and
M. leprae, the pathogens responsible for tuberculosis and leprosy,
respectively. These bacteria have cell walls with high lipid content; in
particular, mycolic acids — a group of branched-chain hydroxy lipids,
which prevent dyes from readily binding to the cells.

M. tuberculosis and M. leprae can be stained by harsh procedures

such as the Ziehl-Neelsen method, which uses heat and phenol to
drive basic fuchsin (primary stain) into the cells. Once basic fuchsin
has penetrated, M. tuberculosis and M. leprae are not easily
decolorized by acidified alcohol (acid-alcohol), and thus, they are said
to be acid-fast.

Nonacid-fast bacteria are decolorized by acid-alcohol and thus are

stained blue by methylene blue counterstain. Red cells are acid-fast ; Blue cells are
non acid-fast
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 18
Staining Specific Structures
Capsule Staining : a negative staining
technique that reveals the presence of capsules, a
network usually made of polysaccharides (polymer)
that surrounds many bacteria and some fungi.

Cells are mixed with India ink or nigrosin dye and

spread out in a thin film on a slide. After air-drying, the
cells appear as lighter bodies in the midst of a blue-
black background because ink/dye particles cannot
penetrate either the cell or its capsule.

The extent of the light region is determined by the size

of the capsule and of the cell itself. The cells can be
counterstained for even greater visibility.

Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 19

Endospore Staining
An endospore is an exceptionally hard and resistant structure
produced by some bacterial genera (e.g., Bacillus and
Clostridium). It is capable of surviving for long periods in an
unfavorable environment (dessication, hot/cold temp.,
drought, nutrient limitation, etc.). It is called an endospore
because it develops within the parent bacterial cell.
Endospores may be spherical to elliptical and either smaller or
larger than the diameter of the parent bacterium.

Endospores are not stained well by most dyes, but once

stained, they strongly resist decolorization. In the Schaeffer-
Fulton procedure, endospores are first stained by heating
bacteria with malachite green (primary stain), which is a very
strong stain that can penetrate endospores. After malachite
green treatment, the cells are washed with water and
counterstained with safranin.
https://upload.wikimedia.org/wikipedia/commons/7/7a/Bacillus_subtilis_Spore.jpg

This technique yields a green endospore resting in a pink to

red cell.
Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 20
Flagella Staining
Prokaryotic flagella are fine, threadlike
organelles of locomotion (movement) -
they can only be seen directly using the
electron microscope.
To observe them under the light
microscope, the thickness of flagella is
increased by coating them with
mordants like tannic acid and
potassium alum, and then staining with
pararosaniline (Leifson method) or
basic fuchsin (Gray method).

Presentation prepared by Prof Sufia K Kazy, NIT Durgapur 21