Basic Techniques In

Microbiology
Course Professor Dr. Nihad Adnan
Guest Teacher Dept. of Microbiology
Stamford University Bangladesh.
Course Code MBO 82A

Credit hour Three (3.0)

Required Texts:
Brock Biology of Microorganisms – M.T. Madigan, K.S.
Bender, D.H. Buckley, D.A Stahl and W.A. Satttley; Pearson
Prentice Hall
Microbiology- M.J. Pelczer Jr, E.C.S. Chan and N.R. Krieg;
McGraw-Hill Book Company Microbiology: Concepts and
Applications - M.J. Pelczer Jr, E.C.S. Chan and N.R. Krieg;
McGraw Hill Inc., New York
Microbiology: An Introduction- G.J. Tortora, B.R. Funke and
C.L. Case; Pearson, Boston
 Syllabus for Class test and Midterm
• Microscopes and Microscopy:
light spectrum, resolving power and magnification power; parts of
microscope, bright-field, dark-field,, phase-contrast, differential
interference contrast microscopes, fluorescence, Electron
microscopes (transmission electron, scanning electron),scanning
tunneling and atomic force microscopy.
• Observation of microorganisms under microscope:
wet-mount and hanging-drop technique: preparation of
microorganisms for staining; chemical properties of stains;
mechanisms of staining; positive and negative staining ,
Observation of microorganisms under microscope: simple,
differential and special staining technique .
• Characterization of microorganisms:
Morphological Characteristics; nutritional and cultural
characteristics, metabolic characteristics; antigenic characteristics,
Pathogenic characteristics and genetic characteristics .
Microscopes and
Microscopy
 History of Microscope
• 1590 –first compound microscope by
Zacharias Janssen.
• 1655 – Robert Hooke used a compound
microscope to observe pores in cork. He
called them “cells”.
• Antoine van Leeuwenhoek (1632-1723)
• 1st to see single-celled organisms in pond
water

Replica of simple microscope made

in 1673 by Leeuwenhoek
TERMS AND DEFINITIONS
Principle
Microscopy is to get a magnified image, in which structures may be
resolved which could not be resolved with the help of an unaided eye.

Magnification
• It is the ratio of the size of an object seen
under microscope to the actual size
observed with unaided eye.
• The total magnification of microscope
is calculated by multiplying the magnifying
power of the objective lens by that of eye piece.
 Total magnification:

➢Most lab microscopes are equipped with 3 objectives, each capable of

a different degree of magnification.

➢These are referred to as the oil-immersion, high-dry and low-power

objectives.

➢The total magnification of the system is determined by multiplying the

magnifying power of the objective by that of the eyepiece.

➢Generally, an eyepiece having a magnification of X10 is used, although

eyepieces of higher or lower magnifications are available.
 Total Magnification (Contd……)

Objective Objective Ocular Total

designation magnification Magnification Magnification
Low power x10 X10 X100

High Power x40 X10 X400

Oil Immersion x100 x10 x1000

TERMS AND DEFINITIONS
Limit of resolution
It is the minimum distance between two points to identify them separately.
It is calculated by Abbé equation.

Limit of resolution

Limit of resolution is inversely proportional to power or resolution.

If the wavelength is shorter then the resolution will be greater.
Working distance
• It is the distance between the objective
and the objective slide.
• The working distance decreases with
increasing magnification.
TERMS AND DEFINITIONS
Numerical aperture(NA)
The numerical aperture of a lens is the ratio of the diameter of the
lens to its focal length.
NA of a lens is an index of the resolving power.
NA can be decreased by decreasing the amount of light that passes through
a lens.

Diameter of the lens

 Resolution, or Resolving power (RP)
• It is the ability to differentiate two close points as separate.
It refers that it has the ability to distinguish images of 2
close objects as separate distinct entities. For e.g. if a
microscope has a resolving power of 0.4 nm, it can
distinguish two points as separate objects if they are at least
0.4 nm apart.

• The resolving power of human eye is 0.25 mm. The light

microscope can separate dots that are 0.25µm apart. The
electron microscope can separate dots that are 0.5nm apart.
•RP is the ability of the lenses to distinguish fine detail and
structure.
 Resolution, or Resolving power (contd…)

• For two points to be seen as separate the light has to be

able to pass between them
• If the points are very close and the light is not able to
pass between, the image will appear as fuzzy.
• Resolution is determined by the wavelength of light
used in the microscope
• The average wavelength of light is 550 nm.
 TERMS AND DEFINITIONS
Refraction and Refractive index:
light is refracted (bent) when passing from one medium to another which
is Refraction. Refractive index means it’s a measure of the relative velocity
at which light passes through a material .

Immersion oil

The light may bend in air so much that it misses the small high-
magnification lens. Immersion oil is used to keep light from
bending. It is used in x100 objective lenses
Relationship between Oil immersion and refractive
index

Because the refractive indexes

of the glass microscope slide
and immersion oil are the
same, the light rays do not
refract when passing from one
to the other when the oil
immersion objective lens is
used.

This method produces images

with better resolution at
magnification greater than
100X.
Microscopes and Microscopy
Microscopes are of two
categories depending upon
the principle on which
magnification is based:
Light (or optical) and
Electron.
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 Function of parts
Subject Function
Ocular lens Re-magnify the images that formed by the objective
lens
Body tubes Transmit the image from the objective lens to the ocular
lens
Arm To hold
Objective lens Primary lenses that magnify the specimens
Stage Holds the microscope slide in position
Condenser Focuses light through specimen
Diaphragm Control the amount of light entering the condenser

Coarse focusing Focus

knob
Illuminator light source
Light microscope

Light microscope, uses a system of lens that

manipulate the path of light beam that travels between
the object that being studied includes:
1.Bright-field
2.Dark-field
3.Fluorescence and
4.Phase-contrast microscopy
Electron Microscope

The Electron Microscope, as the name suggests, uses a

beam of electrons in place of light waves to produce
the image.

Specimens can be examined by either

Transmission or
Scanning electron microscopy.
 Units of Measurement
✓Because microorganisms and their component parts are very small,
they are measured in units that are unfamiliar to many of us in everyday
life.
✓The metric system is generally used and standard unit of length in
metric system is meter.

Table: Metric Units of Length

Metric unit meaning of prefix Metric Equivalent
1 kilometer 1 kilo=1000 1m = 103 mm
1 meter (m) standard unit of length
1 decimeter (dm) deci = 1/10 0.1m = 10-1 m
1centimeter (cm) centi = 1/100 0.01m = 10-2 m
1millimeter (mm) milli = 1/1000 0.001 m = 10-3 m
1 micrometer (µm) micro = 1/1,000,000 0.000001 = 10-6 m
1 nanometer (nm) nano = 1/1,000,000,000 0.000000001 m = 10-9 m
1.Bright-field Microscopy (Compound
light microscopy)

Objects are visible against a bright background.

Light reflected off the specimen does not enter the
objective lens.
A modern compound light microscope has a series of
lenses and uses visible light as its source of illumination.
Bright-field Microscopy (Contd…….)

A series of finely ground lenses forms a clearly forms a

clearly focused image that is many times larger than the
specimen itself. This magnification is achieved when light
rays from an illuminator, the light source, are passed through
a condenser, which has lenses that direct the light rays
through the specimen.
From here, light rays pass into the objective lenses, the
lenses closest to the specimen.
The image of the specimen is magnified again by the ocular
lens, or eyepiece.
Highest magnification in Bright-field
Microscopes
To achieve high magnification (100X) with good resolution, the lens
must be small.
To preserve the direction of light rays at the highest magnification,
immersion oil is placed between the glass slide and the oil immersion
objective lens.
The immersion oil has the same refractive index as glass, so oil
becomes part of optics of the glass of the microscope.
Unless immersion oil is used, light rays are refracted as they enter the
air from the slide, and the objective lens would have to be increased in
diameter to capture them.
 Bright-field Microscopes:
Distinguishing Features

➢Use visible light as a source of illumination

➢Cannot resolve structures smaller than about 0.2µm

➢Specimen appears against a bright background

➢Bright field illumination shows internal structures and the outline of

the transparent sheath.

➢Inexpensive and easy to use

➢are so distorted by staining that their characteristics cannot be identified.
Bright-field Microscopes:
Principal Uses
➢ To observe various stained specimens and to count
microbes

➢ Does not resolve very small specimens, such as

viruses
Pathways of light in bright-field and dark-
field microscopy
2. Dark-field Microscopy
A dark-field microscope is used for examining live microbes
that either
are invisible in the ordinary light microscope,
cannot be stained by standard methods, or

Light objects are visible against a dark background.

Light unreflected off the specimen cannot enter the objective
lens.
Dark-field Microscopes: Distinguishing
Features
Use a special condenser with as opaque disc that blocks
light from entering the objective lens directly.

The only light that reaches the specimen comes in at an

angle; thus, only light reflected by the specimen reaches the
objective lens.

Light reflected by specimen enters the objective lens.

Specimen appears light against a black background.

Dark-field Microscopes:
Principal Uses
❑ To examine living microorganisms that are invisible in
bright field microscopy.

❑ This technique is frequently used to examine unstained

micorbes suspended in liquid.

❑ Frequently used to detect Treponema pallidum in the

diagnosis of syphilis.
3.Phase-Contrast Microscopy
Phase-contrast microscopy is especially useful because it
permits detailed examination of internal structures in living
microorganisms.
The principle of phase-contrast microscopy is based on
slight variations in refractive index.
As rays pass from the light source through the specimen,
their velocity may be altered by differences in the thickness
and physical properties of various portions of the specimen.
Phase-Contrast Microscopy
(contd…)
Light rays passing through the specimen are diffracted
(bent) differently and travel different pathways to reach
the eye of the viewer. These phase differences are seen
through the microscope as different degrees of brightness.

Details of the internal structures of the specimen also

become sharply defined in phase-contrast microscopy.
 Phase-Contrast Microscopy:
Distinguishing Features
❑Use a special condenser containing an annular (ring
shaped) diaphragm

❑The diaphragm allows direct light to pass through the

condenser, focusing light on the specimen and a diffraction
plate in the objective lens

❑Direct and reflected or diffracted light rays are brought

together to produce the image of the specimen on the ocular
lens, containing areas that are relatively light (in phase),
through shades of gray, to black (out of phase).
 Phase-Contrast Microscopy

Principal Uses

To facilitate detailed examination of the

internal structures of living specimen
Differential Interference Contrast
(DIC) Microscopy

DIC microscopy is similar

to phase-contrast
microscopy in that it uses
differences in refractive
indexes.
 Differential Interference Contrast
Microscopy: Distinguishing Features
Like phase-contrast, uses differences in refractive indexes to
produce images.
DIC microscope use two beams of light instead of one separated
by prisms. Prisms split each light beam, adding contrasting colors
to the specimen. Therefore, the resolution of a DIC microscope is
higher than that of a standard phase-contrast microscope.

The specimen appears colored as a result of the prism effect. The

image is brightly colored and appears nearly three-dimensional.

No staining required.
Differential Interference Contrast
Microscopy: Principal Uses
To provide brightly colored nearly three-dimensional
images.

Excellent way to observe living cells.

Thank you