Types of Microscope and

Culture Media
I N S T R U C TO R : E N G R . M U H A M M A D J U N A I D K H A L I D

DEPARTMENT OF ENERGY SYSTEMS ENGINEERING

NFC INSTITUTE OF ENGINEERING AND TECHNOLOGY,
MULTAN, PAKISTAN
 Microscope and Its Types
Light Microscope

 Uses visible light to observe specimen.

Compound Microscope

 Compound light microscope has a series of lenses and uses visible light as its source of illumination.

 They have two types of lenses – the oculars and the objectives.

 These lenses provide a vital function for the microscopes - magnification or enlargement of objects. The total magnification is

the product of the objective lens and of the ocular lens (i.e. multiplication of the two quantities).

Microorganisms are measured in micrometers, µm (10-6), and in nanometers, nm (10-9).

 Microscope and Its Types

 Most microscopes used in microbiology have several objective lenses, including 10X (low power), 40X
(high power), and 100X.

 Microscopes also enable the observer to distinguish structures that are separated by short distances.
This function is called resolution.

 For example, if a microscope has a resolving power of 0.4 nm, it can distinguish two points if they are
at least 0.4 nm apart.


Light Microscopy
 Microscope Type Distinguishing Features Principal Uses

Uses visible light as a source of illumination; To observe various stained specimens and to
Bright-field microscopy cannot resolve structures smaller than about count microbes: does not resolve very small
0.2 µm: specimen appears against a bright specimens, such as viruses
background. Inexpensive and easy to use

Uses a special condenser with an opaque disk To examine living microbes that are invisible in
Dark-field that blocks light from entering the objective bright-field microscopy, do not stain easily, or
microscopy lens directly; light reflected by specimen enters are distorted by staining
the objective lens, and the specimen appears
light against a black background

Uses a special condenser containing an To facilitate detailed examination of the internal

Phase-contrast annular diaphragm. The diaphragm allows structures of living specimens
microscopy 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. No
staining required
 Microscope and Its Types
Differential interference contrast (DIC)

 DIC microscopy is similar to phase-contrast microscopy in that it uses differences in refractive

indexes.

 DlC uses two beams of light instead of one.

 Prism split each light beam, adding contrasting colors to the specimen.

 Resolution of a DIC microscope is higher than that of a standard phase-contrast microscope.

 The image produced is brightly colored and appears nearly three-dimensional.

 Microscope and Its Types
Fluorescence Microscopy

 Principle: Fluorescence

 The ability of a substances to absorb short wavelengths of light (ultra-violet) and give off light at a longer
wavelength (visible).

 Some organisms fluoresce naturally under ultraviolet light; if the specimen to be viewed does not naturally
fluoresce, it is stained with fluorochrome dye.

 Microorganisms stained with fluorochrome appear as luminescent, bright objects against a dark background.
 Microscope and Its Types
Confocal Microscopy

 Uses a single photon to illuminate one plane of a specimen at a time.

To obtain two- and three-dimensional images of cells for biomedical applications.

Confocal microscopy uses a pinhole aperture, so it eliminates the blurring that occurs with
other microscopes.
 Microscope and Its Types
Two-Photon Microscopy

 Two-photon microscopy uses long-wavelength (red) light, and therefore two photons are
needed to excite the fluorochrome to emit light.

 The longer wavelength allows imaging of living cells in tissues up to 1 mm deep.

 It can track the activity of cells in real time. For example, cells of the immune system have
been observed responding to an antigen.
 Microscope and Its Types
Scanning Acoustic Microscopy

 SAM basically consists of interpreting the action of a sound wave sent through a specimen.

 A sound wave of a specific frequency travels through the specimen, and a portion of it is
reflected back every time it hits an interface within the material.

 The resolution is about 1µm.

 SAM is used to study living cells attached to another surface, such as cancer cells, artery plaque,
and bacterial biofilms that foul equipment.
Scanning Acoustic Microscope
Electron Microscopy
 Microscope and Its Types
Electron microscope
 Objects smaller than about 0.2 µm, such as viruses or the internal structures of cells, must be examined with an
electron microscope.

In electron microscopy, a beam of electrons is used instead of light.

The resolving power of the electron microscope is far greater than that of the other microscopes.

Images produced by electron microscopes are always black and white, but they may be colored artificially to
accentuate certain details.

Instead of using glass lenses, an electron microscope uses electromagnetic lenses to focus a beam of electrons
onto a specimen.
 Microscope Type Distinguishing Features Principal Uses

Uses a beam of electrons instead of light; To examine viruses or the internal ultrastructure in
Transmission electrons pass through the specimen; thin sections of cells (usually magnified 10,000-
electron microscopy because of the shorter wavelength of 1,00000X)
electrons, structures smaller than 0.2 µm
can be resolved. The image produced is
two-dimensional

Uses a beam of electrons instead of light; To study the surface features of cells and viruses
Scanning electrons are reflected from the specimen; (usually magnified 1000-10,000X)
electron microscopy because of the shorter wavelength of
electrons, structures smaller than 0.2 µm
can be resolved. The image produced
appears three-dimensional
Scanning Electron Microscope Transmission Electron Microscope
 Microscope Type Distinguishing Features Principal Uses

Uses a thin metal probe that scans a specimen and Provides very detailed view of
Scanned probe produces an image revealing the bumps and molecules inside cells
microscopy depressions of the atoms on the surface of the
specimen. Resolving power is much greater than that of
an electron microscope. No special preparation is
required

Uses a metal-and-diamond probe gently forced down Provides 3 dimensional images of

Atomic force microscopy along the surface of the specimen. Produces a three- biological specimens at high
dimensional image. No special preparation required resolution in nearly atomic detail and
can measure physical properties of
biological specimens and molecular
processes
Scanned probe microscope Atomic force microscope
 Culture Media and Its Types
 The food materials on which the organism is grown is known as culture medium and the
growth of organism is known as culture.

 A culture is an in vitro technique of growing or cultivating microorganisms or other cells in a

suitable nutrients medium.

 Different microorganisms require different nutrient materials. Thus, culture media vary in form
and composition, depending upon the species to be cultivated.

 Glass test tube and glass or plastic petri dishes are used to cultivate microorganisms.
 Test Tubes
Petri dish
 Culture Media and Its Types
Basic Requirements of Culture Medium

 Energy source

 Carbon source

 Nitrogen source

 Salts

 pH

 Adequate oxidation Growth factors

 Culture Media and Its Types
Uses of Culture Medium

To study the characteristics and properties of microorganisms

 Isolate pure cultures by forming colonies

To prepare biological products like vaccines etc.

To identify cause of infection from the clinical sample, so the proper treatment can be given
 Culture Media and Its Types
Common Ingredients of Culture Media

1. Water: It is essential for existence of living cells. They act as source of hydrogen and oxygen.

2. Peptone: Golden granular hygroscopic powder which are obtained from meat, casein fibrin or
soya bean flour. A source of nitrogen source, carbon source.
3. Meat Extract: It contains protein degradation products, carbohydrates, inorganic salts,
enzymes, excites and growth factors that are rich in vitamin B complex.
4. Yeast Extract: It contains proteins, amino acids, growth factors (Vitamin B), Carbohydrates
and inorganic salts like potassium and phosphates.
 Culture Media and Its Types

5. Agar: Dried mucilaginous substance obtained from algae available in powder form; contains

mainly long chain polysaccharides, protein like material and inorganic salts.

6. Fermentable Compounds: Mainly used are sugars, alcohols etc. Act as source of energy,

fermentation reactions are helpful in the identification and classification of organisms.

7. Buffers: Carbonates and phosphates are used as buffer to resist change in pH of the medium.
 Culture Media and Its Types
Types of Media (Based on physical state)

 Liquid media

 Semi solid media

 Solid media

Liquid Media or Broth

 No solidifying agents (e.g. agar) is added while preparing the medium.

 The most commonly used non-synthetic liquid media are nutrient broth, peptone solution, milk, blood,
serum etc.
 Culture Media and Its Types
 Broth is a clear transparent straw coloured fluid prepared from meat extract or peptone.

Advantages of Liquid Media

 For obtaining bacterial growth from blood or water when large volumes have to be tested.

 For preparing bulk cultures for preparation of antigens or vaccines.

Disadvantages of Liquid Media

 It’s difficult to isolate different types of bacteria from mixed population.

 It’s difficult to study colony characteristics.

 Culture Media and Its Types
Semi-Solid Media

 The semi-solid medium remains in the semi-solid condition. It is prepared by adding small
amount of agar (0.5%) or gelatin.

 The semi-solid medium may be selective which promotes the growth of one organism and
retards the growth of another organism.

This type of medium can be used to study bacterial motility and separating motile from non
motile strains.
 Culture Media and Its Types
Solid Media
The solid medium is solid in consistency. It is prepared by adding 2% or 1% gelatin; agar or silica gel is
sometimes an inorganic solidifying agent. It’s used for colony characterization, colony identification, etc.

Types of Media (Based on composition)

 Simple Media
 Complex Media
 Synthetic or defined Media
 Special Media
 Culture Media and Its Types
a) Simple Media
It’s also called Basal media. It consists of meat extract, peptone, Sodium Chloride and water. Nutrient agar

made by adding 2% agar to nutrient broth is the simplest and commonest medium in routine diagnostic

laboratories.

b) Complex Media
These have added ingredients for special purpose or for bringing out certain characteristics or providing

special nutrient required for the growth of certain organisms.

 Culture Media and Its Types
c) Synthetic or defined Media
These media are prepared from pure chemical substances and the exact composition of the medium is known. They

are used for research purpose.

e) Special Media
No single medium or set of conditions can support the growth of all the different types of organisms that occur in

nature. To cultivate, recognize, enumerate and isolate certain types of microorganisms, many special purpose media

are needed on the basis of their application or function, these special media can be classified into different types:
 Culture Media and Its Types
 Enriched Media
 Selective Media
 Indicator Media
 Differential Media
 Anaerobic Media
 Sugar Media
 Transport Media