Microbiology

Practical Exercise 1

Use and Care of the Microscope

Dr. Shijna Kappally
Lecturer
College of Pharmacy, University of Sharjah
Learning Outcomes

1. Distinguish between the different type of microscopes (light and electronic)

2. Demonstrate the correct use of a compound light microscope

3. Name the major parts of a compound light microscope

4. Determine the relative sizes of different microbes

5. Identify the three basic morphologies of bacteria

Light Microscope Fluorescent Microscope

Transmission Electron Scanning Electron

Microscope (TEM) Microscope (SEM)
First microscope Latest microscope

Mini Microscope for iPhone, which attaches to the

camera lens of your iPhone 4 or 4S and offers up to 60x
magnification. The device also includes three LED lights
to illuminate the small object under consideration. 
First microscope
Light Microscope

Electron Microscope
 Introduction

 The most fundamental skill of microbiology is the use of the microscope.

 The definition of a microscope is a device for magnifying objects that are too
small to be seen wit the naked eye.

 Many microscopes have only one ocular, these are termed monocular or two
oculars and are termed binocular
Principal Parts of The Microscope
Light Microscope
Light microscopes use glass lenses to bend and focus light rays and produce
enlarged images of small objects.

Maximum resolution is about 0.2 µm. Bacterial size is measured in micrometers.

(1 Micrometer = 1 µm = 1 micron = 1/1000 mm = 10-6 m).

(1 Nanometer = 1 nm = 1/1000 µm = 10-6 mm = 10-9 m).
(1 Ångstrom unit = 1 Å = 10-1 nm = 10-10 m).
Light Microscope: general characteristics
 With LM, stained tissues are usually examined by means of light that passes through
the specimen.

 The optical components consists of 3 systems of lenses:

- Condenser : Collect and focuses light

- Objective : Enlarge and project the image in the direction of the eyepiece

- Eyepiece: Magnifies and project the image onto the viewer `s retina

Total magnification: (objective magnification X eyepiece magnification).

i.e. 40x total magnification = (4x objective) (10x ocular)
 Light Microscope: general characteristics
 Resolution is the smallest distance between two particles at which they can be
seen as separate objects.

 To magnify an image is to make it larger, and to resolves (Resolution) is the

ability of a lens to separate or distinguish between small objects that are close
together.

 Increasing magnification without improving resolution is not useful.

 The max resolving power of the light microscope is approximately 0.2 um, this
power permits good images magnified 1000-1500 times.

 The frontiers of LM have been redefined by the use of video cameras highly
sensitive to light.
Mechanism of immersion oil of LM

Refractive index and oil-immersion lenses The refractive index of air is 1, and no lens working
in air can have a numerical aperture more than 1. The only practical way to raise the numerical
aperture above 1, and achieve higher resolution, is to increase the refractive index with
immersion oil, a colorless liquid with the same refractive index as glass
Tips while using a Microscope
Microscopes should always be carried with one hand under the base and the other hand
holding the arm of the scope securely, keeping the cord out of the way of feet.
 The lenses should always be wiped down with lens paper and the cord should be kept out
of the way of the working area.
When beginning an examination of a specimen the scope should be on the lowest power
with the stage raised as high as it will go.

 The slide should be placed between the stage clips and all placement of slide and stage
objectives should be done BEFORE looking into the oculars.
 Once all placement is ready, adjustment should be done while looking through the
ocular.
Adjustment should begin with the coarse adjustment, once the specimen is spotted then
the fine tuning adjustment can be used.

Because of parfocality, once the specimen is in focus on the lowest power there
should be no need to adjust the coarse adjustment even with increased
magnification.
Light Microscope
:A variety of light microscopes are commonly used in histology

A. Bright-field microscope

B. Phase contrast and Differential interference microscopy

C. Polarizing microscope

D. Confocal microscope

E. Fluorescence microscope
A. Bright field Microscope

 The ordinary microscope is called a bright field microscope

because it forms dark image against a brighter background.
 E. Fluorescence Microscope
E. Fluorescence Microscope
 Principal:
The fluorescence microscope exposes a specimen to ultraviolet, violet, or blue light and forms
an image of the object with the resulting fluorescent light on a dark background.
E. Fluorescence Microscope
Method:
Specimen should be stained with dye molecules called fluorochromes, that fluoresce
brightly upon exposure to light of specific wavelength.

Cells fluoresce when illuminated with ultraviolet light

Electron Microscope
 Electron microscopes are based on the interaction between electrons and tissue
components.

 Two types of Electron microscopes are known :

1) Transmission Electron Microscope (TEM)

2) Scanning Electron Microscope (SEM)

Transmission Electron Microscope (TEM)
 The transmission electron microscope achieves great resolution (about 0.1 nm), by using
electron beams. This high resolution allows magnifications up to 400.000 times to be
viewed with detail.

 TEM is used to examine the internal structures of microorganisms in great detail, or the
internal details of any section in any specimen.

 TEM uses magnetic lenses to form an image from electrons that have passed through a very
thin section (20-100 nm thick) of a specimen.

 The TEM functions on the principle that a beam of electrons can be deflected by
electromagnetic fields in a manner similar to light deflection in glass lenses.

 To provide good interaction between specimen and electrons, EM requires very thin
sections (40-90 nm) and therefore embedding is performed with a resin that becomes
very hard.

 Freezing techniques allow the examination of tissues by EM without the need of fixation
and embedding.
Scanning Electron Microscope (SEM)
 SEM is used to examine the external surface features of microorganisms.

 SEM permits a pseudo-three dimensional image of the surface of cells, tissues and
organs with a great details.

 SEM produce a very narrow electron beam that is moved sequentially from point to
point across the specimen.

 SEM differs from TEM in generating an image by scanning a fine electron beam over the
surface of specimens rather than projecting electrons that pass through the specimens
as in TEM.
To the practical work
Needed material
 Compound light microscope
 Immersion oil
 Lens paper
 Prepared slides of 3 types of microorganisms:
- Bacteria: E. coli and S. aureus
- Fungi: Aspergillus, Rhizopus, Penicillium, Coprinus comatus, Puccinia graminis
- Protozoa: Amoeba, Paramecium
Procedure
Procedure
http://www.bacteriainphotos.coml
http://www.bacteriainphotos.coml
 Protozoa

Amoeba Paramecium
 Fungi

Rhizopus: Bread mold

Rhizopus_LM
 Fungi

Aspergillus: brown mold Aspergillus_LM

Aspergillus_SEM
 Fungi

Penicillium: The blue Mold

Penicillium_LM
 Fungi

Coprinus comatus, Also called shaggy ink cap and lawyer's wig

 Fungi

Puccinia graminis 

Also called stem black, and cereal rusts

Bacteria
E. Coli
Bacteria
S. aureus
Labster Simulations