Download Microscope

Introduction to the oil immersion compound
microscope
Purpose of microscope

Microscopy is the technology of making very small things visible
to the human eye.

Used in the diagnosis of many diseases.

Used for soil, insects and rocks.

Used in pathological laboratories and medical.

Industrial microscope : Used for metals and various kinds of
measurement .

Educational microscope.

Used for research.
Purpose of microscope in Microbiology

Bacteria is one of the small microorganisms that can’t be seen
with the naked eyes, most bacteria range in size between 0.5-2.0
micrometers (μm) so, there is a need to magnify the bacteria
several times by using a microscope in order to see it.

There are different types of microscopes which are used in
microbial life.

In this lab, you will become familiar with the use of the light
compound microscope (particularly oil immersion microscopy)
and will compare the relative size and shape of various
microorganisms.
Basic component of microscope
Microscope consists of 4 main parts:

frame work.

Adjustment system.

Magnification.

Lighting System.
Frame work
Its includes three parts:
1.
Base
2.
Arm
3.
mechanical stage
Mechanical Stage

Stage- the location of the
specimen to be viewed

Clips- utilized in holding the
specimen in place
Stage Knobs control
Top knob: is to move the forward and
backward.
Bottom knob: is to move the stage right
and left.
Adjustment system
It consists of the following
parts:
1.
Optical tube.
2.
Coarse adjustment.
3.
Fine adjustment.
Focus and Resolution Parts

Course-adjustment knobis the larger of the two
knobs. It is used in bringing
the object into quick focus.

Fine-adjustment knob- is
used for improving the
clarity
of
the
image,
especially when viewing
under high power.
Interpupillary adjustment
To control the distance between the ocular lenses to adapt the
distance between viewers eyes so, the eyepiece lenses will spread
apart or get closer together to fit each individual.
Diopter ring adjustment
Getting in Focus
Magnification
Microscope has two sets of
lenses are :
1.
Ocular lens (eyepiece).
2.
Objetive lenes.
Microscope lenses:

Ocular lens or eyepiece is used for
viewing.

Revolving
nosepiece
contains
objective lenses that are used to
magnify the image in combination with
the ocular lens.
-
The objective of microscopy is not just
to increase magnification, but to do so
while retaining sufficient resolution.
TOTAL MAGNIFICATION
Total magnifigation:
Powers of the eyepiece
(10X) multiplied by
objective lenses
determine total
magnification.
Depth of focus:
thickness of the field;
as magnification increased ,
depth of focus is decreased.
Field of vision:
the surface area of view;
as magnification increased ,
the area of view decreased.
Oil immersion lens
Refraction and Refractive Index

Refraction = bending.

Refractive index: measurement of the extent that the substance
bend light.

Field of view > or equal 500X cause increase in diffraction due to
the differences in refractive index between glass and lenses
(1.51) and air (1) which increase refraction,
and small lenses present in high magnification
so that oil is used in these lenses as it has refractive index
similar to glass, lenses and specimen which equal 1.51 ,as a
result the distortion will decrease.
1.
2.
3.
Resolution
•
The objective of microscopy is not just to increase magnification,
but to do so while retaining sufficient resolution.
•
Resolution: It is the ability of microscope, at specific
magnification to distinguish two separate objects situated close to
one another and the ability of the lens to reveal fine details.
•
The smaller the distance between the two specific objects that
can be distinguished apart, the greater the resolution power of the
microscope.

As magnification increased the resolution also increased.

To achieve high magnification with good resolution the objective
lens must be small.
Shorter wavelengths of light provide greater resolution.
Under the best conditions, with violet light (wavelength = 0.4
um) and a numerical aperture of 1.4, the light microscope can
theoretically achieve a limit of resolution of just under 0.2 pm.
A typical cell is
10-20 um
Light microscope
sees 0.2 um
1 mm=10-3 m
1 um=10-6 m
1 nm=10-9 m
1 A=Angstrom
=10-10 m
Numerical Aperture
Numerical Aperture (N.A.): This is a number that expresses the ability
of a lens to resolve fine detail in an object being observed. It is derived
by a complex mathematical formula and is related to the angular aperture
of the lens and the index of refraction of the medium found between the
lens and the specimen.
Lighting System:
Lighting system consists of
modern microscopes of the
following parts :
i.
ii.
iii.
iv.
v.
vi.
Power supply
Transformer
Variable resistance
light source
sub stage condenser
Aperture diaphragm
Lighting System:

The Condenser (fucous
light through specimen )

Lamp- typically a light
source underneath the
stage
Diaphragmcontrols the amount of
light allowed to pass
through the specimen

How to use Microscope
1.
2.
3.
4.
5.
6.
7.
Plug in microscope and turn on illuminator. Rotate nosepiece to
lock 4X objective in place
Place smear on stage and center it under the 4X objective.
Using the course adjustment knob, move the objective lens to its
lowest point. Look through the ocular and focus upward with the
coarse adjustment until an image comes into view.
Rotate nosepiece to obtain the next objective lens 10x and
repeat step 3.
Rotate nosepiece to obtain the next objective lens 40x .
Look through the ocular and focus upward with the fine
adjustment until an image comes into view.
When using oil immersion lens put a drop of oil on the slide and
Rotate nosepiece and repeat step 6.
Factors that influence the quality and size of
image:

Quality of Microscope and
lenses.

Size of sample.

Type of sample.

Amount of light on the sample.

Quality of sample.
Microscope Maintenance
Microscope Storage


Proper storage of the microscope will prevent or reduce
problems!
Optics and mechanisms of the microscope must be
protected from:



Dust and dirt,
Fungus.
Store the microscope:


Under a protective cover,
In a low humidity environment.
Always carry a microscope with one hand holding the
arm and one hand under the base.
Microscope Cleaning Process

Cleaning the Eyepiece,

Cleaning the Objectives,

Cleaning the Microscope Stage,

Cleaning the Microscope Body,

Cleaning the Condenser.
Cleaning Solutions and Solvents

Soap solution for cleaning of body and stage.

Ether-Alcohol, Alcohol, or Lens Cleaner Solution
for cleaning of lenses.

Refer to manufacturer’s guide for appropriate
organic solvent.
Cleaning Materials

Lint-free cotton gauze pads
 Lint-free cotton swabs
 Lens paper
 Commercial lens tissue for optics

Caution: Do not use paper
towels or other rough paper
products

Alternatives include:



Fine quality tissue paper
Muslin cloth
Silk
Microscope Cleaning Process

Cleaning the Eyepiece,

Cleaning the Objectives,

Cleaning the Microscope Stage,

Cleaning the Microscope Body,

Cleaning the Condenser.
Step 1: Cleaning the Eyepieces
•
Blow to remove dust before
wiping lens.
•
Clean the eyepieces with a
cotton swab moistened
with lens cleaning solution.
•
Clean in a circular motion
inside out.
Step 2: Cleaning the Eyepiece
•
Wipe the eyepieces dry
with lens paper.
•
Repeat cleaning and
drying if required.
Step 3:Cleaning the Objectives

Objectives are cleaned while attached to
microscope





Moisten the lens paper with the cleaning solution
Wipe gently the objective in circular motion from
inside out
Wipe with dry tissue or lens cleaning paper .
Be sure to clean the oil immersion objective after
use.
Objectives should never be removed from the
nosepiece.
Step 4: Cleaning the Microscope Stage
•
Wipe the microscope stage using
the cleaning solution on a soft
cloth.
•
Thoroughly dry the stage.
•
Repeat above steps, if required.
Step 6: Cleaning the Condenser
•
Unplug the microscope from
power source.
•
Clean the condenser lens
and auxiliary lens using lintfree cotton swabs moistened
with lens cleaning solution.
•
Wipe with dry swabs.
Step 5: Cleaning the Microscope Body
i.
Unplug the microscope from power source.
ii. Moisten the cotton pad with a mild cleaning agent .
iii. Wipe the microscope body to remove dust, dirt, and
oil.
iv. Repeat steps 1–3, if required.
Video on how to use Microscope