Microscope

Learning Objectives
Once you have completed the experiments in this section, you
should be:
1. able to know the components of the Microscope
2. understand the use of the component parts of the
Microscope
3. able to visualize the cellular morphology from stained slide
preparations
4. able to visualize living microorganisms using wet mount or
hanging-drop preparation
5. able to know how to take care of the Microscope
Taking Care of Your Microscope
• It is your responsibility to take care of your
Microscope and learn to use it properly
Getting Microscope Out
• When transporting your Microscope,
always hold it with one hand under the Arm
base, and one hand around the arm
Putting Microscope Away
• Clean stage if it is oily and use lens paper to
clean lenses
• Shortest objective lens (the one with the
red band) should be pointing down toward
stage
• Use Course Adjustment Knob to position
stage as low as can go
• Always put your Microscope back in the Base
proper spot in the scope cabinet
Parts of the
Microscope
Parts of the Microscope Ocular lenses/Eyepiece
Head
Body Tube
Revolving Nosepiece

Arm Objective Lenses

Light Source
Fine Adjustment
Knob

Condenser
Adjustment Knob
Base Power Switch
Brightfield Microscope
• This instrument contains two-lens systems for magnifying
specimens
• the Ocular lens in the Eyepiece and
• the Objective lens located in the Nosepiece
• The specimen is illuminated by a beam of light focused on it by a
substage lens called a Condenser; the result is a specimen that
appears dark against a bright background
• A major limitation of this system is the absence of contrast
between the specimen and the surrounding medium, which
makes it difficult to observe living cells
• Most brightfield observations are performed on nonviable,
stained preparations
Pathways of Light in Microscope
Components of the Microscope
Stage - A fixed platform with an opening in the center allows the
passage of light from an illuminating source below to the lens
system above the stage
• This platform provides a surface for the placement of a slide
with its specimen over the central opening
• In addition to the Fixed Stage, most microscopes have a
Mechanical Stage that can be moved vertically or horizontally
by means of adjustment controls
• Less sophisticated microscopes have clips on the fixed stage,
and the slide must be positioned manually over the central
opening
Microscope Slide on the Stage
Illumination
• The light source is positioned in the base of the
instrument. Some microscopes are equipped with a
built-in light source to provide direct illumination
• Others are provided with a reversible mirror that has
one side flat and the other concave.
• An external light source, such as a lamp, is placed in
front of the mirror to direct the light upward into the
lens system.
• The flat side of the mirror is used for artificial light, and
the concave side for sunlight.
Condenser
• This component is found directly under the stage and contains
two sets of lenses that collect and concentrate light as it
passes upward from the light source into the lens systems
• The condenser is equipped with an Iris Diaphragm, a
shutter controlled by a lever that is used to regulate
the amount of light entering the lens system

Iris
Diaphragm Iris
Open Iris Diaphragm Condenser
Diaphragm
Partially open
Closed
Body Tube
• Above the stage and attached to the arm of the microscope is
the body tube. This structure houses the lens system that
magnifies the specimen
• The upper end of the tube contains the ocular or eyepiece
lens. The lower portion consists of a movable nosepiece
containing the objective lenses. Rotation of the nosepiece
positions objectives above the stage opening.
• The body tube may be raised or lowered with the aid of
course-adjustment and fine-adjustment knobs that are
located above or below the stage, depending on the type and
make of the instrument.
Magnification
• Enlargement, or magnification, of a specimen is the function
of a two-lens system; the ocular lens and the objective lens
• These lenses are separated by the body tube. The objective
lens is nearer the specimen and magnifies it, producing the
real image that is projected up into the focal plane and then
magnified by the ocular lens to produce the final image.
• The most commonly used microscopes are equipped with a
revolving nosepiece containing four objective lenses, each
possessing a different degree of magnification. When these
are combined with the magnification of the ocular lens, the
total magnification of the specimen is obtained
Microscope Objective Lenses

Scanning
Oil Immersion
Low Power

High Power
Total Magnification
Total Magnification = Magnification of the Ocular Lens multiplied by the
magnification of the Objective Lens in position
Resolving Power or Resolution
• Although magnification is important, you must be aware that
unlimited enlargement is not possible by merely increasing
the magnifying power of the lenses or by using additional
lenses, because lenses are limited by a property called
resolving power
• Resolving power is how far apart two adjacent objects must
be before a given lens shows them as discrete entities
• When a lens cannot discriminate, that is, when the two
objects appear as one, it has lost resolution
• Increased magnification will not rectify the loss and will, in
fact, blur the object
Bench Rules During Microscopy
Once the microscope is placed on the laboratory bench, observe the
following rules:
1. Remove all unnecessary materials (such as books, papers,
purses, hats etc.) from the laboratory bench
2. Uncoil the microscope’s electric cord and plug it into an
electrical outlet
3. Clean all lens systems; the smallest bit of dust, oil, lint, or
eyelash will decrease the efficiency of the microscope. The
ocular, scanning, low-power, high-power and Oil immersion
lenses may be cleaned by wiping several times with acceptable
Lens Paper. Never use paper toweling or cloth on a lens surface
Correct & Efficient Use of the Microscope
1. Place the microscope slide with the specimen within the stage
clips on the fixed stage. Move the slide to center the specimen
over the opening in the stage directly over the light source.
2. Raise the microscope stage up as far as it will go. Rotate the
scanning lens or low-power lens into position. Lower the
body tube with the coarse-adjustment knob to its lowest
position. Note: Never lower the body tube while looking
through the ocular lens.
3. While looking through the ocular lens, use the fine adjustment
knob, rotating it back and forth slightly, to bring the specimen
into sharp focus.
Correct & Efficient Use of the Microscope (Cont.…)
4. Adjust the substage condenser to achieve optimal focus.
5. Routinely adjust the light source by means of the light-source
transformer setting, and/or the iris diaphragm, for optimum
illumination for each new slide and for each change in
magnification.
6. Most microscopes are parfocal, which means that when one
lens is in focus, other lenses will also have the same focal
length and can be rotated into position without further major
adjustment. In practice, however, usually a half-turn of the
fine-adjustment knob in either direction is necessary for
sharp focus.
Correct & Efficient Use of the Microscope (Cont....)
7. Once you have brought the specimen into sharp focus with a
low-powered lens, preparation may be made for visualizing
the specimen under oil immersion. Place a drop of oil on the
slide directly over the area to be viewed. Rotate the
nosepiece until the oil-immersion objective locks into
position.
Note: Care should be taken not to allow the high-power objective
to touch the drop of oil. The slide is observed from the side as the
objective is rotated slowly into position. This will ensure that the
objective will be properly immersed in the oil. The fine adjustment
knob is readjusted to bring the image into sharp focus.
Correct & Efficient Use of the
Microscope (Cont....)
8. During microscopic examination of
microbial organisms, it is always
necessary to observe several areas of
the preparation. This is accomplished
by scanning the slide without the
application of additional immersion
oil. Note: This will require continuous,
very fine adjustments by the slow,
back-and-forth rotation of the fine-
adjustment knob only.
After Completion of the Laboratory Exercise
Return the microscope to its cabinet in its original condition. The
following steps are recommended:
1. Clean all lenses with dry, clean lens paper
2. Place the low-power objective in position and lower the
body tube completely
3. Center the mechanical stage
4. Coil the electric cord around the body tube and the stage
5. Carry the microscope to its position in its cabinet in the
manner previously described.
 Wet Mount Preparation 1. Microscope Slide

2. Inoculating Loop

3. Microscope
Cover Slip

Wet mount slide ready for

Hanging-drop preparation
Nutrient
Clear/no Nutrient Cloudiness/
Broth cloudiness Turbidity
with Broth
No with
growth Bacterial
growth
Pond Water Hay Infusion
Algae and Protozoa that may be found in Hay Infusion and Pond Water
Algae

Protozoa
Prepares Slides
 Immersion oil
Lens paper dispensing bottle
Staphylococcus aureus Staphylococcus aureus
Wet mount Stained
Bacillus cereus Wet mount Bacillus cereus Stained
Aquaspirillum itersonii Aquaspirillum itersonii
Wet mount Stained
Saccharomyces cerevisiae Saccharomyces cerevisiae
Wet mount Stained
Stained
Human
blood
Smear
Hanging-Drop Preparation
Steps 1–4 are illustrated in the Figure below.
1.With a cotton swab, apply a ring of petroleum jelly around the
concavity of the depression slide.
2.Using aseptic technique, place a loopful of the culture in the center of
a clean coverslip.
3.Place the depression slide, with the concave surface facing down, over
the coverslip so that the depression covers the drop of culture. Press
the slide gently to form a seal between the slide and the coverslip.
4.Quickly turn the slide right side up so that the drop continues to
adhere to the inner surface of the coverslip.
5.For microscopic examination, first focus under the low-power objective
(10*) and reduce the light source by adjusting the condenser. Repeat
using the high-power objective (40*).
Hanging
Drop
Preparation
Wet Mount
A wet mount may be substituted for the hanging drop preparation:
1.With a cotton swab apply a thin layer of petroleum jelly along the
edge of the four sides of a coverslip.
2.Using aseptic technique, place a loopful of the culture in the center of
a clean coverslip.
3.Place a clean glass slide over the coverslip and press the slide gently
to form a seal between the slide and the coverslip.
4.Quickly turn the slide right side up so that the drop continues to
adhere to the inner surface of the coverslip.
5.For microscopic examination, first focus on the drop culture under the
low-power objective (10*) and reduce the light source by adjusting
the condenser. Repeat using the high-power objective (40*).
 1

Flaming an
Inoculating
Loop 2

3
Flaming an
Inoculating
Loop
(Cont..)
4
The same
process can be
followed for
Flaming the
Inoculating
Needle below
5
Amoeba under the microscope
Euglena under the microscope
Algae under the microscope
Cheek cells under the microscope
Paramecium
under the
microscope
Plant cell under the microscope
Yeast under the microscope
Blood cells under the microscope - stained
 Inoculating Inoculating
Needle Loop

Bacterial
Growth on
Plates -
colonies
can be
picked for
staining
Bacterial Growth on slant can be picked for staining
E. coli under the microscope – Stained with Safranin
E. coli under the microscope – Stained with Methylene Blue
Bacillus cereus under the microscope – Stained with Methylene Blue
Staphylococcus under the microscope - Stained