The magnification of an object on the stage
depends on the power of magnification of the
objective lens and the eyepiece.
The total magnification of a microscope is the
magnification of the objective multiplied by the
magnification of the eyepiece.
HOW TO USE, ADJUST AND MAINTAIN A
COMPOUND BINOCULAR MICROSCOPE.
Step #1
 Put the lowest power objective lens into
place.
 Notice that all the objective lenses turn
on a turret and that they click into place.
 As a general rule always use the lowest
power objective lens to find your
specimen.
 On the microscope in front of you, this is
the 4X lens and is the shortest.
 Turn this lens into place.
Step #2
 Now place the specimen on the stage.
 Notice that the slide is held in a clip on
the stage with the specimen side up.
 The clip is adjustable and holds the slide
firmly in place.
 Pull back the clip and slip the glass slide
into place on the stage as shown in the
next slide.
 Make sure that the clip is not on top of
the slide, but holds it firmly in place.
Step # 3
 Now that you have the lens in place and
the specimen centered over the hole in
the stage, gradually raise the stage using
the coarse adjustment until you reach a
stop.
 Watch from the side of the microscope to
make sure that the lens does not hit the
bottom of the slide.
Step #4
 Looking through only the RIGHT
eyepiece, focus by lowering the stage
away from the lens using the coarse
adjustment knob.
 Always remember when focusing to
adjust the stage AWAY from the
objective lens.
 Never raise the stage while looking
through the microscope. You could
smash the slide into the lens and ruin
them both.
 Now focus on your specimen with your
right eye.
Step # 5
 After finding the object with the coarse
adjustment knob, adjust the focus
slightly with the fine adjustment knob
until the specimen is sharp and clear.
Step # 6
 Now you will adjust the binocularity, so
that you can use both eyes to view the
specimen. First move the eyepieces so
that you can look through both eyes at
the same time.
Step # 7
 Now you will adjust the focus of the
eyepieces.
 Note that the right eyepiece is fixed, but
that the left one has telescoping
capability.
 First cover your left eye. Looking through
your right eye, once again bring the
 object into focus by turning the fine
adjustment knob.
 Now cover your right eye. Focus the
specimen with your left eye by adjusting
the telescoping knurled knob on the left
eyepiece.
 The microscope should now be in focus
for you as an individual.
Step # 8
 In preparing to adjust the light on the
specimen, use the stage adjustment
knobs to move the stage forward,
backward or sideways to get a better
view of the specimen.
 Now you will be instructed on how to
adjust the condenser and then the
diaphragm to get reasonably good,
consistent lighting.
Step # 12
Step # 9
 Close the diaphragm as far as possible by
rotating the diaphragm ring.
 Notice that the light decreases but does
not disappear.
Step # 10
 While looking through the microscope,
move the condenser up and down until
you see a “pebbly” or “pearly”
appearance.
 At this point the light from the lamp is
focused on the plane of the specimen.
 Once you have found this pebbly
appearance, adjust the condenser knob
toward you slightly until this disappears
and the light appears constant.
Step # 11
 Once the condenser is adjusted, open the
diaphragm slightly, until the light reaches
a constant illumination across the entire
field.
 Now that the light is adjusted, you might
need to adjust the fine focus again to see
the sharpest image possible.
 Most modern microscopes are par focal.
This means that once the specimen is in
focus with one objective lens, only a
slight turn of the fine focus knob is
required when changing to a higher
power objective.
 It also means that when you change to a
higher power lens, the lens should not hit
the slide. However, just to be sure,
always look at the slide from the side
while you change from one objective lens
to another to make sure that the lens
does not hit the slide or the stage.
 Change to the 10X lens by rotating the
turret 90 degrees and feeling the click as
the objective comes into place.
 Do not move anything else when doing
this.
 Do not adjust the coarse focus knob,
because the slide is now so close to the
lens that movement of the coarse focus
knob could raise the stage enough to
smash the objective lens.
 Now focus using the fine adjustment
knob.
 You might now have to re-center the
specimen using the two stage adjustment
knobs as you did in Step #8.
 You might also need to open the
diaphragm a little more.
 These are the only other adjustments
that you need to make when changing
objective lenses.
  Remember that when you change
objective lenses: do not adjust the lamp
do not adjust the condenser.
 Do adjust if necessary: the fine focus the
diaphragm the mechanical stage.
 Now repeat Step #12 with the “high dry”
40X objective.
USING THE 100X OIL IMMERSION LENS
The last lens is the 100X oil immersion lens. This lens
requires special care. It requires oil between the
slide and the lens, allowing more light to be
gathered by the objective lens.
To apply oil, rotate the oil immersion lens about
halfway into place so that no lens is directly over the
specimen.
USING THE 100X OIL IMMERSION LENS
 Place a drop of immersion oil on the slide at
about the point centered over the stage and
then carefully rotate the 100X oil objective
into place with a click.
 From the side, you should notice a flash as
the lens enters the oil.
When you are ready to clean up, follow the steps
described below.
1. Put the low power objective lens in place.
2. Lower the stage all the way down with the
coarse adjustment knob.
3. Using lens paper, carefully clean all the oil off
of the oil immersion lens using a rotating
motion as shown in the photo below. Be
sure that ALL of the oil is removed!
4. Remove the slide and clean the oil off of the
slide and the stage, using lens paper or a
Kimwipe. Never use Kimwipes on the
objective lenses!
MAGNIFICATION, RESOLUTION, AND WORKING
DISTANCE
Magnification is simply a function of making an
object appear bigger, such as when we use a
hand lens to enlarge printed word. Merely
magnifying an object without a simultaneous
increase in the amount of detail seen will not
provide the viewer with a good image. The
ability of a microscope (or eye) to see detail is a
function of its resolving power. Resolving power
is defined as the minimum distance between
two objects at which the objects can just be
distinguished as separate and is a function of the
wavelength of light used and the quality of the
optics. In general, the shorter the wavelength of
the light source, the higher the resolution of the
microscope.
Working distance is the distance between the
objective lens and the specimen. At low
magnification the working distance is relatively
long. As you increase the magnification the
working distance decreases dramatically. Oil
immersion lenses practically touch the
specimen. Be aware of this change in working
distance with increasing magnification so as to
prevent damage to your specimens.
The resolving power of a microscope is the most
important feature of the optical system and
influences the ability to distinguish between fine
details of a particular specimen.
The term “working distance” is important because it
is a measurement of the free area available to the
viewer to work and manipulate the sample, from the
very bottom of the microscope to the top of the
observed object on the microscope platform or
stage.
The “working distance” of the different objectives
are:
16mm – LPO 4mm – HPO 1.8mm – OIO
Parfocal – it is in focus with one objective, once the
objective is rotated to shift to another objective it
will still remain in focus.
Parcentered – objective in the center of view will
remain in the center when the objective is rotated.
Wet Mount and Hanging Drop
The hanging drop and wet mount techniques allow
for observation of living organisms. The wet mount
tend to dry out quickly under the heat of the
microscope light; it is simpler to perform than the
wet mount, but it is useful for short-term
observation only. The hanging drop is a more
complex technique, but it allows for longer-term
obervation and more reliable observation of
motility.
These techniques are usually performed without
the addition of any stains; therefore, the organisms
can be difficult to see. Reduce the illumination on
your microscope as much as you can while still
allowing yourself enough light to observe the
organism.
If you use these techniques to observe motility, be
sure you can tell the difference between motility
and brownian motion. Vibration of the cell is
caused by the cell colliding with water molecules.
True motility allows the cell to move in different
directions and across larger areas.
WHAT IS A WET MOUNT?
In a wet mount, the specimen is suspended in a
drop of liquid (usually water) located between slide
and cover glass. The water refractive index of the
water improves the image quality and also
supports the specimen. In contrast to permanently
mounted slides, wet mounts cannot be stored over
extended time periods, as the water evaporates.
For this reason, a wet mount is sometimes also
referred to as a “temporary mount” to contrast it
from the “permanent mounts”, which can be
stored over longer times. The permanently
mounted slides use a solidifying mounting medium,
which holds the cover glass in place. The naming
can be a bit problematic, because it is also possible
to make wet mounts that can store over extended
time periods. These are special cases, however.
Different types of wet mounts
Wet mounts can be made using several different
kinds of liquids. Water, immersion oil and glycerin
(glycerol) can be used, with water probably being
the most commonly used. The source of the water
is quite important, especially when observing living
specimens. If you use water with a wrong osmotic
potential (ie. too much or too little salt and mineral
content), then there is the danger of damaging the
specimen. A too high salt content can result in the
specimen to lose too much water. Too low a salt
content, and the specimen may swell and burst.
Brownian movement is a continuous vibrating
motion caused by invisible molecules striking the
bacteria. If the bacteria are truly motile, their
movement will be over greater distances and will
be multi-directional, not just back and forth.
Wet mount:
 Advantages: This method is the simplest
and quickest way to determine motility. It is
also useful for determining cellular shape
and arrangement which is sometimes
destroyed during the staining process.
 Disadvantages: This method is far too risky
to use with highly pathogenic organisms.
Hanging Drop Method
 Advantages: Like the wet mount, the
hanging drop method preserves cell shape
and arrangement. The Vaseline-sealed
depression also slows down the drying-out
process, so the organisms can be observed
for longer periods.
 Disadvantages: The hanging drop method is
also far too risky to use with highly
pathogenic organisms.
True motility is movement in some consistent
direction or path and they may be twisting and
turning. The cells exhibit independent movement
over greater distances than in Brownian
movement.
Brownian motility is movement caused by the
molecules in a liquid striking/hitting against the
particles of an object (eg. Microorganisms) leading
to shaking and vibratio , whereas True motility is
the deliberate/ self-propelled movement of the
organism using its own features such as
flagella,pili/fimbriae.
The hanging drop technique is a well-established
method for examining living, unstained, very small
organisms. The traditional procedure employs a
glass slide with a circular concavity in the centre
into which a drop of fluid, containing the
'microorganisms', hangs from a coverslip.
The iodine stains the cells, especially the nuclei of
the cells and makes them more visible.
Aseptic technique is a method designed to prevent
contamination from microorganisms. It involves
applying the strictest rules and utilizing what is
known about infection prevention to minimize the
risks that you'll experience an infection.
HEAT FIXING THE BACTERIAL SAMPLE
Before staining, the sample must be heat fixed.
This process accomplishes three things:
• kills the bacteria
• firmly attaches the smear to the microscope
slide
• allows the sample to more readily take up
the stain
In order to heat fix a bacterial smear, it is necessary
to first let the bacterial sample air dry. Then either
place the slide in the slide holder of a
microincinerator, or pass the dried slide through
the flame of a Bunsen burner 3 or 4 times, smear
side facing up. Once the slide is heat fixed, it can
then be stained.
This heat fixation step denatures bacterial proteins
causing the cells to stick to the slide while also
killing the bacteria making them safe for the
following steps
the preparation of a smear is required for many
laboratory procedures, including the Gram-stain.
The purpose of making a smear is to fix the bacteria
onto the slide and to prevent the sample from
being lost during a staining procedure. A smear can
be prepared from a solid or broth medium.
An accentuator is any chemical which facilitates
the staining process. Usually the purpose is to
intensify staining, and accentuation with this
meaning is obviously the derivation of the term.
However, it should be noted that inhibition of
staining can also accentuate a structure in
comparison to the background staining. Inhibition
and accentuation of staining are just two sides of a
coin, and will be discussed together.
Accentuators fall into three groups:– pH control,
salts and phenol.
Accentuators= Increase selectivity or stainability -
Does not become part of the dye complex
Why is a mordant used in Gram staining?
The cell walls for Gram-positive microorganisms
have a higher peptidoglycan and lower lipid
content than gram-negative bacteria. Bacteria cell
walls are stained by the crystal violet. Iodine is
subsequently added as a mordant to form the
crystal violet-iodine complex so that the dye
cannot be removed easily.
What is the role of crystal violet in Gram staining?
Conversely, the the outer membrane of Gram
negative bacteria is degraded and the thinner
peptidoglycan layer of Gram negative cells is
unable to retain the crystal violet-iodine complex
and the color is lost. A counterstain, such as the
weakly water soluble safranin, is added to the
sample, staining it red.
How do you determine whether a stain is basic
or acidic?
 If the chromophore is positive, it is basic;
if the chromophore is negative, it is acidic
What is considered a simple stain?
 Staining procedures that use only one
stain
Of what value is a simple stain?
 Determining cell morphology, size, and
arrangement
In heat fixing, what would happen if too much
heat were applied?
 It would damage the cell's structure
Bacteria can be seen without staining. Why then
was Koch's recommendation for fixing and
staining important for microbiology?
 It allows for later re-examination
What does the negative stain technique stain?
 Backgroud
Why doesnt a negative stain stain the bacteria?
 Ionic repulsion; the bacteria and acidic
stain both have negative charges
In which procedure are no heat fixing or strong
chemicals used and why not?
 Negative stain; to keep the bacteria from
getting as distorted
When is the negative stain technique especially
useful?
 When other staining techniques don't
clearly show cell morphology or size
How does bacteria from a negative stain differ
from that of a simple stain?
 It's clearer because only the background
is stained
Why is the size more accurate in a negative stain
than in a simple stain?
 The lack of heat fixing or strong chemicals
keeps from distorting the cell
Could any dye be used in place of nigrosin in a
negative stain?
 No
What type of dye is used for negative staining?
(Acidic or Basic)
 Acidic
What kind of stain is the gram stain? How does
is allow you to classify bacteria?
 Differential; as either gram positive or
gram negative
In a gram stain, what are bacteria called when
they are easily decolorized?
 Gram negative
In a gram stain, what are bacteria called when
they retain the primary stain?
 Gram positive
Why do bacteria stain differently in a gram
stain?
 Chemical and physical differences in their
cell walls
In a gram stain, what type of cell does this
describe?
Crystal violet is picked up by the cell. Iodine
reacts with the dye in the cytoplasm to form CV-
I. The decolorizing agent dissolves the outer
lipopolysaccharide layer, and the CV-I washes
out through the thin layer of peptidoglycan.
  Gram negative
When is the gram stain most consistent?
 When done on cultures less than 24
hours old
List the steps of the gram stain technique.
 1-Cover smear with crystal violet for 30
seconds and rinse, 2-Cover smear with
Gram's iodine for 10 seconds and rinse,
3-Decolorize with alcohol and rinse, 4-
Cover smear with safranin for 30 seconds
and rinse
Name some gram positive bacteria.
 S. aeru, S. epi, S. pyogenes, B. sub, B, meg
Name some gram negative bacteria.
 E. coli, P. vulg, Neisseria
Why will old gram positive cells stain gram
negative?
 They can't retain the primary stain
because with time the lipid layer
degrades allowing the stain to reach the
peptidoglycan
Can iodine be added before the primary stain in
a Gram stain?
 No
What color does Crystal Violet turn each of the
cells in a gram stain?
 Purple
What color does Gram's iodine turn each of the
cells in a gram stain?
 Purple
What color does ethyl alcohol turn each of the
cells in a gram stain?
 Gram positive-purple; Gram negative-
clear
What color does safranin turn each of the cells
in a gram stain?
 Gram positive-purple; Gram negative-red
Since you can't identify bacteria from a gram
stain, why might a physician perform a gram
stain on a sample before perscribing an
antibiotic?
 Because only certain antibiotics work
depending on if the bacteria is gram
positive or negative
What type of antibiotics work on gram positive
bacteria?
 Penicillin
What type of antibiotics work on gram negative
bacteria?
 Tetracycline
If you gram stained human cells, what would
happen?
 Primary stain would wash away because
humans do not have cell walls
What type of stain is the acid-fast stain?
 Differential
What is the value of the acid-fast stain?
 Allows diagnosing of Mycobacterium and
Nocardia, which do not decolorize with
acid alcohol
The cell walls of acidfast organisms have a wax-
like lipid called mycolic acid, which does what?
 Makes the cell wall impermeable to most
stains
Name an acid-fast negative bacteria
  E. coli  Both are gram positive
Name an acid-fast positive bacteria You can see endospores by simple staining. Why
not use this technique?
 M. phlei
 It would be more difficult to pick them
What is the decolorizing agent in the Gram
out because they would be transparent
stain? In the acid-fast stain?
What controls the amount of light reaching the
 Ethyl alcohol; Acid-alcohol
ocular lens?
What diseases are diagnosed using the acid-fast
 Diaphragm
staining technique?
Name two ways in which you can enhance the
 Tuberculosis and Leprosy
resolving power of a microscope.
Why are endospores called resting bodies?
 Decrease the wavelength of light and
 Because they do not metabolize and are increase the numerical aperture
resistant to many harsh conditions
What would occur if water were accidentally
When are endospores formed? used in place of immersion oil?
 When essential nutrients or water are  It would not be as clear because water
not available does not have the same refractive index
as glass
What happens to the cell when an endospore is
formed? What advantages does the low power objective
have over the oil immersion objective for
 It disintegrates
viewing fungi?
Because of a capsules nonionic nature, _____
 Fungi cells are large so you can see more
stains will not adhere to it.
of the cell
 Simple
How must a capsule be stained in order to view
it under a microscope?
 The background must be stained, leaving
the capsules unstained
These thin proteinaceous structures that
originate in the cytoplasm and project out from
the cell wall are the most common means of
motility in bacteria
 Flagella
What are the gram reactions of Clostridium and
Bacillus?