INTRODUCTION TO MICROSCOPY AND SIMPLE STAINING

1. MI-INTRODUCTION TO MICROSCOPY AND SIMPLE STAINING
Purpose
To observe the microorganisms in microscope
Theory:
A microscope is an instrument used to see objects that are too small to be seen by the naked eye.
The most familiar type of microscope is the optical, or light, microscope, in which glass lenses are
used to form the image. Optical microscopes can be simple, consisting of a single lens, or compound,
consisting of several optical components in line. The hand magnifying glass can magnify about 3 to
20%. Single-lensed simple microscopes can magnify up to 300×-and are capable of revealing bacteria-
while compound microscopes can magnify up to 2,000x. A simple microscope can resolve below 1
micrometre (μm; one millionth of a metre); a compound microscope can resolve down to about
0.2μm.
The magnifying power of a microscope is an expression of the number of times the object being
examined appears to be enlarged and is a dimensionless ratio. It is usually expressed in the form 10x
Part of the microscope
Eyepiece Lens (Ocular lens): the lens at the top that you look through. They are usually 10X or 20X
power.
Tube: Connects the eyepiece to the objective lenses
Arm: Supports the tube and connects it to the base
Base: The bottomof the microscope, used for support
Illuminator: A steady light source (110 volts) used in place of a mirror. If your microscope has a mirror,
it is used to reflect light from an external light source up through the bottom of the stage.

2. Stage: The flat platform where you place your slides. Stage clips hold the slides in place. If your
microscope has a mechanical stage, you will be able to move the slide around by turning two knobs.
One moves it left and right, the other moves it up and down.
Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can be
rotated to easily change power.
Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always
consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens,
we get total magnifications of 40X (4X times 10X), 100X, 400X and 1000X. The shortest lens is the
lowest power, the longest one is the lens with the greatest power. Lenses are color coded and if built
to DIN standards are interchangeable between microscopes.
Rack Stop: This is an adjustment that determines how close the objective lens can get to the slide. It
is set at the factory and keeps students from cranking the high power objective lens down into the
slide and breaking things. You would only need to adjust this if you were using very thin slides and
you weren't able to focus on the specimen at high power. (Tip: If you are using thin slides and can't
focus, rather than adjust the rack stop, place a clear glass slide under the original slide to raise it
a bit higher)
Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen.
Condenser lenses are most useful at the highest powers. Microscopes with in stage condenser lenses
render a sharper image than those with no lens.
Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This diaphragm has
different sized holes and is used to vary the intensity and size of the cone of light that is projected
upward into the slide. There is no set rule regarding which setting to use for a particular power.
Rather, the setting is a function of the transparency of the specimen, the degree of contrast you
desire and the particular objective lens in use.

3. How to Focus Your Microscope: The proper way to focus a microscope is to start with the lowest
power objective lens first and while looking from the side, crank the lens down as close to the
specimen as possible without touching it. Now, look through the eyepiece lens and focus upward only
until the image is sharp. If you can't get it in focus, repeat the process again. Once the image is sharp
with the low power lens, you should be able to simply click in the next power lens and do minor
adjustments with the focus knob. If your microscope has a fine focus adjustment, turning it a bit
should be all that's necessary. Continue with subsequent objective lenses and fine focus each time.

4. Staining
Microbial cells are mostly transparent and difficult to detect under microscope due to the lack of
contrast from their surrounding. In order to increase the contrast between cell and surrounding,
either cell or surface could be stained by a staining agent. Staining agent (coloring solutions)
containing a coloring molecule called chromophore which give the characteristic color of the stains.
Cell are stained when the stain molecules have attached/adsorbed to cell surface cell via ionic bond.
Recall the chemistry knowledge in order to create an ionic bond the cell molecule and the stain
should have opposite electronic charges.
Figure 1: Demonstration of staining principle. Cells are mostly negatively charged and the positively charged stain molecules
attached to the cell [1].
Mostly microbial cell have a negative (-) charge. So stain molecule should have a positively (+)
charged chromophore which are called cationic dyes. The main cationic dyes used in microbiology is
methylene blue which have a dark blue color
Figure 2: Methlene blue molecule. The chromophore part have an ionic bond with CT. When dissolved, positively charged
chromophore released from stain molecule.

5. Stain molecules are foreign molecules (unwanted, may be toxic) for cells. Living cells try to avoid the
staislecule attached and entered the cell. In order to protect themself from stain molecule, Semip
cable cell membrane, the active transport system of microbial cell are used as defance mechanism
which caused the cell not to stain.
In order to prevent this obstacle, cells are heat fixed. Heat fixing procedure denature the proteins in
the cell resulted to both kill and attached the cells to the microscope slide. Due to heat fixing, also
attached cells do not wash away while performing staining procedure.
Size and shape of the microorganisms:
In microbial world there are enormous variation in size and type among
microbes. Microorganisms size range from 0,5- 300 μm in generals. For
instance while an amoeba have a 0,3 mm diameter, yeast have a diameter
range from 2-8 um and bacteria have 0,2 µm diameter.In addition many
bacteria can be differentiated by the help of their morphological shapes.
Some of them are spherical are called Coccus, some of them have a rod
shape are called Bacil.

6. Procedure:
1. Preperations of smear
> Prepare Methylene blue solution, washingbottle, microscope, microscope slide and analysed
sample, inoculation loop
> Clean the microscopeslide
> Transfer some microbial sample and disperse it in to the slide. If the samples comes from agar or
other solid particles are used, disperse it by the help of a few drop of water. > Wait till the water
evaporate.
2. Heat fixing:
Holding the smear on the corner and pass it through the flame by quick motions. Avoid putting the
slide to long to fire. Quick and repeat movements should performed. In heat fixing procedure the
microbial proteins denature and stick to the microscope slide.
3. Staining
 >2-3 drop of staining solution (methylene blue) put on to the heat fixed smear and wait 1
minute.
 Stain molecules are attached to the cell surface and stain them.
 After 1 min rinse the smear to remove excess methlene blue solution.
 Blot between sheets of bibulous paper.
 Wait till the excess water evaporate.
 Your sample are ready to microscopic examination.

7. Figure: Simple staining procedure.
Questions:
1.Does every type of the microbial cell should stain in order to make a microscopic
examination or are there any exceptions ? Why?
2.In case of forgetting the fixing part while performing staining procedure, what would
happen?
3. What does the microscopic view of E.coli, Bacillus, Staphylococcus and yeast cell look like?
4. What does the microorganisms size?