EXERCISE NO.

MICROSCOPY: USE OF MICROSCOPES

The compound microscope is an optical instrument used to see small things beyond
ordinary vision. The microscope is one of the basic tools of a microbiologist and absolutely
essential to any microbiology laboratories.
Since this course deals with the study of microorganisms it is necessary that one should
be familiar with the parts of the microscope. Understanding the proper function of each part,
constant and proper use of the microscope will increase one’s proficiency.
Microscopes have proven its usefulness both in science and technology. It is by far the most
invaluable tool in every laboratory and the most important discovery of mankind. The usefulness
of this equipment is more than we could imagine since the use of microscopes has been around
to every field of interest.
Microscope techniques are applied to make sure that every specimen is examined
thoroughly under the lens of a microscope. It played a major role in education and research with
its power to magnify even the tiniest specimen.
You will be using an assigned light microscope for a variety of lab exercises through the
semester, everything from viewing pond water to identification of your unknown bacterium.
Therefore, it is extremely important that you understand how to use the microscope effectively
and how to use different types of microscopy—brightfield, phase-contract, and darkfield.
You will also get your first exposure to the preparation of a bacterial smear and fungal
molds and subsequent staining of it. However, you are making a simple stain using only one dye.
Everything on the slide will be the same color, but you can distinguish among shapes, sizes, and
arrangements of bacteria.

Objectives:
At this activity, the students should be able to know the following:
 Different types of microscopes and their parts;
 Different functions of the parts of microscope; and
 Proper way of using and handling microscope

Materials:
1. Microscope
2. Glassware
 Microscope slides
 Cover slips
 Medicine dropper
 3. Specimens
 Bacteria
 Fungi 1
4. Staining Solution
 Methylene blue
 Crystal violet
5. Tissue paper/paper towels

General Procedures:
1. Make sure all backpacks and junk are out of the aisles.
2. Plug your microscope into the extension cords. Each row of desks uses the same
cord.
3. Always start and end with the Scanning Objective. Do not remove slides with the
high power objective into place—this will scratch the lens!
4. Always wrap electric cords and cover microscopes before returning them to the
cabinet. Microscopes should be stored with the scanning objective clicked into
place.
5. Always carry microscopes by the arm and set them flat on your desk.

Focusing Specimens:
1. Always start with the scanning objective. Odds are, you will be able to see
something on this setting. Use the coarse knob to focus, image may be small at
this magnification, but you won’t be able to find it on the higher powers without
this first step. Do not use stage clips, try moving the slide around until you find
something.
2. Once you’ve focused on scanning, switch to low power. Use the coarse knob to
refocus. Again, if you haven’t focused on this level, you will not be able to move
to the next level.
3. Now switch to high power. (If you have a thick slide, or a slide without a cover,
do NOT use the HPO.) At this point, ONLY use the fine adjustment knob to focus
specimens.
4. If the specimen is too light, try adjusting the diaphragm.
5. If you see a line in your viewing field, try twisting the eyepiece, the line should
move. That’s because it’s a pointer, and is useful for pointing out things to your
lab partner or teacher.

Drawing Specimens:
1. Use pencil—you can erase and shade areas
 2. All drawings should include clear and proper labels (and be large enough to view
details). Drawings should be labeled with the specimen name and magnification.
3. Labels should be written on the outside of the circle. The circle indicates the
viewing field as seen through the eyepiece, specimens should be drawn to scale—
i.e., if your specimen takes up the whole viewing field, make sure your drawing
reflects that.

2
Making a Wet Mount:
1. Gather a thin slice/piece of whatever your specimen is. If your specimen is too
thick, then the coverslip will wobble on top of the sample like a see-saw, and you
will not be able to view it under high power.
2. Place ONE drop of water directly over the specimen. If you put too much water,
then the coverslip will float on top, making it hard to draw the specimen, because
they might actually float away. (Too much water is messy.)
3. Place the coverslip at a 45 degree angle (approximately) with one edge touching
the water drop and then gently let go. Performed correctly, the coverslip will
perfectly fall over the specimen.

How to Stain a Slide:

1. Place one drop of stain (iodine, methylene blue, etc.) on the edge of the coverslip.
2. Place the flat edge of a piece of paper towel on the opposite side of the cover slip.
The paper towel will draw the water out from under the cover slip, and the
cohesion of water will draw the stain under the slide.
3. As soon as the stain has covered the area containing the specimen, you are
finished. The stain does not need to be under the entire cover slip. If the stain does
not cover as needed, get a new piece of paper towel and add more stain until it
does.
4. Be sure to wipe off the excess stain with a paper towel.

Placing Microscopes Back into the Cabinets

 YOU are responsible for your assigned microscope! There is only one person in
each lab who is assigned that particular microscope, so if someone else complains
about it being left with oil or a slide on stage, you or another person who is
assigned that particular scope will be reprimanded.
 Wrap the cord around the cord holder on the arm.
 Make sure that 10x low power lens is in place—not the 100x oil immersion lens.
The lens could hit against the stage and get scratched.
 Turn the coarse adjustment knobs so that is far from the lens.
  PLACE YOUR MICROSCOPE BACK IN ITS NUMBERED POSITION IN
THE CORRECT CABINET.

3
QUESTION:
1. What are the common problems in using microscope? Give at least five.

2. Which objective lens is also called the oil-immersion lens?

3. What is an oil-immersion?

4. How do the functions of the sub-stage condenser and the iris diaphragm within the
condenser differ?

5. Define bright field, dark field and phase-contrast microscopy.

6. What condenser setting value do you want when you are using 100x objective lens?

7. What is a parfocal lens?

8. How to get the total magnification?

Define the following and give the function of each:

4

1. Coarse adjustment knob

2. Fine adjustment knob

3. Arm

4. Power switch/brightness control

5. Base

6. Condenser knob

7. Iris diaphragm lever

8. Objective lens

9. Revolving nosepiece
10. Ocular eyepiece lens

11. Stage clips

12. Stage Motion Knobs

13. Stage

LABEL THE PARTS OF THE MICROSCOPE

5
How do I find if a compound is ionic or covalent by just looking at its chemical formula?

If you are very familiar with the periodic table of elements, you will notice that elements are
grouped in vertical column called groups and horizontal rows called period.

The first three columns to the left (ie groups 1, 2 and 3) are classified as metals due to their very
low electronegative values, while columns far right with exception to the very last column (ie
groups 5,6 and 7) are classified as non metals due to their high electronegative values.

Now to go straight to your question on how to easily identify if a compound is ionic or covalent
just by inspection. What you have to do is to look at the compound this way:

1. if the compound is made of just two elements, if one is a metal (ie belongs to any of
groups 1, 2 or 3) and the other element a non metal, (ie belongs to group 5, 6 or 7) then
the compound is most likely to be an ionic compound. For example NaCl, MgO
2. If the compound is made of identical non metalic elements as in O2, Cl2 then the
compound is covalent
3. If the compoud is made of just two elements that are both non metals such as in
SO2, CO, NO, CCl4, the compound is covalent
4. If the compound is made up of more than two elements, such as in HNO3, Na2CO3,
CuSO4.5H2O, you may need to break the compound into dissociating parts. You will
see that, the compounds are ionic.
5. Hydrocarbons, compounds containing only hydrogen and carbon of varying
molecular size are all covalent. Examples are C2H6, C2H4, C2H2,
6. Note that there could be some little exceptions to the examples given. Mostly with
first members of every group because of their small size which make them show
substantial deviations from group behavior. For example HCl is covalent not ionic.

Chapter 4: Calculations Used in Analytical Chemistry

4A Some important units of measurement 4A-1 SI Units SI is the acronym for the French
“Système International d’Unités.” The International System of Units (SI) is based on 7
fundamental base units. Numerous other useful units, such as volts, hertz, coulombs, and
joules, are derived from these base units. To express small or large measured quantities in
terms of a few simple digits, pre-fixes are used with these base units and other derived units.
The ångstrom unit Å is a non-SI unit of length widely used to express the wavelength of very
short radiation such as X-rays (1 Å = 0.1 nm). Thus, typical X-radiation lies in the range of 0.1 to
10 Å.
Metric units of kilograms (kg), grams (g), milligrams (mg), or micrograms (µg) are used in the
SI system. Volumes of liquids are measured in units of liters (L), milliliters (mL), microliters
(µL), and sometimes nanoliters (nL).
The liter, the SI unit of volume, is defined as exactly 10-3 m3 . The milliliter is defined as 10-6
m3 , or 1 cm3 .
 Physical Quantity Name Of Unit Abbreviation
Mass Kilogram Kg
Length Meter m
Time Second s
Temperature Kelvin K
Amount of Substance Mole Mol
Electric Current Ampere A
Luminous Intensity Candela cd
S.I Basic Units

Prefixes for Units

Prefix Abbreviation Multiplier

yotta-
zetta-
exa-
peta-
tera-
giga-
mega-
kilo-
hecto-
deca-
deci-
centi-
milli-
micro-
nano-
femto-
atto-
zepto
yocto-
4A-2 The Distinction Between Mass and Weight Mass is an invariant measure of the
quantity of matter in an object.
1. Weight is the force of attraction between an object and its surroundings, principally the
earth. Because gravitational attraction varies with geographical location, the weight of an
object depends.
2. Weight and mass are related by the familiar expression

w = mg

w is the weight of an object

m is its mass,
and g is the acceleration due to gravity.
Analytical data are based on mass rather than weight.
A balance is used to compare the mass of an object with the mass of one or more standard
masses.
g affects both unknown and known equally, hence, the mass of the object is identical to the
standard masses with which it is compared.
4A-3 The Mole

 The mole (abbreviated mol) is the SI unit for the amount of a chemical substance.
 It is always associated with specific microscopic entities such as atoms, molecules, ions,
electrons, other particles, or specified groups of such particles as represented by a
chemical formula.
 It is the amount of the specified substance that contains the same number of particles as
the number of carbon atoms in exactly 12 grams of 12C.
 This is Avogadro’s number NA= 6.022 x 10^23 .
 The molar mass M of a substance is the mass in grams of 1 mole of that substance.
4B Solutions and their concentrations
4B-1 Concentration of Solutions
The molar concentration Cx of a solution of a solute species X is the number of moles of that
species that is contained in 1 liter of the solution (not 1 L of the solvent). n, number of moles of
solute and V, the volume of solution.

The unit of molar concentration is molar, symbolized by M, which has the dimensions of mol/L,
or mol L^-1. Molar concentration is also the number of millimoles of solute per milliliter of
solution.

There are two ways of expressing molar concentration:

Molar analytical concentration is the total number of moles of a solute, regardless of its
chemical state, in 1 L of solution. The molar analytical concentration describes how a solution of
a given concentration can be prepared.

The molar equilibrium concentration, or just equilibrium concentration, refers to the molar
concentration of a particular species in a solution at equilibrium.

To specify the molar equilibrium concentration of a species, it is

necessary to know how the solute behaves when it is dissolved in a
solvent. They are usually symbolized by placing square brackets around the chemical formula for
the species. Ex., [H2SO4] = 0.00 M; [H+] = 1.01 M.

The IUPAC recommends the general term “concentration” to express the composition of a
solution with respect to its volume, with four sub terms: amount concentration, mass
concentration, volume concentration, and number concentration.

Molar concentration, molar analytical concentration, and molar equilibrium concentration are all
amount concentrations by this definition.

The molar analytical concentration of H2SO4 is given by CH2SO4 = [SO4^-2] + [HSO42]

because SO4^-2 and HSO4^-2 are the only two sulfate-containing species in the solution.

The molar equilibrium concentrations are [SO4^-2] and [HSO4^-2].

Percent Concentration

In IUPAC terminology, weight percent is mass concentration and volume percent is volume
concentration.

Weight percent is often used to express the concentration of commercial aqueous reagents. Volume
percent is commonly used to specify the concentration of a solution prepared by diluting a pure liquid
compound with another liquid.

Weight or volume percent is often used to indicate the composition of dilute aqueous solutions of
solid reagents.
Parts per million and parts per billion

In IUPAC terminology, parts per billion, parts per million, and parts per thousand are mass
concentrations. For very dilute solutions, parts per million (ppm) is a convenient way to express
concentration.

For even more dilute solutions, 109 ppb rather than 106 ppm is used in the previous equation to
give the results in parts per billion (ppb). The term parts per thousand (ppt) is also used,
especially in oceanography.