St.

Paul University Manila 1

Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

EXPERIMENT NO. 1

MICROSCOPE: MAKING AN INVISIBLE WORLD VISIBLE

Objectives:

After performing this experiment, students should be able to:

1. Explain the principles of microscopy by:

1.1 Differentiating between resolution and magnification;
1.2 Determining the resolving power of a lens;
1.3 Explaining the increased resolution of the oil immersion lens;
1.4 Defining parfocal working distance; and
1.5 Describing the primary characteristics of specialized microscopes.
2. Convert between units ranging from centimeter to angstrom;
3. Identify the parts of the brightfield microscope and their function; and
4. Correctly use and clean the laboratory microscope.

The scientific method requires evidence for the determination of facts. If our senses are limited,
we need tools to increase the power and accuracy of our observations and thus, our knowledge of the
world. For biologists, light and electron microscopes have become indispensible as "eyes" for discovery
about the detailed structure of cells.

Microscopes extend the limits of human vision by (a) magnifying an image, enlarging it so that
it can be seen by the human eye, and (b) increasing resolution, the detail provided by magnification
which reflects the ability of a viewing instrument (the eye, a camera, a microscope) to distinguish
adjacent objects as separate rather than as one larger object. For example, how close can two lines get
before you will no longer be able to see both of them? How thin can a single line become and still be
visible?

The Realm of the Ultra - Small

Because of their size, the dimensions of microorganisms, cells and their component parts are
expressed as fractions of a meter in units such as micrometer, nanometer and angstrom.

Fraction of Equiv. in
Unit Representative object
a meter Inch
Centimeter (cm) 1/100 2.54 Diameter of human heart (8 cm)
Millimeter 1/1000 25.4 Diameter of an artery (6 mm)
(mm)
Micrometer 1/1,000,000 25,4000 Diameter of red blood cell (8 µm);
Diameter of a bacterium (1µm)
Nanometer 1/1,000,000,000 25,400,000 Diameter of cell membrane (6
(nm) nm);
Herpes virus (130 nm)
Angstrom (Å) 1/10,000,000,000 254,000,000 Hemoglobin molecule (10 Å)

The lens of the human eye has a resolving power of approximately 100 µm. This means that the
eye will see two objects as distinct if the space between them is at least 100 µm. If the space is 90 µm,
they will appear as a single object. The best light microscopes have resolving powers of about 1 nm.
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

They will "see" objects or spaces between objects as small as 1 nm. If you look at the period at the end
of this sentence you will see a dot. Imagine that it is a cell composed of many parts. If you magnified the
dot 100 fold without increasing resolution (as in photographs) you would have a larger image but not
greater detail. (This is called empty magnification.) Magnifying the dot 100 fold with a microscope,
however, resolves ultra-fine structures of the image. Thus, the useful magnification of a viewing system
depends on its resolving power.

The two most important factors that determine resolving power are the (1) quality of the lens
system and the (2) wavelength of the source of illumination. Resolution varies inversely with
wavelength; a shorter wavelength allows for greater resolution.

Parts of the Microscope

(www.microscope-microscope.org)

 Eyepiece Lens:  the lens at the top that you look through.  They are usually 10x or 15x power.

 Tube:  Connects the eyepiece to the objective lenses.

 Arm:  Supports the tube and connects it to the base.

 Base:  The bottom of the microscope, used for support.

 Illuminator:  A steady light source (110 volts) used in place of a mirror.  If the microscope has a
mirror, it is used to reflect light from an external light source up through the bottom of the stage.

 Stage:  The flat platform where the slides are placed.  Stage clips hold the slides in place.  If the
microscope has a mechanical stage, the observer 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 there are 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, total magnifications of 40x (4x times 10x), 100x, 400x and 1000x may be
obtained. 

 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. 

 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 (400x and above).  Microscopes with in
stage condenser lenses render a sharper image than those with no lens (at 400x). 

 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 contrasts the observer desires and the particular objective lens in use.
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

Oil Immersion

Resolving power (RP) varies with wavelength of light and quality (light gathering capacity) of a
lens. The light gathering property is termed numeric al aperture (NA).

RP = Wavelength/ 2 x NA

The brightfield microscope often uses visible light with a wavelength of 550 nm. The NA of
most low power objectives is 0.25. Thus the smallest object that can be seen through the lens is 1100
nm. In order to increase resolution, either the wavelength of light transmitted through the specimen must
be decreased or the NA of the objective must be increased. Many brightfield microscopes use a blue
filter over the light source to reduce the wavelength of transmitted light to 475 nm. NA depends on the
configuration of the lens and the material between the lens and the light source. Some light is lost when
it passes from the specimen on a glass slide through air to the objective lens. This occurs because the
refractive index (ability to bend light) of glass and air differ. The air space can be replaced by a drop of
immersion oil which has a higher refractive index, closer to that of glass. When the objective lens is
lowered into the oil, more light is transmitted to the lens. Check your microscope to determine the NA
of your oil immersion lens. It is usually imprinted on the objective. What is the resolving power of the
oil immersion lens?

Because of the increased resolving power, most bacteriological microscopy uses the oil
immersion lens. It is important to be aware that the working distance, the distance between the specimen
and the objective lens, decreases with higher magnification. The 97x lens has an average working
distance of approximately 0.1 mm. Care must be taken not to break the slide or the cover slip when
viewing at this magnification. The observer should become familiar with the brightfield microscope.
Use the low and high power dry lenses and the oil immersion on each slide. Quality brightfield
instruments are parfocal and parcentric. This means that the specimen remains in focus and at the center
of the viewing field when objective lenses are rotated. Compare what you can see at each magnification.
(Do not worry if you do not see anything clearly at low power.) Describe your observations in your lab
notebook.

Care and Use of the Microscope

1. Always carry the microscope upright and with two hands - one hand should grip the arm and the
other hand should be placed firmly underneath the base. Be careful not to bump or drop the
instrument. Make sure that your lab bench is clear before you bring the microscope.
2. Use only optically safe le ns paper to clean the objective, ocular lenses and the condenser before
and after each lab period. If you should find a microscope that has not been cleaned, please
notify your instructor. Do not use any solvents or soap without the permission of your instructor.
3. Remove all immersion oil from the objective. Make sure that the oil is not used on the dry
objectives.
4. Before returning the microscope to its designated space storage cabinet, perform the following:
a. remove any clipped slide;
b. clean the optical system;
c. rotate the lowest power objective into position;
d. center the mechanical stage;
e. wrap the light cord around the base; and
f. cover
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

EXPERIMENT NO. 1
MICROSCOPE: MAKING AN INVISIBLE WORLD VISIBLE
Group No. Name Course, Year & Section Date Score

Label the parts of a compound microscope:

Eye piece

Revolving nose
piece
Light
Objectives
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

The compound microscope

Materials:

Equipments Reagents
toothpick xylol / xyline
lens paper methylene blue
glass slides safranin
gloves cedar wood oil
microscope

Procedure:
1. Review instructions for the use of microscope, giving special attention to the use of the oil –
immersion objective.
2. Prepare a cheek cell smear. With the aid of a clean toothpick, gently scrape the inner lining of your
cheek.
3. Place the scraped material at the middle part of the glass slide. Air dry.
4. Put a drop of methylene blue on the specimen and allow it to stay for three minutes.
5. Incline the glass slide at 45 degrees; rinse the glass slide by gently splash water using the wash
bottle.
6. Wipe off excess water on the lateral and bottom part of the slide.
7. Place the cover slip over the specimen then examine it under low - power, high - power and oil
immersion objectives.
8. On the second slide repeat the Procedure 3 – 8 however, use safranin instead of methylene blue.

Cheek Cell
Smear
(Methylene
Blue)
Label and
identify all
prominent
parts

Low – Power Objective High – Power Objective Oil – Immersion Objective

_________________ _________________ _________________
Total Magnification Total Magnification
Total Magnification
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

Allium cepa
Label and identify all prominent parts

Low – Power Objective High – Power Objective Oil – Immersion Objective

_________________ _________________ _________________
Total Magnification Total Magnification
Total Magnification

Blood Smear
Label and
identify all
prominent
parts

Low – Power Objective High – Power Objective Oil – Immersion Objective

_________________ _________________ _________________
Total Magnification Total Magnification
Total Magnification
 St. Paul University Manila 7
Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

Skeletal Muscle
Label and identify all prominent parts

Oil – Immersion Objective

Low – Power Objective High – Power Objective
_________________ _________________ _________________
Total Magnification Total Magnification
Total Magnification

REVIEW QUESTIONS
1. Explain why the body tube of the microscope should be lowered while looking through the ocular
lens.

_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________

2. For what purpose would you adjust each of the following microscope components during a
microscopy exercise?

a. Coarse Adjustment
________________________________________________________________________
________________________________________________________________________

b. Fine Adjustment
________________________________________________________________________
________________________________________________________________________

c. Iris Diaphragm
________________________________________________________________________
________________________________________________________________________

d. Condenser
________________________________________________________________________
________________________________________________________________________
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

3. Discuss the procedure used to maintain the optical system of the microscope properly
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________

Compute for the total magnification of the following:

Ocular Objectives Total

Magnification
5x Scanner
5x Low Power
10x Scanner
10x High Power
10x Oil Immersion
15x Scanner
15x Low Power
15x Oil Immersion

Write T (True) or F (False) for the following statements.

______1. The objective lens is the one nearest to the observer's eye.
______2. Only special lens paper should be used to clean the objectives, oculars,
and the condenser of the microscope.
______3. The working distance is the distance between the specimen and the objective
lens.
______4. Magnifying a blurred image generally reveals further details.
______5. When a microscope is parfocal, you should not have to use the coarse
adjustment at higher magnification.
______6. The total magnification of the object seen at high dry power is approximately
40X.
______7. Immersion oil can be used to increase resolution of all the objective lenses in a
brightfield microscope.
______8. Always be sure to oil your microscope lenses before returning the instrument
to its designated space.
______9. Animals will not survive without mitochondria because they will not be able
to make ATP.
______10. The use of normal saline solution (NSS) in viewing cheek cells is to prevent
lysis or crenation of cells.

EXPERIMENT NO. 2
THE CELL

Objectives:

At the end of the activity, students should be able to:

1. Name the three main parts of a human cell;
2. Describe the structure and function of the following:
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

a. plasma membrane and nucleus;

b. endoplasmic reticulum and the Golgi apparatus in the cytoplasm;
c. lysosomes and the role of these organelles in the breakdown of molecules;
d. mitochondria and their role in producing ATP; and
e. centrioles, cilia, and flagella and their roles in cellular movement.

Cells are the smallest living subunits of a multicellular organism such as a human being. A cell is
a living complex arrangement of chemicals which carries out specific activities. Microorganisms, such
as amoebas and bacteria, are single cells that function independently. Human cells, however, must work
together and function interdependently. Homeostasis depends upon the contributions of all of the
different kinds of cells. Human cells vary in size, shape, and function. Most human cells are so small
they can only be seen with the aid of a microscope and are measured in units called micrometers
(formerly called microns). One micrometer is equal to 1/1,000,000 of a meter or 1/25,000 of an inch.
One exception is the human ovum or egg cell, which is about 1 millimeter in diameter, just visible to the
unaided eye.

Some nerve cells, although microscopic in diameter, may be quite long. Those in our arms and
legs, for example, are at least 2 feet (60 cm) long. With respect to shape, human cells vary greatly. Some
are round or spherical, others rectangular, still others irregular. White blood cells even change shape as
they move.
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

EXPERIMENT NO. 2
THE CELL
Group No. Name Course, Year & Section Date Score

I. Label and use colored pens or pencils to shade in the cell parts below. Use contrasting colors
for different parts
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

II. Match the organelles in the key to the functions listed in questions 1-5.

a. mitochondria d. rough ER
b. nucleus e. centrioles
c. Golgi apparatus

____________1. packaging and secretion ____________4. protein synthesis

____________2. cell division ____________5. control center for the cell

____________3. powerhouses of the cell

III. Fill in the blanks.

6. The fluid-mosaic model of membrane structure says that _______________molecules drift about
within a double layer of _________________molecules.

7. Rough ER has __________________, but smooth ER does not.

8. Basal bodies that organize the microtubules within cilia and flagella are believed to be derived from
___________________.

9. Water will enter a cell when it is placed in a ________________solution.

10. Active transport requires a protein ______________and _______________ for energy.

11. Vesicle formation occurs when a cell takes in material by _________________.

12. At the conclusion of mitosis, each newly formed cell in humans contains ____chromosomes.
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

13. The ________________, which is the substance outside the nucleus of a cell, contains bodies called
_________________, each with a specific structure and function.

IV. Match the organelles in the key to the functions listed in questions 14-17.

a. DNA c. tRNA
b. mRNA d. rRNA

_______14. Joins with proteins to form subunits of a ribosome

_______15. Contains codes that determine the sequence of amino acids in a polypeptide

_______16. Contains a code and serves as a template for the production of RNA.

_______17. Brings amino acids to the ribosomes during the process of transcription

Review Questions:
1. Antibiotics have three known mechanisms of action: (1) disruption of protein synthesis within
bacteria, (2) blockage of bacterial DNA synthesis and cell division; and (3) inhibition of bacterial
cell wall synthesis. Considering that all antibiotics are equally effective, which do you think will an
ordinary person believe to be the reason for the effect? Justify.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

2. A new laboratory instructor wanted his students to see living cells. He placed a drop of his own
blood on a glass slide; added two drops of distilled water “so the cells will be spread out and easier
to see,” placed a cover glass on top of the specimen and viewed the slide under a microscope on high
power. He invited his students to see living red blood cells. The students claimed that they cannot
see any cells. Explain what happened.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

3. A friend asks you how DNA can be used to identify someone, and why it is called a “DNA
fingerprint.” What simple explanation can you give?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

4. Give a brief explanation of the functions of the rough ER, Golgi apparatus, microvilli and
mitochondria.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

5. A bacterial toxin is found to cause harm by first fitting into a receptor on human cell membranes;
once the toxin fits, the cell will be destroyed. Medications that may be given to stop this toxin can
work in one of these two ways: by blocking the receptors to prevent the toxin from fitting in, or the
drug can act as decoy molecules shaped like the receptors. Which one of these might be better, and
why?
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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

________________________________________________________________________
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