TECHNICAL UNIVERSITY OF MOMBASA

FACULTY OF APPLIED AND HEALTH SCIENCES

DEPARTMENT OF PURE & APPLIED SCIENCES
UNIVERSITY EXAMINATION FOR:
DIPLOMA IN LABORATORY TECHNOLOGY
AAB 2209: BIOMEDICAL TECHNIQUES – CELL BIOLOGY and
HISTOLOGY
END OF SEMESTER EXAMINATION
SERIES: DEC 2022
TIME: 2HOURS
DATE: Pick Date Select Month Pick Year
Instructions to Candidates
You should have the following for this examination
-Answer Booklet, examination pass and student ID
This paper consists of FIVE questions. Attempt question ONE (Compulsory) and any other TWO questions.
Do not write on the question paper.

SECTION A:
Question ONE

a) Define the following terms (5 marks)

i. Cell- the basic unit of life of an organism.

ii. Nucleotides- are the building blocks of nucleic acids composed of nitrogen bases, sugars and phosphate
iii. Histology- Histology (microanatomy) is the study of the human body at a tissue or sometimes at a cellular level
iv. Sumoylation-SUMOylation involves the covalent attachment of a member of the SUMO (small ubiquitin-
like modifier) family of proteins to lysine residues in specific target proteins via an enzymatic cascade
analogous to, but distinct from, the ubiquitination pathway.

v. Histones- is a protein that provides structural support for a chromosome

b) Outline the universal principles of living cells (5 marks)

i. Genetic information stored in the chemical sequence of DNA is duplicated and passed on to daughter cells.

ii. Linear chemical sequences stored in DNA code for both the linear sequences and three-dimensional
structures of RNAs and proteins.
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iii. Macromolecular structures assemble from subunits

iv. Membranes grow by expansion of preexisting membranes

v. Cellular constituents move by diffusion, pumps and Motors.

vi. Signal-receptor interactions target cellular constituents to their correct locations.

vii. Receptors and signaling mechanisms allow cells to adapt to environmental conditions

c) State FIVE classes of histone proteins (5 marks)

H1, H2A, H2B, H3 and H4

d) Differentiate between the following terms (6 marks)

i. Nucleoplasm and cytoplasm

ii. Nucleoside and Nucleotide

iii. Staining and Decalcification

e) Outline the characteristics of connective tissues (5 marks)

i. Eukaryotic cells have the nucleus enclosed within the nuclear membrane.

ii. The cell has mitochondria.

iii. Flagella and cilia are the locomotory organs in a eukaryotic cell.

iv. A cell wall is the outermost layer of the eukaryotic cells.

v. The cells divide by a process called mitosis.

SECTION B:

Question TWO

a) Differentiate between positive and negative feedback mechanisms (4 marks)

POSITIVE FEEDBACK & NEGATIVE FEEDBACK; Positive: is deviation from the optimum which causes
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changes resulting in an even greater deviation from thenorm e.g. Giving birth. Very rare because they’re hard
tocontrol. Negative: body’s mechanism for reversing change so it’s returned to optimum.

b) With example,discuss the JANUS stain used in cytological studies (6 marks)

c) Floaters are problems in tissue processing,explain .(10 marks)

"Floaters" are small pieces of tissue that appear on a slide that do not belong there--they have floated in during
processing. Floaters may arise from sloppy procedure on the cutting bench-- dirty towels, instruments, or gloves
can have tissue that is carried over to the next case. Therefore, it is essential that you do only one specimen at a
time and clean thoroughly before opening the container of the next case.

The best way to guard against unrecognized floaters is to always separate like specimens in the numbering
sequence. For example, if you have three cases with prostate chips, separate them in accessioning with totally
different specimens such as uterus or stomach. That way, if numbers are transposed or labels written wrong or
tissue carried over, then you will have an obvious mismatch. Carrying over one prostate to another, or
transposing the numbers of identical tissues may never be recognized.

If reusable cassettes are employed, you must be aware that tissue may potentially be carried over and appear as
"floaters" even several days later, when the cassette is re-used. The problem arises when, during embedding, not
all the tissue is removed from the cassette. Then, in the cleaning process, not all of the wax is removed. Then, the
next person using the cassette does not pay attention to the fact that there is tissue already in the cassette and puts
his specimen in it. The floater that appears on the slide will look well-preserved--it should, because it was
processed to paraffin.

Always be sure that you properly identify the tissue! This means that you make sure that the patient label on the
specimen container matches that of the request slip. An accession number is given to the specimen. This number
must appear with the tissue at all times. You must never submit a cassette of tissue without a label. You must
never submit a cassette of tissue with the wrong label. Mislabelling or unlabelling of tissues is courting disaster

Question THREE

a) Discuss the Watson and Crick Model of DNA structure (10 marks)

Features of Watson-Crick model of DNA

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1. Right handed double helix

DNA consists of two polydeoxy ribonucleotide chains twisted around one another in a right handed double helix
similar to a spiral stair case. The sugar and phosphate groups comprise the handrail and the bases jutting inside
represent the steps of the staircase. The bases are located perpendicular to the helix axis, whereas the sugars are
nearly at right angles to the axis.

2. The base pairing rule

Always the two strands are complementary to each other. So, the adenine of one strand will pair with thymine of
the opposite strand, while guanine will pair with cytosine. The base pairing (A with T; G with C) is called
Chargaff's rule, which states that the number of purines is equal to the number of pyrimidines.

3. Hydrogen bonding

The DNA strands are held together mainly by hydrogen bonds between the purine and pyrimidin bases. There
are two hydrogen bonds between Aand T while there are three hydrogen bonds between C and G. The GC bond
is therefore stronger than the AT bond.

4. Antiparallel

The two strands in a DNA molecule run antiparallel, which means that one strand runs in the 5' to 3' direction,
while the other is in the 3' to 5' direction. This is similar to a road divided into two,

each half carrying traffic in the opposite direction

b) Differentiate between the DNA and RNA (10 marks)

Parameter DNA RNA

DNA is a double-stranded
It is a single-stranded helix
molecule consisting of a long
Structure consisting of a short chain of
chain of nucleotides. B type of
nucleotides. A type of helix.
helix.

Function Transmits genetic information to It transfers the genetic code from

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 make other cells and new
organisms. Long-term storage of
genetic information
the nucleus to the ribosomes to
make proteins.
 

DNA is self-replicating.

Propagation   Synthesized from DNA.

Deoxyribose sugar
Ribose sugar
phosphate backbone
phosphate backbone
Composition adenine, guanine,
adenine, guanine,
cytosine, thymine
cytosine, uracil bases.
bases.

 Located in the nucleus of a cell Located in the cytoplasm,

Location
and in the mitochondria. nucleus, and in the ribosome.

Nitrogenous
GC(Guanine pairs with Cytosine) GC(Guanine pairs with Cytosine)
Bases and
A-T(Adenine pairs with Thymine). A-U(Adenine pairs with Uracil)
Pairing

Molecular
2 to 6 million 25,000 to 2 million
Weight

DNA is a more stable molecule Much more reactive than DNA

Stability than RNA. DNA is stable under and is not stable in alkaline
alkaline conditions. conditions.

Ultraviolet
DNA is vulnerable to damage by Much more resistant to damage
(UV)
ultraviolet light. from UV light than DNA.
Sensitivity

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Question FOUR

Describe the cell cycle (20 marks)

The cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. Non-
dividing cells not considered to be in the cell cycle. The stages are G1-S-G2-M. The G1 stage stands for "GAP
1". The S stage stands for "Synthesis". This is the stage when DNA replication occurs. The G2 stage stands for
"GAP 2". The M stage stands for "mitosis", and is when nuclear (chromosomes separate) and cytoplasmic
(cytokinesis) division occur.

Regulation of the cell cycle

How cell division (and thus tissue growth) is controlled is very complex. The following terms are some of the
features that are important in regulation, and places where errors can lead to cancer. Cancer is a disease where
regulation of the cell cycle goes awry and normal cell growth and behavior is lost.

• Cdk (cyclin dependent kinase, adds phosphate to a protein), along with cyclins, are major control switches
for the cell cycle, causing the cell to move from G1 to S or G2 to M.

• MPF (Maturation Promoting Factor) includes the CdK and cyclins that triggers progression through the
cell cycle.

• p53 is a protein that functions to block the cell cycle if the DNA is damaged. If the damage is severe this
protein can cause apoptosis (cell death).

o p53 levels are increased in damaged cells. This allows time to repair DNA by blocking the cell cycle.

o A p53 mutation is the most frequent mutation leading to cancer. An extreme case of this is Li Fraumeni
syndrome, where a genetic a defect in p53 leads to a high frequency of cancer in affected individuals.

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p27 is a protein that binds to cyclin and CdK blocking entry into S phase. suggests that breast cancer prognosis is
determined by p27 levels. Reduced levels of p27 predict a poor outcome for breast cancer patients.

MITOSIS

Mitosis is nuclear division plus cytokinesis, and produces two identical daughter cells during prophase,
prometaphase, metaphase, anaphase, and telophase. Interphase is often included in discussions of mitosis, but
interphase is technically not part of mitosis, but rather encompasses stages G1, S, and G2 of the cell cycle.

Interphase

The cell is engaged in metabolic activity and performing its prepare for mitosis (the next four phases that lead up
to and include nuclear division). Chromosomes are not clearly discerned in the nucleus, although a dark spot
called the nucleolus may be visible. The cell may contain a pair of centrioles (or microtubule organizing centers
in plants) both of which are organizational sites for microtubules.

Prophase

Chromatin in the nucleus begins to condense and becomes visible in the light microscope as chromosomes. The
nucleolus disappears. Centrioles begin moving to opposite ends of the cell and fibers extend from the
centromeres. Some fibers cross the cell to form the mitotic spindle.

Prometaphase

The nuclear membrane dissolves, marking the beginning of prometaphase. Proteins attach to the centromeres
creating the kinetochores. Microtubules attach at the kinetochores and the chromosomes begin moving.

Metaphase

Spindle fibers align the chromosomes along the middle of the cell nucleus. This line is referred to as the
metaphase plate. This organization helps to ensure that in the next phase, when the chromosomes are separated,
each new nucleus will receive one copy of each chromosome.
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Anaphase

The paired chromosomes separate at the kinetochores and move to opposite sides of the cell. Motion results from
a combination of kinetochore movement along the spindle microtubules and through the physical interaction of
polar microtubules.

Telophase

Chromatids arrive at opposite poles of cell, and new membranes form around the daughter nuclei. The
chromosomes disperse and are no longer visible under the light microscope. The spindle fibers disperse, and
cytokinesis or the partitioning of the cell may also begin during this stage.

Cytokinesis

In animal cells, cytokinesis results when a fiber ring composed of a protein called actin around the center of the
cell contracts pinching the cell into two daughter cells, each with one nucleus. In plant cells, the rigid wall
requires that a cell plate be synthesized between the two daughter cells.

Question FIVE

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 a) Discuss the groups of fixatives used in cytological studies (10 marks)

There are five major groups of fixatives, classified according to mechanism of action:

 Aldehydes
 Mercurials
 Alcohols
 Oxidizing agents
 Picrates

Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by cross-linkages

formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the
structure of proteins greatly, so that antigenicity is not lost. Therefore, formaldehyde is good for
immunohistochemical techniques. Formalin penetrates tissue well, but is relatively slow. The standard
solution is 10% neutral buffered formalin. A buffer prevents acidity that would promote autolysis and
cause precipitation of formol-heme pigment in the tissues.

Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not good for

immunohistochemical staining. However, it fixes very quickly so is good for electron microscopy. It
penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. The standard solution is a
2% buffered glutaraldehyde

Mercurials fix tissue by an unknown mechanism. They contain mercuric chloride and include such well-
known fixatives as B-5 and Zenker's. These fixatives penetrate relatively poorly and cause some tissue
hardness, but are fast and give excellent nuclear detail. Their best application is for fixation of
hematopoietic and reticuloendothelial tissues. Since they contain mercury, they must be disposed of
carefully.

Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and
are not used routinely for tissues because they cause too much brittleness and hardness. However,
they are very good for cytologic smears because they act quickly and give good nuclear detail. Spray
cans of alcohol fixatives are marketed to physicians doing PAP smears, but cheap hairsprays do just as
well.

Oxidizing agents include permanganate fixatives (potassium permanganate), dichromate fixatives

(potassium dichromate), and osmium tetroxide. They cross-link proteins, but cause extensive
denaturation. Some of them have specialized applications, but are used very infrequently.

Picrates include fixatives with picric acid. Foremost among these is Bouin's solution. It has an unknown
mechanism of action. It does almost as well as mercurials with nuclear detail but does not cause as
much hardness. Picric acid is an explosion hazard in dry form. As a solution, it stains everything it
touches yellow, including skin.

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 b) Discuss the factors affecting fixation (10 marks)

There are a number of factors that will affect the fixation process:

 Buffering
 Penetration
 Volume
 Temperature
 Concentration
 Time interval

Fixation is best carried out close to neutral pH, in the range of 6-8. Hypoxia of tissues lowers the pH, so
there must be buffering capacity in the fixative to prevent excessive acidity. Acidity favors formation of
formalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include
phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered with phosphate at a
pH of 7.

Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant.
Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are
somewhere in between. One way to get around this problem is sectioning the tissues thinly (2 to 3 mm).
Penetration into a thin section will occur more rapidly than for a thick section.

The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Obviously, we
often get away with less than this, but may not get ideal fixation. One way to partially solve the problem
is to change the fixative at intervals to avoid exhaustion of the fixative. Agitation of the specimen in the
fixative will also enhance fixation.

Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as
you don't cook the tissue. Hot formalin will fix tissues faster, and this is often the first step on an
automated tissue processor.

Concentration of fixative should be adjusted down to the lowest level possible, because you will expend
less money for the fixative. Formalin is best at 10%; glutaraldehyde is generally made up at 0.25% to
4%. Too high a concentration may adversely affect the tissues and produce artefact similar to
excessive heat.

Also very important is time interval from of removal of tissues to fixation. The faster you can get the
tissue and fix it, the better. Artefact will be introduced by drying, so if tissue is left out, please keep it
moist with saline. The longer you wait, the more cellular organelles will be lost and the more nuclear
shrinkage and artefactual clumping will occur.

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