Download Labratory Examination Questions

Answers to Laboratory exam questions in Biology
2005/2006
Labratory Examination Questions
1. Structure and operation of the light microscope
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Two stage magnifying system (  objective lens and eyepiece lens)
Total magnification is obtained by multiplying the magnifying power of the
objective and the ocular lens.
Condenser:
Concentrate and focus the light of the specimen.
Light travels through the specimen, and is magnified in 2 steps:
1. By the objective lens
2. By the ocular lens
Image is viewed directly, without image formation on screen etc.
Abbe’s formula
Resolution = ______
n x sin 
= wavelength of light
n x sin  = aperture of objective
Resolution power: The ability of a microscope to render too closely point as distinct.
Object on specimen smaller than the resolution power  invisible to investigator.
Highest resolution: requires use of oil immersion lens of maximum numerical aperture 
space between Specimen and objective is filled with a material of high refractive index.
2. Structure and operation of the electron microscope
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Permits higher resolution than a light microscope
Electron beam is used for illumination to produce an image  electron beam
is directed by a system of electromagnetic waves.
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Because of the short wavelength of the electron beam, the electron
microscope has much better resolution than a light microscope.
- Operated in high vacuum (electrons could collide with air molecules)
- Two types:
1. Transmission electron microscope ( practical resolution 1-2 nm)
2. Scanning electron microscope ( resolution : 10 nm)
Transmission electron microscope:
 Illuminating system
Source of electrons:
- Tungsten cathode
- Potential difference between Cathode and Anode (metallic plate with small
hole in the center)
- Emitted electrons are accelerated from cathode to anode  accelerating
voltage ( determined speed and wavelength of the electron beam)
Condenser light:
- Focuses the beam on the specimen
Objective lens:
- Electrons transmitted through the specimen focused by objective lens
magnified image.
Projector lens
- Further enlarged by projector lens.
Scanning electron microscope:
Scattering of electrons are detected by the detector, and made visible on a TV screen , as
3D images.
3. Sample preparation and contrasting methods for light- and electron microscopy
Electron microcopy
- Cell and tissue components are treated with salt of heavy metal such as
Osmium , lead and uranium increased electron density
- Double fixation:
1. Fixation with buffered gluteralaldehyde cross linked proteins
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2. Fixation with buffered osmium tetroxide solution. increased contrast
- Dehydration , and embedding material ( impregnation)
- Sections are stained with solutions of heavy metal compounds:
Uranyl acatate and lead citrate.
Metal shadowing:
- Increases the contrast.
- Specimen coated with thin layer of evaporated metal thicker in some
places than others, do to the spraying from an angle shadow effect
giving the specimen 3D appearance.
Rotary shadowing:
- variation of shadow tecnique2 sample is rotated during evaporation of
metal high contrast of all sides of specimen
Freeze – fracture, freeze etching:
- No sample preparation required.
1. Freeze fracture:
- Specimen frozen at temperature of liquid nitrogen in presence of
glycerol fractured with a cutting device.
Freeze – etching:
- Specimen frozen as in freeze fracture  cracked with a knife blade 
sublimation of frozen water in the surface,
Light microscopy:
Fixation:
Kills cell, with structural and chemical composition of living cell preserved.
Ex ethanol, formalaldehyde
Dehydration
Gradual removal of water from tissue block, by immersing it into increasing
concentrations of ethyl alcohol.
Clearing
Removal of dehydrating agent
Embedding:
Evaporation of solvent space filled with paraffin.
Sectioning:
Sectioning by a microtome: 4 -10 m thick
Staining and mounting:
Paraffin removed with xylene  tissue rehydrated by a series of decreasing concentration
of alcohol.
Stained with appropriate dye solution  passed through alcohol of increasing
concentration to remove all water again. Finally immersed in xylene  mounted in
mounting medium soluble in xylene.
4. Radioactive isotopes in molecular cell biology
Radioisotopes:
Have unstable nuclei  random disintegration results in emission of easily detectable
radiation. Can be used to study intracellular processes.
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Radioactive precursors:
Labeled molecules that can be incorporated into macromolecules of interest, and then
traced.
Radioisotop
Emitted radiation
3H
 particle
14C
 particle
35S
 particle
32P
 particle
131I
 particle
Example of application
(labeled precursor/ process
studied)
3H  amino
acid/proteinsynthesis
3H  Sugar /glycosylation
3H 
methionine/proteinsynthesis
- 32P  ATP/In vitro protein
phosph.
In vitro labelling
5. Homogenisation, cell fractionation
Sedimentation of particles is fluenced by their size, shape and density, by centrifugal
force appealed, and by density and viscosity of the medium in witch particles are
centrifuged.
1. Tissue or cell suspension homogenized in aqueous solution:
- cell membrane destroyed by osmotic shock, sonication or mechanical force.
2. Homogenate subjected to a series of centrifugation steps increased velocity
 Nuclear fraction
 Mitochondrial fraction (+ lysosomes and perioxisomes)
 Microsome fraction (fragmentations of ER)
 Ribosome fraction (free ribosomes)
Supernatant: Cytosol containing soluble components of cytoplasm.
6. Hypopicnic and isopicnic gradient centrifugation
Hypopicnic:
Separate macromolecules of different size
1. Centrifuge tube is filled with solution of increasing density towards the bottom.
(Sucrose, glucose etc)
2. Sample is loaded on the top of gradient  centrifuged until its components are
separated.
3. Drops from the gradient collected in fractions of identical volume.
4. Concentration of molecules determined by UV absorption (proteins: 280 nm, nucleic
acids: 260 nm), radioactivity, enzyme activity etc.
Isopicnic:
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Separation on macromolecules of different buoyant density  use gradients of high
density solutions (e.g. CsChl)
1. Sample mixed with gradient solution  centrifuged in ultracentrifuge.
2. Particles of the sample migrate to the layers of the gradient with the same density,
3. Collection of fractions, identification of separated components.
7. Gel filtration
Gel filtration = Chromatographic fractionation
Solution of molecules to be fractionated (mobile phase) is run through a solid matrix
(stationary phase)  if solutes have different interactions with matrix (ex: size), they can
be separated from each other.
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Matrix is filled into a glass aqueous suspension.
Sample is loaded on the top of solid phase  fractions are collected through a outlet
of the column.
Matrix is washed with elution buffer  carried the sample through the column
If molecules travel with different speed  collected in different fractions
Analyzed by a chromatogram.
Depending on solid phase, chromatography can be performed by paper, thin layer and
column chromatography
Gel filtration:
- Consist of small porous gel beads prepared from agarose,
polyacrylamide or dextran.
- Large molecules of sample are excluded from pores of beads and pass
through the matrix rapidly - small molecules penetrates the pores, are
distributed in larger solvent volume, and appear later in the
chromatography fractions.
- Gels can be prepared from beads of various size  gel filtration uses in a
wide molecular weight range to fractionate molecules of different size
(E.g. proteins)
- Gel filtration column is best characterized by its :
Exclution limit molecular weight above Kd = 0.
range of fraction  molecular weight range within witch molecule of different
size can be separated from each other.
Principle of gel chromatography fractionation:
Ve=Vo +Kd x Vi
Ve= Elution volume amount of solvent that carries the molecules through the column to
maximal concentration.
Vo =Void volume amount of buffer between the beads.
Vi = Internal volume amount of buffer in the pores of the beads.
Kd = Distribution coefficient number between 0 and 1, represent the fraction of the
internal volume accessible for a given molecule,
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Ex.
Kd = 1 the molecule or ion occupies the total mobile phase of the column. Ve will be the
sum of Vo and Vi.
Kd = 0, the molecules are excluded from the beads because the Ve will be equal to Vo .
8. Ion exchange and affinity chromatography
Ion exchange
Ion exchange columns are filled with a suspension of gel beads that are positively
(anion exchanger) or negatively (cation exchanger) charged molecules of the sample
with the opposite charge are bound to the ion exchanger, others pass rapidly through
the column. Molecules absorbed on the beads can be eluted from the column by
increasing the ionic strength (salt concentration) or changing the pH of the elution
buffer. Ion exchange is widely used to separate proteins of different charge, but can
also be employed to fractionate other molecules (e.g. single- and double stranded
nucleic acids can be separated by hydroxyl apatit chromatography).
Affinity chromatography
The chromatographic matrix and molecules in the sample can have biologically
specific interactions. A ligand is covalently attached to the gel beads that can be
recognised and specifically bound by the macromolecules to be purified: other
components of the mixture pass through the column without binding. The bound
molecules can subsequently be eluted from the matrix. The highly specific nature of
this interaction makes affinity chromatography the most powerful purification
technique: the molecule of interest can be highly enriched in the sample by a single
chromatographic step. Affinity chromatography can be used for a wide range of
purposes: for example, enzymes can be purified with substrates, antibodies with
antigens, DNA binding proteins with oligonucleotides, poly(A)+ mRNAs with
oligo(dT) as ligands.
9. Protein electrophoresis
Polyacrylamide gel electrophoresis
Polymerization of acrylamide and bisacrylamide yields a covalently cross-linked
electrophoretic matrix with excellent physical properties. The pore size of the gel can
be regulated by the concentration of the monomers. Polyacrylamide gels are usually
prepared as gel slabs between two glass plates and used in vertical electrophoresis.
SDS-polyacrylamide gel electrophoresis is the most popular method used for protein
fractionation. The sample is boiled in the presence of sodium dodesulphate (SDS) and
metacapoethanol. SDS is a strong ionic detergent: an ester of a long carbon chain
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alcohol and a sulphuric acid. Its molecules bind to hydrophobic amino acids with their
nonpolar aliphatic chain and the sulphate groups of SDS molecules make the proteins
uniformly negative charged. The repulsive effect of the negative charges makes the
polypeptide chain extended and dissociates it from other macromolecules. Disulphide
bonds within or between polypeptide chains are disrupted by metacapoethanol. The
negatively charged proteins migrate during electrophoresis according to their size,
SDS-polyacrylamide gel electrophoresis can thus be used to determine the molecular
weight of proteins, using molecular weight markers.
Separation of proteins using SDS-polyacrylamide gel electrophoresis
10. Nucleic acid electrophoresis
Agarose gel electrophoresis
Agarose is a polysaccharide; its aqueous suspension forms a gel upon heating and
cooling: the network is stabilizing by H-bonds between the agarose chains. The pose
size of the gel depends on the concentration of agarose.
Agarose gels are used in horizontal electrophoretic tanks to fractionate nucleic acids:
small molecules migrate faster than large ones. DNA fragments can be fractionated
by agarose gel electrophoresis. Formaldehyde-containing gels are used to separate
RNA molecules: in such gels RNA strands are denatured, fully extended and their rate
of migration is determined by their size.
Nucleic acids are visualized in the gel by staining with a fluorescent dye (e.g. ethium
bromide)
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Fractionation of mammalian rRNAs and mRNAs by formaldehyde/agarose gel electrophoresis
11. Isolation of mammalian DNA
Molecular biologists developed a wide range of methods to study mammalian DNA
and RNA. Some of these techniques are very sensitive to protein contaminations:
others require the isolation of intact, high molecular weight DNA. To serve the
different needs of the techniques there are a number of different DNA/RNA isolation
methods available.
Cell fractionation begins with homogenization. To inhibit cellular hydrolyses and
other enzymes it is useful to cool the sample on ice during this process. After
homogenization, the first step of cell fractionation is carried out in order to isolate
nuclear fraction and post nuclear supernatant.
DNA is found in nucleus as a DNA-protein complex. The first extraction buffer used
NaCl/SDS/EDTA solution) has to destroy the nuclear membrane and loosen proteinDNA interactions. SDS, the detergent helps with both terms: dissolves the lipid
membrane and denatures chromatin proteins. EDTA – by chelating Ca++ and Mg++
ions that stabilize the chromatin – loosens the structure, while the concentrated NaCl
solution destroys the ionic bonds between DNA and proteins.
During the next step, chloroform precipitates the proteins, while DNA remains in the
solution. After centrifugation, the upper, aqueous layer contains the DNA, the middle
layer consists of precipitated proteins and the third layer is chloroform. Next DNA is
precipitated with ethanol. The long DNA molecules form a fibrous precipitate.
To measure the concentration of our DNA solution, DNA must be hydrolyzed.
Trichloroacetic acid (TCA) hydrolyses DNA, so diphenylamine can bind to
deoxyribose. This reaction gives a blue colour. Light absorption is measured by
photometer at 532 nm. To determine the concentration of our DNA solution, DNA
standard curve has to be used. The DNA standard curve was prepared by measuring
the light absorption of several DNA solutions with a known concentration.
(Concentration of a pure DNA solution can also be measured with UV light as 260 nm
- for that DNA does not have to be hydrolyzed).
12. Isolation of mammalian RNA
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Total cytoplasmic RNA is located from the post nuclear supernatant. The isolation
procedure is similar to DNA isolation, chloroform is used to precipitate proteins.
(Generally, both chloroform and phenol can be used to precipitate proteins during
DNA and RNA isolation). The RNA pellet is finer than the DNA pellet; therefore
centrifugation should be used to collect it. RNA concentration is measured after
hydrolysis of RNA, the orcin reactions shows the amount of ribose in the solution.
The amount of isolated RNA can be calculated.
13. Plasmids, plasmid isolation
Plasmids are small, circular, double stranded DNA molecules, located extrachromasomally in various types of bacteria. Their size ranges from 1 to 200 kilobases
(1 kb = 1000 bases). They may contain genes that are beneficial for the host cell, e.g.
coding for antibiotic resistance factors, enzymes necessary for the digestion of organic
molecules or restriction-modification enzymes, which are a special set of enzymes to
protect the host cell genome in prokaryotes. One of the most important practical
aspects of plasmids is that they can be used for cloning, a process in which a single
cell grows into a colony of cells, having the same genetic material. If a plasmid is
properly introduced into the host cell, it multiplies independently from the
chromosomal DNA of the bacteria. As a result, we obtain a multiplied series of
plasmids in the host colony, from which the plasmids can be isolated, and used for
various purposes e.g. transformation (= introduction of foreign DNA into
prokaryotes), transfection (= same process carried out in eukaryotes) and many other
gene manipulating procedures.
Plasmids can be used for cloning if they:
- are smaller than 10 kb
- have some kind of a selection marker (= a special sequence that makes it easy
to isolate host cells containing the plasmid)
- have one or more special restriction cleavage site where a foreign sequence
can be built in by the help of restriction enzymes and ligases.
There are two main groups of vectors: expression and insertions vectors.
Expression vectors contain sequences that drive transcription and translation of the
inserted gene in the host cells. They are:
- bacterial replication origo (ori), which drives
- plasmid replication
- promoter region (P), which directs transcription of
- inserted DNA in the host cell
- multiple cloning site (MCS), contains unique restriction
- endonuclease cleavage sites to clone the inserted DNA
- polyA region, which signals polyA formation
- selection marker e.g. antibiotic resistance gene (Amp`).
Insertion vectors do not contain promoter region and polyA
region, so there is no transcription and translation from the
General structure of an expression vector
inserted DNA.
The main steps of plasmid isolation:
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14. Histochemistry of nucleic acids
Histochemistry and cytochemistry are morphological/microscopic methods that aim to
identify and localize specific chemical components in cells and tissues by specific
staining reactions. Histochemistry deals with localization of specific substances in and
around cells, with the identification of intra-and extra cellular materials of a tissue.
Cytochemistry – a branch of histochemistry – analyzes individual cells, e.g. cells
growing in a cell culture, blood cells or cells of diagnostic specimens etc.
The identification of macromolecules in macroscopic preparations is achieved by
substance-specific chemical reactions that result in colourful products easily
detectable in the light microscope, or fluorescent dyes for fluorescent microscopy.
These techniques are qualitative and quantitative as well, since they detect a specific
molecule and the intensity of the colour depends on the amount of molecules studied.
Fixation and specimen preparation must proceed the cytochemical and histochemical
staining procedure. It might involve specific procedures according to the chemical
reactions to be used (e.g. preparation for a lipid-specific staining should not include
lipid-solvents like xylol) the general idea of preparation is always the same; to
immobilize the molecules and maintain the structure of the cells.
Several histochemical methods are in daily use in pathological laboratories, thus they
have a paramount medical importance.
Feulgen reaction (stains only DNA)
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The method is named after a German scientist Feulgen.
The 2 main steps of chemical reaction are:
1) Generation of free aldehyde groups of the deoxribose of DNA by removing the
Purine (A, G) bases with mild acid hydrolysis. ( Excessive hydrolysis can lead
to cleavage of the phosphdiester bond of DNA)
2) Reaction of the free aldehyde of depuriated DNA and Schiff’s reagent.
The reagent contains Fuchin dye in sulfurous acid. It contains a series of double
bond that causes the purple colour of Fuchin. Fuchin in a complex of with
sulfurous acid loses its double bond and form in a colorless reagent called
Leukofuchin. A Chemical reaction between Leukofuchin and the free aldehyde
groups of depurinated DNA reform the double bonds, and fuchsin regains its
original colour.
Methyl-green-pyronin staining (methyl-green stains DNA, pyronin stains RNA)
Methyl green - pyronin staining detects the nuclear DNA and cytoplasmic RNA.
These two basic dyes are usually used in a mixture. The sterochemical
configuration (degree of coiling) and the polymerization state of the nucleic acid
molecules seem to define the different affinities to each of the stains: Methyl
green: great affinity to highly polymerized DNA, Pyronin: bind to the less
polymerized RNA
Gallocyanine-chromalusum staining (stains both)
This method results in simultaneous detection of DNA and RNA. Cellular
components containing DNA or RNA stained greyish blue. This method is
performed with a complex of gallocyanine and chromalaum. The complex will
bind to phosphate groups of DNA and RNA chains, to render this specific method
either to DNA or RNA, one of them must be destroyed, e.g. with a specific
enzyme, with RNase or DNase.
15. Histochemistry of the cytoplasm
Detection of lipids:
The term lipid stands for a group of bio molecules which are based on their solubility in
organic solvents and their hydrophobic nature. For this reason it is important to avoid the
use of organic solvents during histological detection of lipids. Lipids exist in two forms in
the body:
Free lipids, usually aggregate into droplets of various sizes in the cytoplasm of fat, while
Bound lipids can be found in membrane structures of the cell or its membrane bound
organelles, often as lipoprotein complexes. For the detection of lipids, staining of lipid
containing structures with lipid soluble dyes such as Sudan-B. The blackish-blue dye
accumulates in, and outlines parts of the tissue that are rich in free lipids. The technique
however requires substantial amount of free lipids because of its low sensitivity. They can
also be detected with chemical methods, such as osmium tetroxide. The C=C bond will
participate in a chemical reaction forming a black end product.
Detection of carbohydrates:
Pure polysaccharides occur as glycogen with storage form of glucose. Glycogen is soluble
in water; therefore water based solution should be avoided during histological detection.
Mucopolysacharides are polysaccharides containing hexosamine; they usually esterified
with sulphuric acids. Mucoproteins, also called proteoglycans are mucopolysacharides
chemically bound to proteins. They are common in saliva, where they interact with
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collagen to form gel-like networks. Glycoproteins are very similar in composition, but
their hexosamine content is less than 4%.
In glycolipids the carbohydrate component is attached to a lipid molecule, cerebrosides
are abundant in glycolipids of the nervous tissue.
Glycolipids are detected with PAS reaction.
16. Phase – contrast microscopy
17. Polarizing microscope
18. Enzyme histochemistry and immunohistochemistry
Histochemistry of proteins and enzymes:
This type of detection is mainly based on the detection of the reactive groups of their
amino acids. Ninhidrine-inducedoxidation of amino acids leads to the formation of
aldehyde groups which can be detected with Shiffs reagent. Sulfhydril (-SH) groups and
disulfide (-S-S-) can also be shown quite easily by tetrasolium reaction. Amino-acid
containing aromatic groups react with benzidine yielding a colour end product (benzidinereaction)
Identifying and localizing enzymes in the cell is the detection by the end-product of the
reaction catalyzed by them. The fixation is therefore crucial- such as freeze-drying. Or by
using alkaline and acid phosphatase, ATPases, nucleases etc are all common targets for
enzyme histochemistry.
Immunohistochemistry:
Use of labelled antibodies as specific reagents to localize specific constituents, such as
antigens usually the proteins in tissues or cells. The antibody-antigen complex is very
specific, therefore the target protein is easily found. This is a specific reaction as all
antibody-antigen reactions. To detect the complex, electron dense labels such as colloidal
gold particles can be used to label the end product, and visualize by electron microscope.
19. Plasmolysis and haemolysis
Plasmolysis: is a phenomenon based on shrinkage caused by fluid loss of plant cells when
they are put in hypertonic solution. Since the cell wall is a rigid structure it does not
shrink with the rest of the membrane bound cytoplasm. This results in the separation of
the cell membrane bounded cytoplasm that results in the cell wall separation from the
plasma membranes. Plasmolysis exists in many forms based on its appearance.
(Draw pics. page 78 and 79)
Convex plasmolysis:
When plant cells are put in hypertonic solution that contains Na+ or K+ ions. These ions
“soften” in the cytoplasm by reducing the viscosity so that it maintains nicely rounded
boundaries by surface tension while shrinking. The vacuole in the cell will also shrink
proportionally, indicating that the tonplast = the membrane surrounding the vacuole is
also semi permeable.
Concave plasmolysis:
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It is elicited in hypertonic solution that contains Ca2+ ions. The reason for shrinkage is
hypertonic environment around the cell, but the Ca2+ harden the cytoplasm by increasing
the viscosity, the detachment of the plasma membrane from the rigid wall is uneven,
resulting in the serrated outlined of the cytoplasm under such conditions.
Convex and concave plasmolysis can be explained by the differences in the structure of
hydrated state of potassium and calcium ions. The number of protons is one is higher in
Ca (20) than in the potassium (19). Having one more positive charge in its nucleus attracts
the electrons better and gives the cell a more compact structure overall, so it has a smaller
diameter than that of potassium.
Tonplast and cap plasmolysis:
Tonplast plasmolysis is a special stage of convex plasmolysis, when the hypertonic
solution around the cell contains SCN- (rhodanide) besides K+ ions. The process starts
with a convex plasmolysis due to the K+ ions. At the end of it the cytoplasm is shrunken
around the vacuole. Rhodanide ions gradually destroy the semipermeability of the plasma
membrane, but do not damage that of the tonplast membrane, as a result the shrunken
cytoplasm starts to as well= deplasmolysis. His resembles the two caps sitting on top of
the vacuole = cap plasmolysis. Finally the plasma membrane that has become completely
permeable reaches the cell wall again. Meanwhile the vacuole continues to loose fluid and
shrinks (tonplast plasmolysis).
Haemolysis:



In an isotonic solution they have a bi-concave disc. (e.g. 0.9% NaCl)
In hypotonic solution the cells swell take up a rounded shape because of
the intake of water. After a while the membrane burst. And cell content is
released into the environment- osmotic haemolysis. (e.g. 0,01 % NaCl)
In hypertonic solution the opposite happens, the cells shrink and water
flows out. (e.g. 5% NaCl)
20. Methods of cytogenetics
Deals with the structure and function of chromosomes, including chromosomal
abnormalities. Karyotype analysis- technique which describes the set of chromosomes of
a cell. Karyotyping is usually performed using lymphocytes of peripheral blood. They are
stimulated to divide with mitogen phytohaemagglutinin and then arrested in the
metaphase with colchisine. These are then separated into groups based on size and shape;
metacentric, submetacentric and acrocentric.
Banding techniques: Using special staining procedures it is possible to stain different
regions of the chromosome with different intensities. The banding pattern is highly
specific and reproducible for each chromosome. Most used techniques are the Giemsa
Banding: loosely bound chromosomal techniques are removed by digestion with trypsine
and stain with Giemsa which visualizes the heterochromatin region.
Fluorescence in situ hybridisation (FISH): Hybridisation with metaphase chromosome
spreads with probes labelled with fluorescence dyes to visualize
1. centromeres,
2. telomeres,
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3. whole chromosomes or
4. individual genes.
Using FISH aneuploidy = (Having an abnormal number of chromosomes not an exact
multiple of the haploid number, as contrasted with abnormal numbers of complete haploid
sets of chromosomes, such as diploid, triploid, etc.) or structural chromosome
abnormalities can be detected even in non-dividing cells (interphase cytogenetics).
Comparative Genomic Hybridisation (CGH): New FISH technique developed in which
deletions, duplications (or amplifications) of unknown localisation can be identified. CGH
is mostly used to detect chromosome abnormalities in tumor cells.
- DNA isolated from tumor cells and labelled with fluorescent dye of different
colours.
- Mixture of 2 probes is then used for in situ hybridisation with a normal set of
chromosomes. Hybridisation = (nucleic acid probes to cellular DNA for detection
by autoradiography. Under proper laboratory conditions, the binding process
occurs spontaneously. In situ hybridization constitutes a key step in DNA
fingerprinting).
- During hybridization there is a competition between 2 probes – chromosomes with
equally represented probes will give a homogenous fluorescence.
- Regions with deleted in the cancer will preferentially hybridize with control DNA
and genes amplified in the tumor will mostly hybridize the DNA of the cancer
cells.
- The fluorescence can then be measured and quantitated by digital image analysis
which can give the extent of deletion or amplification.
Flow cytometry:
Metaphase chromosomes stained with a fluorescent dye can be fractionated by using
FACS (Fluorescence activated cell sorter). The technique is suitable for rapid karyotyping
or for isolation of individual chromosomes.
21. Observation of prokaryotic cells by immersion objective
22. Determination of cell diameter by light microscopy
Calibration of a measuring eyepiece.
A) Graduated glassplate
- Special type of eyepiece, contain graduated glass plate that serve as a
measuring scale.
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Determined the measurements of the cell or tissue components if stained
microscopic preparation.
B) Hemocytometer (Burker’s chamber)
- thick glass slide having two main counting chamber
- each chamber is divided into squares by engraved parallel lines witch
consist of two lines system:
1. parallel lines in 50 m distance
2. parallel lines in 200 m distance
Place the Hemocytometer in stage of the microscopy; bring it to focus in 10x objective
lens. Remove one eyepiece of the microscope and place the measuring eyepiece in the
tube. Rotate the measuring eyepiece to the 50 m line system until the scale of measuring
eyepiece reaches parallel orientation of the hematocytometer  count the nr of units of
the measurement scale that cover the 50 m distance and determined the distance between
two units in m. (If 50 m line system is covered by 8 units, 50/ 8 = 6,3 m)
23. Separation of starch and Cl- by gel filtration – analysis of the diagram
Aim of the experiment:
An aqueous solution of sodium chloride and soluble starch is to be fractionated in this
experiment by column chromatography through a dextran gel (Molselect G-25; exclusion
limit: 25000 dalton). Starch and chloride ions are detected in the chromatographic
fractions and the elution volumes of the two solutes are determined.
Experimental protocol. A glass column filled with approximately 25 ml of waterswollen
Molselect G-25 gel is used.
Outline of the order and aim of the steps:
a. Remove excess water from the surface of the gel using a plastic pipette.
b. Layer 0.5 ml of starch-NaCl solution on the gel.
c. Let the sample sink into the gel by opening and closing the outflow of the column.
Collect the drops of water in the first test tube.
d. Fill the column with distilled water, open the outflow and collect 2 ml fractions in the
test tubes. Close the outflow after washing the column with about 25 ml of water and
add a few more ml of water to prevent drying of the gel.
e. Divide the fractions into two. Perform the iodine test with one half of each fraction (it
stains starch blue) and the chloride test with the other half (add 2 drops of AgNO3
solution: white AgCI precipitate will form in NaCl-containing fractions).
f. Determine the void volume ( V0 ) of the gel and the elution volumes Ve1 and Ve2 ) of
starch and chloride ions.
Analysis of the diagram:
- Large molecules of sample are excluded from pores of beads and pass
through the matrix rapidly - small molecules penetrates the pores, are
distributed in larger solvent volume, and appear later in the
chromatography fractions.
Result:
Observations:
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Ve starch = 9  2ml = 18
Ve NaCl = 19  2ml =38
Starch is a bigger molecule than NaCl, and will be excluded from the pores of beads, pass
rapidly through the matrix and appear as the first peak in the diagram.
Ve  V0  Kd *Vi
Kd tells how many of the molecules that will travel through the pores in the gel.
Kd=1  All molecules will travel through the pores
Kd=0  No molecules will travel through the pores
For starch, Ve = V0 because Kd=0
For NaCl : Vi = Ve - Vo = _38 - 18_ = 10 ml
Kd
1
24. Operation of the photometer – determination of DNA and RNA concentration
The photometer measures light absorption at 532 nm. Standard curve of DNA was
prepared by measuring light absorption of several DNA solutions with known
concentration  use this standard curve to determined concentration of RNA and
DNA
25. Analysis of protein gels and Western blots
SDS-polyacrylamide gel electrophoresis of proteins.
Aim of the experiment:
Proteins of different size can be separated from each other by this method. A precast gel is
used for the electrophoresis. Proteins are stained with Coomasie Blue after separation (see
protein electrophoresis).
Transfer of proteins from the gel to a nitrocellulose gel.
Aim of the experiment:
In order to make the proteins detectable to antibodies during the Western blot procedure,
they must be transferred from the polyacrylamide gel to a nitrocellulose membrane. This
is called blotting. The movement of molecules during the blot is forced by an electric field
in witch uniformly negatively charged proteins migrate towards the anode.
Detection of proteins on the membrane: Ponceau staining
Aim of the experiment: Detection of the transfer of proteins by staining using a red
coloured dye specific for proteins. It doesn’t affect the antigen-antibody reaction
supposed to be carried out on the membrane in the next step.
Examination of the final result of a western blotting.
In western blotting, protein of interest are detected by antibodies.
First the antibody binds to the target protein on the surface of the filter, and then a
labelled antibody binds to the first antibody. The antibody can be detected e.g. with
autoradiography.
Facts: Western blotting
 Immunoblotting
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Variation of southern blotting
Immunological technique.
Steps:
protein mixture
Gel: SDS Page
- gel electrophoresis / separated by size
- SDS POLYACRYLAMIDE GEL  neg. Charged detergent. Used to
migrate the protein through. Each protein binds to the detergent mol. Witch
denature the protein, and gives it an NEG. CHARGE
Protein migrated toward POS electrode
C) Filter: Blotting
-Transferred to a filter membrane
D) Imunnoblotting
- Treated with an antibody
- Bind the gene to a filter membrane
- Visualized by radiolabelling, fluorescent, colour

1)
2)
3)
4)
5)
Imunnoprecipitation
Radiolabeled protein incubated with antibody
Antibody complex form complex with antigen ( protein of interest)
Boiling – dissociate the antigen - antibody complex
Recovered protein is analyzed by SDS electrophoresis.
The radioactive protein is detected by autoradiography.
26. Analysis of histochemistric preparations – cytoplasm
Theory:
Histological detection of lipids:
Lipids and steroids are stained with Sudan –B, osmium tetroxide.
The term lipid stands for a group of bio molecules which are based on their solubility in
organic solvents and their hydrophobicity. For this reason it is important to avoid the use
of organic solvents during histological detection of lipids. Lipids exist in two forms in the
body: Free lipids, usually aggregate into droplets of various sizes in the cytoplasm of fat,
while Bound lipids can be found in membrane structures of the cell or its membrane
bound organelles, often as lipoprotein complexes. For the detection of lipids staining of
lipid containing structures with lipid soluble dyes such as Sudan-B. The blackish-blue dye
accumulates in, and outlines parts of the tissue that are rich in free lipids. The technique
however requires substantial amount of free lipids because of its low sensitivity. They can
also be detected with chemical methods, such as osmium tetroxide.
Detection of carbohydrates:
Carbohydrates are stained with PAS Specific for the aldehyde groups of
carbohydrate components.
Pure polysaccharides occur as glycogen with storage form of glucose. Glycogen is soluble
in water; therefore water based solution should be avoided during histological detection.
Mucopolysacharides are polysaccharides containing hexosamine; they usually esterified
with sulphuric acids. Mucoproteins, also called proteoglycans are mucopolysacharides
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chemically bound to proteins. They are common in saliva, where they interact with
collagen to form gel-like networks. Glycoprotein’s are very similar in composition, but
their hexosamine content is less than 4%.
In glycolipids the carbohydrate component is attached to a lipid molecule, cerebrosides
are abundant in glycolipids of the nervous tissue. Glycolipids are detected with PAS
reaction.
Histochemistry of proteins and enzymes:
Proteins have different reactions, depending on what reactive group of the amino acids
we want to detect.
This type of detection is mainly based on the detection of the reactive groups of their
amino acids. Ninhidrine-inducedoxidation of amino acids leads to the formation of
aldehyde groups which can be detected with Shiffs reagent. Sulfhydril (-SH) groups and
disulfide (-S-S-) can also be shown quite easily by tetrasolium reaction. Amino-acid
containing aromatic groups react with benzidine yielding a colour end product (benzidinereaction)
Identifying and localizing enzymes in the cell is the detection by the end-product of the
reaction catalyzed by them. The fixation is therefore crucial- such as freeze-drying. Or by
using alkaline and acid phosphatase, ATPases, nucleases.
Immunocytochemistry:
Use of labelled antibodies as specific reagents to localize specific constituents, such as
antigens or proteins. The antibody-antigen complex is very specific, therefore the target
protein is easily found. To detect the complex, electron dense labels such as colloidal gold
particles can be used to label the end product, and visualize by electron microscope.
Adrenal gland: cortex (Sudan B)
Rat liver: PAS  glycogen containing cytoplasm
Lysosomes: Acid phosphatase (due to enzymes only present in the lysosomes)
Insulin and glycogen producing cells:
Immunocytochemistry.
27. Identification of nuclear components on electron microscopic pictures
TEM
Item 3 – outer nuclear membrane
Item 4 – inner nuclear membrane
Item 5 – perinuclear space
Item 6 – heterochromatin
Item 7 – euchromatin
Item 8 – interchromatin granules
Item 9 – perichromatin granules
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TEM
Item 10 – peripheral heterochromatin
Item 11 – nucleolus associated h.ch
Item 12 – euchromatin
Item 13 - nucleolus
28. Analysis of agarose gels after plasmid electrophoresis and restriction mapping
Restriction endonucleases are enzymes, isolated mainly from prokaryotes that
recognise specific sequences within double-stranded DNA. The recognition sequence
is typically 4-8 nucleotides long and has the same sequence on the two antiparallel
strands, read in 5`to 3` direction (palindrome sequence). Restriction endonuclease cut
phosphodiester bonds in both strands at the same position. Some enzymes produce
blunt-ended fragments by cleaving at the axis of the palindrome, others produce
protruding ends by cleaving apart from the axis. Those belonging to the latter group
are especially important for molecular biologists, because any of these termini (called
cohesive or sticky ends) can form base pairs with any other, so DNA molecules
containing such recognition sites can be joined to form recombinant DNA molecules.
Each enzyme requires specific ion concentrations and pH values for its action, so
different buffers should be used for the different enzymes. The fragments are mixed
with a sample buffer containing a blue dye so the position of the fragments can be
monitored during the electrophoresis. The agarose gel contains ethidium bromide and
the DNA fragments can be visualized in UV-light.
Restriction endonucleases can be used for restriction mapping, a procedure to
determine the relative positions of the cleavage sites of one or more enzymes in a
given DNA molecule. To make a restriction map, DNA is treated with the enzymes
both separately and in combination and the fragments are separated by
electrophoresis. The size of the fragments is determined by using molecular weight
markers.
Uncut plasmid DNA and restriction fragments separated by agarose gel electrophoresis
29. Analysis of histochemistric preparations – nucleic acids
See lab nr 7, your own results!
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30. Operation of the polarizing microscope
Polarizing microscope is a modification of the light microscopic techniques. It is
suitable to study birefringent (norsk: dobbeltbrytende) structures. In contrast to the
LM the PM have two additional polarizing devices: the polarizer and the analyzer,
both of them made from polarizing filters. The polarizer is located below the
condenser of the microscope and transmits only plan polarized light that vibrates in
one direction (normal light vibrates in all directions). The analyzer, a similar filter
system is placed above the objective lens (the filters can be made from Nicol prisms
of calcite). When the polarizer is rotated 360 degrees, the field of view of the
microscope alternates between bright and dark at every 180-degree turn. The two
positions of maximum light transmission are obtained when the main optical axis of
analyzer and polarizer are parallel. When the axis of polarizer is placed perpendicular
position to the axis of the analyzer no light can pass through because the polarized
light emerging from the polarizer is blocked by the analyzer structure, therefore the
field of view is dark. If, however microscopic preparations containing oriented
molecules (such as hair, collagen, microtubules etc) are placed between the crossed
polarizer and analyzer, their repetitive structure allows them to rotate the axis of the
polarized light emerging from the polarizer. This appears as bright structures against
dark background. The ability to rotate the direction of vibration of polarized light is
called birefringence and is present in crystalline substances and well-ordered
(oriented) fibrous structures (birefringent or anisotropic materials). When the
birefringent sample is parallel to the plane of polarizer, the polarized light emerging
from the sample is blocked by the analyzer, because the optical axis of analyzer is
perpendicular to the optical axis of the birefringent structure. The result of this
position is a dark field of view.
Polarization microscope is an important tool among microscopical techniques because
it gives information about the birefringent properties of biological structures which are
related to molecular and macromolecular organization.
Basic principles of polarization microscopy
A:
Polarizer and analyzer are parallel; the field of view is bright
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Answers to Laboratory exam questions in Biology
B:
C:
D:
2005/2006
Polarizer and analyzer are perpendicular (crossed): the field of view is dark
A specimen manifests its birefringent by appearing bright when placed at
angle between crossed polarizer and analyzer.
Polarizer and analyzer are crossed, optical axis of birefringent sample is
parallel to the plane of polarization, and the field of view is dark.
31. Centring adjustment and the operation of the phase-contrast microscope
The phase contrast microscope is a modified LM which has been developed for
observation of unstained biological samples and living cells. The transparent
structures of the cell (e.g. nucleus, mitochondria etc) have low contrast because they
absorb light poorly. However, light is retarded as it passes through these structures so
that its phase is altered (phase retardation) compared to light that has passed through
the surrounding cytoplasm. This phase difference and retardation is a result of small
differences in refractive index and thickness of different parts of the biological
specimen. These differences cannot be seen by LM. The phase contrast microscopy
converts this small phase differences in refractive index into differences in intensity
that is into contrast differences which are visible by the human eye.
This conversion requires special components in the phase contrast microscope as
follows: phase contrast objectives containing phase plates, phase condenser containing
phase ring (annular diagram), telescope microscope and green light filter.
In essence the light waves initially retarded by a cell
component is further retarded by a transparent ring-shaped
phase-shifting plate difference are converted into changes
in amplitude (changes in intensity). The phase-contrast
objective lenses are constructed such that they have a phase
plate at their back focal plane that corresponds geometrically
to the position of the phase ring of direct light from the
phase-condenser. The telescope microscope is an optical
device serving for centring of phase plate and phase
annulus. The green filter is a standard part of the phase
contrast microscope producing an optimum level of contrast.
If the phase plate and the phase ring are covering one
another in the optical axis of microscope, the unstained and
living objects can be visualized with useful amount of contrast.
A: image before centring
B: image during centring
C: image after centring
adjustment
32. Identification of cytoplasmic organelles on electron microscopic pictures
TEM
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Item 1
Item 2
Item 3
Item 4
Item 5
Item 6
Item 7
Golgi membrane
Rough Endoplasmatic reticulum
Free ribosomes
Small vesicles
Transport vesicle
Secretory granules
Secondary lysosome
TEM
Item 8 Smooth endoplasmatic reticulum
Item 9
Item 10 Secondary lysosome
33. Lymph node from Burkitt lymphoma – identification of mitotic figures
Look for Burkitt lymphoma in the lab room! Or maybe on intranet 
LIGHT MICROSCOPE
(human metaphasic chromosomes)
Item 14 – one of the chromosomes
Item 15 - labelled telomere
TEM
(traditional staining)
Item 16 - chromosome
TEM
(chromosomes during M-phase)
Item 17 – chromosome
Item 18 – kinethocor proteins
Item 19 – kinetohcor microtubules
Item 20 – broken membrane
fragment of nuclear envelope
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SEM
(three dimensional appearance)
Item 21 – chromosome containing
both of its chromatids
34. Analysis of light microscopic autoradiograpic preparations
Use of radioactive isotopes. Isotopes are variants of the same number of protons but
different number of neutrons. The cell can however not distinguish these from each other
and incorporates them into its macromolecules. Radioisotopes have unstable nuclei and
their random disintegration results in the emission of easily detectable radioactive
elements. - Labelled molecules incorporated intomacromolecules of interest are called
radioactive precursors.
AutoradiographyLight and electron microscopic auto can be used to detect labelled precursors containing
low emitting isotopes. The cells or tissue is covered by a thin photo emulsion and covered
for some time. The molecules incorporated into cellular molecules produce photochemical
reactions and this leads to the black silver grains on the developed autoradiogram. The
synthesis of molecules can be studied this way:
- DNA replication can be analyzed by labelling with [3H]thymidine,
- RNA with [3H] uridine,
- Protein synthesis with [3H] leucine,
- Glycosylation with [3H] mannose etc.
35. Identification of normal and cancer cells on PAP – smears
Cancer of the cervix is one of the most common cancers among females. The fastest and
most effective method of the early detection of this is by extraction of cells from the
cervix, stained according to Papanicolau and then subjected to microscopic examination.
Due to the presence of potentially harmful cells the vaginal smear can be classified from I
to V, I is normal (contains only normal epithelial cells), where No. V refers to the late
stage of carcinoma invasion in which tumor cells are apparent.
Samples contain a range of normal to abnormal sample slides.
Normal cells: Flat, quite large, pale stained, variably shaped and contain small nuclei.
Tumor cells: Uniformly round with a prominent, enlarged dark stained nucleus. (The
more number of those type of cells the more developed or severe the cancer is.
36. Identification of inheritance patterns on pedigrees
Observation of the matter where a particular trait is transmitted from one generation to
another, and how diseases appear in several family members. Drawing up family tree
always start with the affected person first found to have the trait and through whom the
family came to attention of the investigator. This person is called proband and indicated
by an arrow on the pedigree. Affected persons are presented with dark symbols, healthy
with open ones, males with squares, females with circles. Persons in the pedigree are
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identified by the generation (designated by Roman numbers) and from their location
(Arabic numbers)
Dominant Trait is one of the manifests itself in the heterozygote. The dominant allele
usually generated by gain of function mutation from a normal one. Dominantly inherited
diseases are rare; therefore affected persons are usually heterozygotes, having a normal
homozygote and a heterozygotic parent.
Y-linked inheritance implies only males affected and that a male parent transmits the
disease to his son and not daughter.
X-linked dominant inheritance is in the heterozygotic female as well as in males having
a mutant allele on his X-chromosome. An affected male transmits only to his daughters,
not sons. However both sons and daughters of an affected mother have 50% chance to
both be affected. Usually an excess of affected females are seen in these families.
Autosomal dominant inheritance affects both male and females equally. These include
diseases such as Huntington’s chorea, osteogenesis imperfecta and achondroplasia.
Brachydactilia is also inherited this way.
Recessive Inheritance only manifest when both of the alleles are present in double dosehomozygotes. The recessive allele is generated by a loss- of-function mutation; in
heterozygotes the normal functioning of one healthy allele usually ensures the normal
function and individual being perfectly healthy.
X-linked recessive inheritance manifests only in females when both alleles are present
in double dose, in homozygous state. The mutant in males will always only be present in
one copy because of the Y-chromosomes, thus giving it an allele to counteract it. The
diseases are usually transmitted by healthy female carriers or affected males called
hemizygotes, Duchene’s muscular dystrophy and haemophilia as well as partial colour
blindness are inherited in X-linked recessive manner.
37. Analysis of chromosomal abnormalities
Photographs of normal and abnormal metaphase chromosomal study the chromosomes
and determine karyotypes using following scheme:
a) Count nr of chromosomes
b) Encircle the large acrocentric chromosomes and all the chromosomes smaller than
that group.
c) Encircle the small acrocentric chromosomes and identify the Y-chromosome
d) Encircle chromosomes of group E
e) Encircle small metacentric chromosomes (Group F)
f) Label largest chromosomes by numbers (Groups A & B)
g) Count unlabelled chromosomes (Group C). Identify the X-chromosomes. Based
on the karyotypes give a diagnosis (e.g. normal male, female with Down’s
syndrome etc.)
Deals with the structure and function of chromosomes, including chromosomal
abnormalities. Karyotype analysis- technique which describes the set of chromosomes of
a cell. Karyotyping is usually performed using lymphocytes of peripheral blood. They are
stimulated to divide with mitogen phytohaemagglutinin and then arrested in the
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metaphase with colchisine. These are then separated into groups based on size and shape;
metacentric, submetacentric and acrocentric.
Picture page 92
38. Observations of histochemistric preparations
PC12 cells, derived from a rat adrenal gland tumor, provide the most widely used model
system for the investigation of neuronal differentiation. In response to NGF (nerve GF)
treatment, these cells start to grow neuritis and, along with many other biochemical
changes undergo a complete differentiation of nerve cells becoming similar to
sympathetic-like neurons.
39. Identification of undifferentiated and apoptotic PC12 cells
Immunolocalization of MAPK and CREB proteins in PC12 cells
Several protein kinases are known to play crucial role in the different signal transduction
pathways in many different signal transduction pathways in many different cell types.
Mitogen-activated protein kinase (MAPK) enzymes form one of the best known families
of these enzymes, taking part in a great variety of signal mechanism. In PC12 cells, as
elements of a cytoplasmic protein kinase cascade, they are involved in the pathway
leading to neuronal differentiation.
40. Analysis of chromosome preparations.
Comparison of a normal lymph node to one of a patient suffering from Burkitt’s
lymphoma
Burkitt’s lymphoma
Is a malignant tumor derived B cells of lymphatic tissues with quite high frequency in
some regions of Africa. Among the causative agents both the role of viruses and
activation of oncogenes are suspected; Epstein-Barr virus infection, for instance, is known
to be associated often with this type of human malignancy. The role of the myc from
chromosome 8 to immunoglobin heavy-chain locus on chromosome 14 results in
abnormal Myc expression leading to the development of the tumor from lymph nodes.
Normal lymph nodes are covered by fibrous connective tissue of which projections extend
inward dividing the organ into smaller compartment.
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