Labratory Examination Questions - Free



Labratory Examination Questions

1. Structure and operation of the light microscope

- 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 = _(_____ (= wavelength of light |

|n x sin ( 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.

[pic] [pic]

2. Structure and operation of the electron microscope

- 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.

- 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)

[pic]

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

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.

Radioactive precursors:

Labeled molecules that can be incorporated into macromolecules of interest, and then traced.

|Radioisotop |Emitted radiation |Example of application |

| | |(labeled precursor/ process studied) |

|3H |( particle |(3H ( amino acid/proteinsynthesis |

|14C |( particle |(3H ( Sugar /glycosylation |

|35S |( particle |(3H ( methionine/proteinsynthesis |

|32P |( particle |((- 32P ( ATP/In vitro protein phosph. |

|131I |( particle |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:

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.

- 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, |

|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.

[pic]

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 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.

[pic]

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)

[pic]

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 protein-DNA 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

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 extra-chromasomally 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

inserted DNA.

The main steps of plasmid isolation:

[pic]

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)

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 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 (benzidine-reaction)

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:

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,

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.

- 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 ([pic]) of the gel and the elution volumes[pic] and [pic]) 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: |

| |

| |

|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. |

| |

|[pic] |

|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, [pic]= [pic] 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

- Variation of southern blotting

- Immunological technique.

Steps:

protein mixture

1 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

• Imunnoprecipitation

1) Radiolabeled protein incubated with antibody

2) Antibody complex form complex with antigen ( protein of interest)

3) Boiling – dissociate the antigen - antibody complex

4) Recovered protein is analyzed by SDS electrophoresis.

5) 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 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 (benzidine-reaction)

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 |

|[pic] | |

| |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 |

|[pic] |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.

[pic]

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!

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.

[pic]

Basic principles of polarization microscopy

A: Polarizer and analyzer are parallel; the field of view is bright

B: Polarizer and analyzer are perpendicular (crossed): the field of view is dark

C: A specimen manifests its birefringent by appearing bright when placed at angle between crossed polarizer and analyzer.

D: 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

| | |

|[pic] |TEM |

| | |

| |Item 1 Golgi membrane |

| |Item 2 Rough Endoplasmatic reticulum |

| |Item 3 Free ribosomes |

| |Item 4 Small vesicles |

| |Item 5 Transport vesicle |

| |Item 6 Secretory granules |

| |Item 7 Secondary lysosome |

|[pic] | |

| |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 |[pic] |

|(human metaphasic chromosomes) | |

| | |

|Item 14 – one of the chromosomes | |

| | |

|Item 15 - labelled telomere | |

|TEM |[pic] |

|(traditional staining) | |

| | |

|Item 16 - chromosome | |

|TEM |[pic] |

|(chromosomes during M-phase) | |

| | |

|Item 17 – chromosome | |

|Item 18 – kinethocor proteins | |

|Item 19 – kinetohcor microtubules | |

|Item 20 – broken membrane fragment of nuclear | |

|envelope | |

|SEM |[pic] |

| | |

|(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.

Autoradiography-

Light 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 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 dose-homozygotes. 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 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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General structure of an expression vector

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