The Cytoskeleton, Cilia & Undulipodia + Homogenization, fractionation and cytology Flashcards

Question

What are the centrioles and the centrosome?

Answer 1

- In many cells microtubules grow out from a centrosome near the nucleus - The centrosome is a microtubule organizing centre - In animal cells the centrosome has a pair of centrioles each with 9 triplets of microtubules arranged in a ring DO NOT CONFUSE CENTROMERE WITH CENTROSOME THE CENTROME IS A REGION OF THE CHROMOSOME WHILE THE CENTROSOME IS A MICROTUBULE ORGANIZING CENTRE

Answer 2

- In anaphase, sister chromatids separate and move along the kinetochore microtubules toward opposite ends of the cell - The microtubules shorten by depolymerising at their kinetochore ends - Microtubules are dismantled by the kinetochore to release tubulin subunits

Answer 3

Axonal transport of vesicles along microtubules in nerve axons involves kinesin and cytosolic dynein motor proteins: vesicle can be transported 250 – 400 mm/day Kinesin /dynein head proteins have ATPase activity and tails that bind to vesicles: kinesin mediates anterograde movement of vesicles towards synapses along singlet microtubules cytosolic dynein mediates retrograde movement to the cell body for recycling along singlet microtubules * In synaptic endings motor proteins can transport neurotransmitter vesicles along actin microfilaments

Answer 4

Kynesin - Anterograde transport (towards + end of microtubule - Transport of cellular cargo - Important role in depolymerising microtubule minus ends at centrosomes during anaphase

Answer 5

Dynein - Retrograde transport (towards - end of microtubule - Transport of cellular cargo - Cytoplasmic dynein: organelle function, helps to position the Golgi complex and other organelles in the cell, centrosome assembly - Axonemal dynein: cilia/flagella movement

Answer 6

- Flagellum undulate in a snake like motion driving the cell forward e.g. human sperm - Cilia move back and forth with a rapid power stroke and slower recover stroke

Answer 7

- Cilia and flagella share a common ultrastructure: - A core of microtubules sheathed by the plasma membrane - A basal body that anchors the cilium or flagellum - A motor protein called dynein which drives the bending movements of a cilium or flagellum

Answer 8

Dyneins are arranged in doublets on the inside flagellum/cilium Dynein ‘walk’ along microtubules, carrying cargo (microtubule) Dyneins release from microtubule, swing forward, and re-bind ATP required Inhibitory signals on either side of flagella - switch inhibition mechanism As one side is inhibited, other side is active - beating motion Dyneins release from microtubule, swing forward, and re-bind

Answer 9

Sliding Forces exerted by dynein arms cause doublets to curve, bending the cilium or flagellum

Answer 10

To obtain precise information about the cells organelles, it is necessary to isolate them free from contamination organelles

Answer 11

It involves 3 steps: Extraction, Homogenisation and fractionation

Answer 12

It is the first step toward isolating any sub-cellular structures. In order to maintain the biological activity of organelles and biomolecules. The cells or tissues are suspended in a solution of appropriate pH and salt content, usually isotonic sucrose (0.25 mol/L) at 4°C.

Answer 13

* Homogenisation just means to “break open”. * Homogenisation is concerned only with rupture of the membrane.

Answer 14

The principal aim is to achieve: * highest degree of cell breakage * minimum of disruptive forces * without damage to any of the organelles

Answer 15

1. Volume/number of cells to be homogenised? 2. Number of samples to be homogenised? 3. How difficult are the cells to homogenised? 4. What impact will the method have on the desired product? 5. How stable is the product being isolated? 6. How dangerous are the cells, products and methods used? 7. Cost of method? The choice of method should take these factors into consideration and to ultimately achieve the best product yield and quality.

Answer 16

- Bead mill homogenisers are mechanical solid shear homogenisers that agitate beads at high speeds to break up cells * Agitators are fitted with the shaft which provide kinetic energy to the small beads present in the chamber * Disruption takes place due to the grinding action of the rolling beads and the impact resulting from the cascading ones. * The choice of bead size and weight is greatly dependent on the type of cells

Answer 17

* Ultrasonic homogenisers mechanical liquid shear work by inducing vibration in a titanium probe combined with an aspirator device that is immersed in the cell suspension. * Homogenisation occurs both directly through the ultrasonic forces and cavitation - the rapid formation and collapse of bubbles producing a shockwave and disrupting cell walls by pressure change. * This combination of forces make ultrasonic homogenisers excellent for cell disruption.

Answer 18

* Perfect when sample is mostly liquid or consists of small particles in a liquid. * Unsuitable for applications where sample is mostly solid and / or not immersed in a liquid buffer.

Answer 19

* mechanical liquid shear which consists of a hydraulic pump that drives a piston. The piston forces the liquid sample through a tiny valve under high pressure. * This pressure could be between 10,000-50,000psi * As the sample passes through the valve, the cells experience shear stress, resulting in cellular disruption. * As the cells move through the valve, they experience decompression and subsequently expand and rupture

Answer 20

* Non-mechanical physical which mainly used for bacteria. * Involves heating the cells (heat shock) to 50-55C to disrupt the outer membrane and release periplasmic proteins (Gram –ve) * or up to 95C to break the cell wall and release cytoplasmic contents (E coli, Gram +ve). * Relatively easy and cheap but used only for heat stable products

Answer 21

* Osmotic shock is a non-mechanical physical, it is osmotic stress which is physiologic dysfunction caused by a sudden change in the solute concentration around a cell, * This causes a rapid change in the movement of water across its cell membrane (in/out) result in cell burst. * Note that this method can only work with animal cells and protozoa, since they do not have cell walls (bacteria).

Answer 22

* It is a non-mechanical (chemical) mainly used in plant cells. Organic solvents such as toluene, ether, benzene, methanol, and DMSO (Dimethyl sulphoxide) can be used to permeate cell walls. * EDTA can be used specifically to disrupt the cell walls of gram negative bacteria, whose cell walls contain lipopolysaccharides. EDTA will chelate the cations leaving holes in the cell walls.

Answer 23

* It is non-mechanical (chemical) , which directly causes damage to the cell wall or membrane. * Its mechanism of action is to solubilise membrane proteins and lipids, thereby causing the cell to lyse * Detergents can be anionic, cationic and non-ionic detergents. * Most commonly used anionic detergent is sodium dodecyl sulfate (SDS) which disturbs protein-protein interactions.

Answer 24

* It is non-mechanical enzymatic which degrade the cell wall which will lead to release the intracellular components. * Enzymes that are commonly used for degradation of cell wall of plants (cellulases), yeast (zymolyases), Grame –ve/+ve (lysozomes), and animal cells (collagenase and proteinase K). * The enzyme’s high price and limited availability limits their utilization in large scale processes

Answer 25

It is the process used to separate cellular components/organelles while preserving individual functions of each component

Answer 26

Mainly carried our using a centrifuge which is a spinning device which applies high-speed centrifugal forces to separate materials based on density/size.

Answer 27

* Particles suspended in a solution are pulled downward by Earth’s gravitational force. * In a solution, particles whose mass or density is higher than that of the solvent sink or sediment and particles that are lighter than they float on the top

Answer 28

* Separation by – Differential centrifugation * Separation by– Density gradient centrifugation

Answer 29

1. Differential centrifugation: separates particles based on difference in sedimentation rate, which reflect differences in sizes and densities of organelles

Answer 30

1-The preparation (homogenate) is initially centrifuged at low speeds to completely sediment the largest and heaviest sub-cellular component. 2- The supernatant is carefully decanted and is again centrifuged at a higher speeds till the desired portion of cell lysate is obtained

Answer 31

Density gradient centrifugation: separates particles in which the sample is centrifuged in a medium that gradually increases in density from top to bottom.

Answer 32

Sucrose (5-40%) is commonly used for dentistry gradient

Answer 33

* The homogenate is applied in a thin zone at the top of the centrifuge tube on a density gradient (sucrose). * If the density of the organelle at any point in the gradient is same to that of the gradient, then these organelles will stop otherwise it will move downward towards more denser region

Answer 34

* An enzyme that is known to be localised exclusively in the particular organelle. - Marker enzymes also provide information on the biochemical purity of the fractionated organelles. The presence of unwanted marker enzyme activity in the preparation indicates the level of contamination by other organelles. Allows for one to monitor the fractionation of organelle protocol

Answer 35

Is the microscopic examination of cells obtained from the body by aspiration or scraping for diagnostic purposes,

Answer 36

The main advantage of this test is that the collection of cytological specimens does not require anaesthesia, is painless and there is no bleeding neither inflammation.

Answer 37

1-Stage: flat platform where slides are placed 2- Ocular Lens (10x): where specimen viewed though 3-Objective lens: Lens closest to the specimen; changes magnification 4-Base: support microscope and contain light source 5-Diaphragm (Iris): Adjust the amount of light that reaches specimen. 6-Coarse Focus: will bring specimen into a general focus. 7-Fine Focus: will fine tune the focus, increasing the detail. 8-Light Source: white light source found on base. 9-Condenser: focus light onto specimen. 10-Mechanical stage arms: move stage L, R, up and down

Answer 38

* Major Function: Localisation of specific cellular molecules – example proteins Fluorescent dyes or proteins (Flurochromes) * flurochromes may be indirectly or directly associated with the cellular molecule * Multiple flurochromes may be used simultaneously

Answer 39

* Sensitivity: "glow” against dark background * Specificity: immunofluorescence * Cells may be fixed or living

Answer 40

* Sample collections * Reception of the specimen and request form. * Preparation of slides for microscopic. * Staining * Screening and reporting the slides.

Answer 41

1-Exfoliative cytology 2-Fine needle Aspiration Cytology (FNAC) 3-Body fluids

Answer 42

Microscopic examination of cells that have been shed or removed from the epithelial surface (mucous membrane) of various organs.

Answer 43

is a diagnostic procedure used to investigate lumps or masses (a way of taking a sample of cells from the breast tissue). A thin (23–25 gauge), hollow needle is inserted into the mass for sampling of cells

Answer 44

is a diagnostic procedure in which a thin (23–25 gauge), needle is inserted into the organ cavity for sampling of cells. Pleural and pericardia fluid, CSF and ascitic fluid

Answer 45

1. peritoneal, pericardial and pleural fluids 2. CSF 3. Nipple discharge 4. Bronchial brushings / washings 5. Sputum 6. Gastric washings 7. Urine sediment 8. Prostatic secretions 9. Cervicovaginal (paps) smear

Answer 46

*Details on the specimen and form are checked. *The specimen and form must have at least three patient identifiers (out-of-name, surname, date of birth, NHS number, hospital number). *Laboratory reference number assigned.

Answer 47

* Line smear technique * Squash technique

Answer 48

* Place a drop of fluid near the end of a slide and drag a spreader slide backward into the drop. * The drop spreads along the junction of the two slides. * When the spreader slide reaches no more than 2/3 the length of the slide, lift it directly upward.

Answer 49

Place a drop of fluid near the frosted end of the slide. Gently place a second (spreader at 90 degree) slide on top of the drop slowly pull the spreader slide across the first slide

Answer 50

* prevention of degeneration of cells and tissue * preservation of cells as close as possible to the living state * specific periods of time * changes the physical and chemical state of the cells

Answer 51

* Penetrate cells rapidly * Minimize cell shrinkage * Maintain morphologic integrity * Deactivate autolytic enzymes * Replace cellular water * Facilitate diffusion of dyes across cell boundaries * Help cells adhere to a glass surface * Provide consistent results over time

Answer 52

* 95% Ethyl alcohol * 100% Ethanol * 100% methanol * 80% isopropanol

Answer 53

involves treating specimens with a mild detergents (such as saponin and digitonin, Tween 20) to dissolve lipids from the cell membrane, thereby allowing stains to dye intracellular components and organelles

Answer 54

- Basic (+ve) = stain the acidic components of the cell (nucleus) - Acidic dye (-ve) stains the basic components of the cell (cytoplasm)

Answer 55

*Fixation *Permeabilisation *Hydration: to prepare the cell sample for uptake the nuclear dye *Staining *Clearing: cells are not transparent while the smear is in the staining or alcohol solutions. During clearing, Xylene replaces the alcohol , which is also miscible in the mounting medium. *Mounting: slides are cover slipped with a mountant that bonds them and protects the sample from air drying, oxidation, or fading of the stain.

Answer 56

Nuclear staining: is done by using a hematoxylin stain. Dehydration to prepare the cells for cytoplasmic staining Cytoplasmic staining: cytoplasmic stains are Eosin, OG-6/EA-50.

Answer 57

It was developed by Dr Papanicolaou by 1940s It is a polychrome stain which to this day is used It is one of the most used staining procedures in cytology

Answer 58

For routine diagnosis, the use of H & E is by far preferred for viewing cellular and tissue structure detail by pathologists.

Answer 59

Eosin is an acidic dye: it is negatively charged. It stains basic (or acidophilic) structures red or pink.

Answer 60

Hematoxylins is a basic dye, it is positively charged used to stain acidic (or basophilic structures nucleus) blue.

Answer 61

It is known as Romanowsky stain. is based on a modification of the Wright-Giemsa stain Is used for cytoplasmic element detail, elements such as mucins, fat droplets and neurosecretory granules. Diff-Quik stain is often used for initial screening of cytopathology specimens. Diff-Quik allows the pathologist to quickly decide whether or not additional staining is required

Answer 62

* Detection of surface antigens (markers) on isolated cells. * The detection is based on specific antigen-antibody binding (immunoreactions). * A specific antibody identifies an epitope with 8-15 length amino acid sequence in a protein (epitopes).

Answer 63

* Tumour diagnostic/classification * Prognostic/Predictor Markers * Target Therapy

Answer 64

Cervical screening

The Cytoskeleton, Cilia & Undulipodia + Homogenization, fractionation and cytology Flashcards

(89 cards)