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Cell Biology Lab
| Question | Answer |
|---|---|
| Principle of Magnification | An object is magnified in size so that it becomes visible to the observer |
| What is the max resolving power of a light microscope? | 0.2 micro meters |
| What does phase contrast optics do for inverted microscopes? | Monitor tissue cultures directly without stains or enhancements |
| What does phase contrast microscopy do? | Uses lens system that produces visible images from transparent objects |
| Principle of phase contrast microscopy | Light changes its speed and direction when passing through cellular and extracellular structures with different refractive indices. These changes cause the structures to appear lighter or darker relative to each other |
| What is Fluorescence Microscopy used for? | Used to detect structures, molecules or proteins within the cell |
| What is the practical value of parfocal positioning of objective lenses? | Clearer image, don't need to refocus the sample |
| What is the function of the iris diaphragm of the condenser lens? | To control light onto the image/adjust the aperture of light |
| What are the advantages of phase contrast microscopy? | Exploits the refractive index of the cells, larger live cells, view unstained cells |
| What are the advantages of using fluorescence microscopy? | Label target molecules, to detect structures in the cell, stain different components, identify location of structures |
| Characteristics of TEM | 2-D, better resolution, higher magnification, electrons transmitted through thinner section |
| Characteristics of SEM | 3-D, electrons scattered, thicker section |
| Three things required for viewing cells in light microscope | Bright light must be focused onto specimen by lenses in condenser, the specimen must be carefully prepared to allow light to pass through, an appropriate lens must be arranged to focus an image of the specimen in the eye |
| Principle of transmission of electron microscopy | Uses a beam of electrons whose wavelength is very short and uses magnetic coils to focus the beam |
| What does fixation do for a sample of cell tissue? | Used to avoid tissue digestion and to preserve tissue morphology and molecular composition |
| Examples of chemical substances used to preserve tissues | formaldehyde, glutaraldehyde, paraformaldehyde |
| How do you obtain thin tissue sections? | Tissues must be infiltrated with embedding substances that impart a rigid consistency to tissue, making tissues more resistant to sectioning |
| Name the embedding materials used | paraffin and plastic resins that penetrate all intercellular spaces and even into cells |
| Steps for preparing of tissues for microscopic examination | Fixation, dehydration, clearing, embedding, sectioning |
| Dehydration process | Replaces tissue water with organic solvents, water extracted by bathing tissue in graded series of ethanol |
| Clearing step in tissue preparation | As tissues become infiltrated with solvent, usually become transparent |
| Why were methods of staining tissues made? | Make various tissue components conspicuous and to permit distinctions to be made between various tissue components |
| Basophilic meaning | Tissue components that stain more readily with basic dyes |
| Acidophilic meaning | Tissue components that stain more readily with acidic dyes |
| Example of basic dye | Haematoxylin |
| Example of acidic dye | Eosin |
| Haemotoxylin stains... | acidic structures such as the nucleus, DNA and RNA blue/purple |
| Eosin stains... | basic structures such as the cytoplasm, muscle and collagen pink/red |
| Stains used in the Masson Trichrome technique | Celestine blue, Mayer's haematoxylin, Ponceau red, acid fuchsin and light green |
| What does Celestine blue stain? | Nuclear portion of the cell |
| What does acid fuchsin stain? | The red blood cells |
| What does the ponceau red stain? | The muscle and epithelium |
| What does light green stain? | The collagen fibres |
| What is FRAP? | Fluorescence recovery after photobleaching |
| What does FRAP do? | Involves uniformly labeling proteins across the cell surface, bleaching the label from a small region in this fluorescent sea, and then seeing how quickly the surrounding labeled proteins seep into this bleached patch of membrane |
| What protein is used for FRAP? | Green fluorescent protein (GFP) |
| What is the limitation of FRAP? | It is impossible to see what individual molecules are doing |
| What technique was made to fix the limitation of the FRAP technique? | Single-particle tracking microscopy |
| What type of protein recovers quickly after bleaching? | A freely diffusing protein |
| What is Trypan blue used for? | Used to assess the viability of cells by staining the membrane |
| Principle of Trypan blue staining? | Live cells possess intact cell membranes and exclude the dye whereas dead cells do not |
| Diffusion definition | The movement of dissolved molecules from a region of high concentration to a region of low concentration |
| Osmosis definition | The movement of water molecules across a semi permeable membrane |
| Tonicity definition | The ability of a solution to cause a cell to gain or lose water/the effect of an extracellular environment on the volume of the cell |
| What happens to a RBC in a hypertonic solution? | The hypertonicity of the solution causes the cell to shrivel and die (crenation) |
| What happens to RBC in a hypotonic solution? | The hypotonicity of the solution causes the cell to swell and lyse (haemolysis) |
| Why is a mature RBC ideal to use for the study of a membrane? | Because it has no nucleus or other internal membranes |
| How do Paramecium feed? | Absorbing food through their oral groove |
| What inhibits the action/movement of Paramecium? | Methyl cellulose solution or vaseline |
| What dye is used during the Paramecium experiment? | Congo red |
| What colour and shape were the yeast cells in Paramecium initially? | Red, meaning the yeast cells were basic |
| What colour change occurs in the yeast cells when stained with Congo red? | Red to blue to black |
| Why is it important to handle intracellular organelles with care? | So the membranes don't rupture |
| Why are all the steps carried out in an isotonic buffer? | To prevent osmotic damage |
| Why must the materials must be kept in ice? | To inhibit the activity of proteases |
| Homogenate definition | A thick extract that contains large and small molecules from the cytosol, such as enzymes, ribosomes, metabolites and membrane-enclosed organelles |
| What is centrifugation? | A procedure used to separate a homogenate into different parts or fractions through high speed rotation |
| Why do the centrifuge chambers need to be refrigerated? | To have the air evacuated so that the friction does not heat up the homogenate |
| Principle of centrifugation | Separates cell components on the basis of size and density |
| What does the solution buffer contain? | Sucrose, EDTA and Tris-HCl |
| Where is the porin in the mitochondria found? | Outer membrane |
| Where is the mitochondrial genome found? | The matrix |
| Where are the citric acid cycle enzymes located in the mitochondria? | The matrix |
| Where are the proteins of the electron transport chain located in the mitochondria? | The inner membrane |
| Where does ATP synthase occur in the mitochondria? | The inner membrane |
| Supernatant definition | Liquid above the pellet, made of less dense compounds |
| What is the SDH assay and what does it do? | Succinate Dehydrogenase Assay detects the activity of a marker enzyme, SDH, which is confined to and unique to mitochondria |
| What is SDH? | An inner mitochondrial membrane protein involved in the citric acid cycle which takes place in the matrix |
| What does the absorbance value tell you about SDH activity? | How high or low the activity or SDH is, low absorbance means high SDH activity which means more mitochondria is intact |
| What is happening in the tube if you see a drop in absorbance? | Decrease in functioning mitochondria, colour change from blue to colourless, DCPIP is reduced if SDH is present - electron transfer |
Created by:
tarajdaly16
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