Section 2 - Cells: 3. Cell structure Flashcards
Steps to prepare a microscope slide.
- Cut a piece of onion and peel off one thin layer using tweezers
- this gives one layer of cells, so it is thin enough to be used on a light microscope. - Place the layer of onion on the slide and add two drops of the necessary solution.
- Plant cells = Iodine, to stain nucleus, cytoplasm and cell walls.
- Animal cells = Methylene Blue
- Already stained (eg, red onion) = water. - Gently lower a coverslip over the specimen using a mounted needle.
- Angle avoids air bubbles (artifacts). - Use a paper towel to pat dry and excess liquid.
Steps to setting up a microscope.
- Place the slide on the stage at the lowest point
- altered with the coarse focusing wheel. - Set the objective lens to the lowest magnification
- For a greater field of view. - Focus the image with the course, then fine focusing wheels.
- Change the magnification and repeat focusing
- Total magnification = objective x eyepiece
Magnification
How many times larger the image is than the actual object
What is Resolution in microscopy
The minimum distance apart that two objects must be to appear as separate.
Depends on the wavelength of the image used (0.2micrometers for visible light)
Microscopes have a limit of resolution, magnification beyond which makes the image appear blurry.
What is the equation to calculate magnification
Magnification = Image / Actual
How to do a scientific drawing (7 key features)
1) Examine significant features to include
2) Only draw what you see
3) Draw in pencil
4) Use single lines (no shading)
5) Give clear title (+ Magnification)
6) Label with straight lines
7) Underline scientific names
What is a light microscope (optical microscope)
Uses visible light as a form of radiation to view specimens. The light passes through the specimen on the slide and through a set of lenses to the eyepiece, where the image can be viewed.
- x40 / x100 / x400 magnification
What are the limitations of a light microscope
- The specimen must be thin to allow light to pass through.
- The longer wavelength of light mean that it has a limited resolution.
What is a Scanning electron microscope
Uses a beam of electrons as a form of radiation. The electrons a directed onto the specimen from above, and computer analysis allows a 3D image to be built based on the scattered electrons.
- Resolving power = 20nm
What are the limitations of a SEM
- Specimen must be dead (in a vacuum)
- Complex staining process is required, although image is not in colour.
- Only shows the outer surface of the specimen
What is a Transmission electron microscope
Uses a beam of electrons as a form of radiation. The electrons are passed through the specimen from below, some of which are absorbed by parts of the specimen, appearing darker on the image. 2D image (photomicrograph) is produced that can be layered into 3D but it is complicated and time consuming to do.
- Resolving power = 0.1nm (greatest of all)
What are the limitations of a TEM
- Specimen must be dead (in a vacuum)
- High resolving power may not be achieved
(due to difficulty preparing the specimen and high energy electrons may destroy specimen) - Complicated staining process needed
- Specimen must be extremely thin
(microtome needed to prep)
What is an eye piece graticule and how do you calibrate it
An eyepiece graticule is a ruler in the eyepiece of a microscope
used to measure specimens.
To calibrate:
- Set the microscope to the desired magnification.
- Place the stage micrometre on the stage and count the amount of divisions for a given length, and divide to give the real length of one division.
- Use this to measure the length of specimens and of the field of view by multiplying the number of divisions by the scale.
Remember to recalibrate for every magnification.
What is cell fractionation and why is it needed
Cell fractionation is a method to separate organelles from cells, allowing them to be studied.
Cells are broken down and the different organelles are separated.
What is homogenation
When cells are broken down with a homogeniser (blender) to release the organelles inside it
What is a homogenate
Fluid produced by homogenisation, containing the organelles form inside a cell
What is ultracentrifugation
The process where fragments in the homogenate are separated by a centrifuge (spun round)
What is the supernatant in cell fractionation
The fluid that is removed and re-spun in the centrifuge during ultracentrifugation, to remove lighter organelles
What is the sediment in cell fractionation
The layer at the bottom of the tube containing the heaviest organelles after homogenisation (each sediment produced will contain a different group of organelles of similar weights)
What is the process of cell fractionation, ultracentrifugation and homogenization
1) Chop up the fresh tissue (eg. liver) and place in an ice cold, isotonic buffer solution.
2) put chopped tissue in a homogenizer (blender) to break open cells
3) Filter the mixture to remove debris
4) spin the mixture in a centrifuge
5) the supernatant (liquid at the top) is removed to be re-spun, and the pellet (sediments at the bottom) is removed, containing the largest organelles, such as the nucleus.
6) This process is repeated, spinning the supernatant at higher and higher speeds, separating into different organelles of different sizes.
Why is an ice cold, isotonic buffer solution used in cell fractionation
Ice cold:
Reduces enzyme activity
Isotonic:
Prevents osmosis
Buffer:
Maintain pH
What is a eukaryotic cell
A cell present in complex living organisms, containing a nucleus and membrane bound organelles
What are the main components of a eukaryotic cell
- Nucleus
- Mitochondria
- Endoplasmic reticulum (SER/RER)
- Golgi apparatus
- Lysosomes
- Ribosomes
- Chloroplast (only plant)
- Cell wall (only plant)
- Vacuoles (only plant)
What is the function of the nucleus
- Stores the DNA
- Site of DNA transcription leading to protein synthesis (production of mRNA)
- Helps cells produce ribosomes (rRNA)
