Details about microscope slide preparation - 

• Specimens can be seen under a light microscope. This would allow some information about cellular material to be witnessed.

• Pre-prepared permanent slides could be viewed. These slides are produced by cutting very thin layers of tissue which are stained and permanently mounted on a glass slide for repeated use.

• Various methods can be used to view different types of specimen. For instance: temporary slide preparations can be produced in the school laboratory as explained in the steps below - 

 

Preparing a slide using a liquid specimen - 

1.  Add a few drops which contain the liquid sample to a clean slide by using a pipette.

2. Lower a coverslip over the specimen. Then, lightly press down to remove air bubbles. Coverslips are effective as they will protect the microscope lens from liquids and help to prevent them from drying out.  - 

 

Preparing a microscope slide through the use of a solid specimen - 

- Use scissors or a scalpel so you can cut a small sample of tissue.

- Next, peel away or cut a very thin layer of cells from the tissue sample. This is because the preparation method always needs to ensure that samples are thin enough in order for light to pass through.

- Later, place the sample onto a slide. A drop of water can be added at this particular point.

- After this, apply iodine stain. In the end, slowly lower a coverslip over the specimen and press down. This will remove any air bubbles.

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Preparing a microscope slide using onion cells diagram - 

- First, we take an onion. Using tweezers, we can remove the epidermal tissue. 

- Then, add a drop of water to a clean slide.

- Keep the epidermal tissue into water that is on the slide.

- Add a drop of iodine to stain the cell. This will ensure that the structures within the cell can be seen clearly. 

- Put a cover slip on the top of the specimen.

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Preparing a slide using human cells - 

- Brush your teeth properly by using a regular toothbrush and toothpaste. This will remove bacteria from the teeth. So they will not distract the view of what can be seen in the cheek cells.

- Take a sterile cotton swab and slowly scrape the inside cheek surface of the mouth for about 5 to 10 seconds.

- Place the cotton swab on the centre of the microscope slide for 2 to 3 seconds.

- Add a drop of methylene blue solution. This is because methylene blue stains negatively charged molecules in the cell (this includes DNA and RNA). Thus, in turn, causes the nucleus and mitochondria to appear darker than their surroundings.

- Place a coverslip on the top. Place the coverslip down at one edge and then slowly lower the other edge until it is flat. This will reduce bubble formation under the coverslip.

- Absorb any excess solution by making a paper towel to touch one side of the coverslip.

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Staining specimens - 

 

 - ​It is important to note that the cytoplasm and other cell structures could be transparent or difficult to differentiate. In saying that, stains allow them to be clearly viewed when it is placed under a light microscope.

- In regards to the type of preparation that is required, the type of stain which is used is dependent on the specimen that is being viewed.

Common microscope stains and uses table

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Iodine: stains starch blue-black, and colours nuclei and plant cell walls pale yellow

Crystal violet: stains cell walls purple

Methylene blue: stains animal cell nuclei dark blue

Congo red: is not absorbed by cells and stains the background red, so it provides contrast with any cells which are present.

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Drawing Cells

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To record observations that are witnessed under a microscope, a labelled biological drawing is often made. Biological drawings consists of line drawings which show specific features, elements and characteristics that have been noticed when the specimen was viewed.

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There are different rules and conventions that must be followed when making a biological drawing. This includes:

1. The drawing must have an appropriate title.

2. A scale bar could be used

• A sharp pencil is needed

• Drawings should be on plain and clear white paper

• Lines should be clear and there should be single lines without sketching

• No shading needs to be done

• The drawing should take up as much space on the page as needed

• Well-defined structures should be drawn

• Only visible structures should be drawn, and the drawing should look like the specimen

• Drawings should be made with proper proportions

• Structures should be clearly labelled with label lines so they:

  • Do not cross

  • Do not have arrowheads

  • Connect directly to the part of the drawing being labelled

  • Are on one side of the drawing

  • Are drawn with a ruler

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• Drawings of cells are usually made when visualising cells at a higher magnification power. On the other hand, plan drawings are made of tissues which are viewed under lower magnifications. Also - individual cells are never drawn in a plan diagram. 

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Understanding Magnification

 

Magnification in microscopy is the process of magnifying the visual appearance of an object as it is seen through a microscope. It is significant for studying minute biological structures which cannot be seen to the naked eye.

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Details about calculating magnification - 

1. ​Identifying Magnification Factor: this refers to the ratio of the image size as it is observed through the microscope in comparison to the actual size of the object which is being observed.

2. Formula for Magnification is: Magnification = Image Size / Actual Size.

3. Utilising Microscope Scale: most microscopes have a built-in scale or a way to calibrate magnification, which is important in terms of determining the magnification factor accurately.

 

How is magnification important in Biological Studies?

1. Allows accurate observations: high magnification encourages and enables in-depth study of cellular structures. This is essential for understanding biological processes, as well as recognising a variety of microscopic organisms.

2. Determining cell size: knowing cell size is important in many areas of biology. For instance: from genetics to pathology.

3.  Quantitative Analysis: is important in studies which requite exact measurements of cell dimensions or comparison of cell sizes.

4. Comparative Studies: allows researchers to accurately compare cells from various samples or conditions.

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Measuring actual size from images:

Accurate measurement of the original size of cells and structures which are observed under a microscope is essential for scientific accuracy and recognising biological functions.

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Steps for Measurement - 

1. Getting image Size: This involves measuring the size of the image as it appears through the microscope, often using the microscope's scale.

2. Apply Formula: To find the actual size, use the formula Actual Size = Image Size / Magnification.

3. Consistency in Units: It's critical to ensure that all measurements are in the same units (mm, µm, or nm) to maintain accuracy.

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Types of microscopes

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Light Microscope:

• These use light to form an image.

• Using light reduces light microscopes resolution because it is impossible to distinguish between two objects (resolve) that are closer than half the wavelength of light.

The wavelength of visible light is between 500-650 nanometres (nm), so a light microscope cannot be used to distinguish between objects less than half of this value

• Light microscopes have a maximum resolution of around 0.2 micrometres (µm) or 200 nm (Nanometers) and a maximum useful magnification of about ×1500.

 

• They can be used to observe eukaryotic cells structure, their larger organelles such as nuclei as well as sometimes mitochondria and chloroplasts.

Smaller organelles such as ribosomes, the endoplasmic reticulum or lysosomes cannot be viewed.

 

Electron microscopes:

• These utilize electrons to form an image.

• Using electrons provides a clearer image by increasing  the resolution of electron microscope. The reason for this is a beam of electrons has a much smaller wavelength than light, so an electron microscope can resolve two close together objects.

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• Electron microscopes have a maximum resolution of around 0.0002 µm or 0.2 nm (1000 times greater than light  microscopes resolution) and the maximum useful magnification of electron microscopes is about ×1,500,000

  • This means electron microscopes can be used to observe small organelles that light microscopes cannot such as lysosomes, centrioles and ribosomes 

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• There are two types of electron microscopes:

  • Transmission electron microscopes (TEMs)

  • Scanning electron microscopes (SEMs)

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Transmission electron microscopes (TEMs):

• TEMs use electromagnets to focus a beam of electrons which are transmitted through the specimen.

• Denser parts of the specimen absorb more electrons meaning they appear darker on the final image produced. This effect forms clear contrast based on density between different parts of the object being observed.

• Advantages of TEMs:

  • They give high-resolution images (which means: more detail)

  • This allows the internal structures within cells (or even within organelles) to be seen.

• Disadvantages of TEMs:

  • They can only be used with very thin specimens or thin sections of the object being observed.

  • They cannot be used to observe live specimens.

    • As there is a vacuum inside a TEM, all the water must be removed from the specimen and so living cells cannot be observed, meaning that specimens must be dead. Optical microscopes can be used to observe live specimens.

  • The lengthy treatment required to prepare specimens means that artefacts can be introduced.

    • Artefacts look like real structures but are actually the results of preserving and staining.

  • They do not produce a colour image.

    • Unlike optical microscopes that produce a colour image.

 

Scanning electron microscopes (SEMs):

• SEMs scan a beam of electrons across the specimen.

• This beam bounces off the surface of the specimen, and the electrons are detected, forming an image.

  • This means SEMs can produce three-dimensional images that show the surface of specimens.

 

• Advantages of SEMs:

  • They can be used on thick or 3-D specimens.

  • They allow the external, 3-D structure of specimens to be observed.

• Disadvantages of SEMs:

  • They give lower resolution images (less detail) than TEMs.

  • They cannot be used to observe live specimens.

  • They do not produce a colour image.

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Comparison of the electron microscope and light microscope:

• Light microscopes are used for specimens above 200 nm.

  • Light microscopes shine light through the specimen. This light is then passed through an objective lens (which can be changed) and an eyepiece lens (x10) which magnifies the specimen to give an image that can be seen by the naked eye.

  • The specimens can be living (and therefore can be moving), or dead.

  • Light microscopes are useful for looking at whole cells, small plant and animal organisms, tissues within organs such as in leaves or skin.

• Electron microscopes, both scanning and transmission, are used for specimens above 0.5 nm.

  • Electron microscopes fire a beam of electrons at the specimen either a broad static beam (transmission) or a small beam that moves across the specimen (scanning).

  • Due to the higher frequency of electron waves (a much shorter wavelength) compared to visible light, the magnification and resolution of an electron microscope is much higher than a light microscope.

  • Electron microscopes are useful for looking at organelles, viruses and DNA as well as looking at whole cells in more detail.

  • Electron microscopy requires the specimen to be dead. However, this can provide a snapshot in time of what is occurring in a cell. For instance: DNA can be seen replicating and chromosome position within the stages of mitosis are visible.

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       Cells as the basic units of living organisms

Students should be able to:

1) Recognise organelles and other cell structures found in eukaryotic cells and outline their structures and functions

2) describe and interpret photomicrographs, electron micrographs and drawings of typical plant and animal cells 3) compare the structure of typical plant and animal cells

4) state that cells use ATP from respiration for energy-requiring processes

5) outline key structural features of a prokaryotic cell as found in a typical bacterium

6) compare the structure of a prokaryotic cell as found in a typical bacterium with the structures of typical eukaryotic cells in plants and animals

7) state that all viruses are non-cellular structures with a nucleic acid core (either DNA or RNA) and a capsid made of protein, and that some viruses have an outer envelope made of phospholipids

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Eukaryotic cells:

Eukaryotic cells are complex cells with many specialised areas called organelles, and a clearly defined nucleus separated from the rest of the cytoplasm by 1 or more membranes (membrane bound organelles).

 

• ribosomes- these are small granules made of protein and rRNA, synthesizing proteins through translating MRNA into polypeptide chains of amino acids, forming a protein. The rRNA catalyses the peptidyl transferase reaction forming peptide bonds between these amino acids. Ribosomes are 80S in size in the cytoplasm and 70S in chloroplasts and mitochondria.

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• Nuclear envelope- A structure comprising of a double membrane with fluid in between them called nucleoplasm. It allows substances to pass from inside the nucleus to the cytoplasm through pores and separates the contents of the nucleus from the cytoplasm due to incompatible reactions taking place there and different PH levels. The nuclear envelope also provides the structural framework of the nucleus

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• Plasma membrane- This is made up of a phospholipid bilayer in a fluid mosaic structure. This structure is formed as hydrophobic fatty acid tails repel water while the hydrophilic phosphate heads are attracted to water so configure in a way which limits water contact with the tails. It has glycolipids and glycoproteins on the surface, with cholesterol imbedded between the fatty acids. 

          Its function is regulating the movement of substances into and out of the cells

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• Lysosomes- These specialised vesicles are small round membrane bound sacs. They contain hydrolytic enzymes surrounded by membrane and get rid of worn or senile cells and organelles which no longer have benefits

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• Golgi apparatus- A series/stack of fluid filled sacs. They are membrane bound and surrounded by small hollow vesicles called golgi vesicles. This organelle modifies proteins and packs them into vesicles to be transported either to a different part of the cell or secreted

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• Vacuole- The organelle is found permanently in plants, but also in animal cells. Its a single membrane sac filled with liquid containing salts, sugars, water and amino acids. Its used for maintaining cell shape and also for storage of these molecules. The membrane around the vacuole is called tonoplast 

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• Nucleus- The nucleus carries genetic information and controls the shape as well as structure of the cell. DNA replication and transcription also occurs inside. It contains a transparent gelatinous fluid called nucleoplasm, with minute tangled threads coiled around themselves called chromatin. 

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• Rough endoplasmic reticulum- This consists of sheets of membranes linked to the nucleus, surrounded by flattened sacs called cisternae. They have ribosomes attached to their surface and are used for the synthesis and transport of proteins in the cell. In the cisternae they're folded into their correct tertiary structures. 

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• Nucleolus- This organelle Has fibrillar centre where ribosomal proteins form. Its a site of ribosome production

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• Centrioles and microtubules- Centrioles are cylindrical structures made up of protein tubes called microtubules in triplets. They organise mitotic spindle fibres, pulling apart the chromosomes in mitosis and meiosis.

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• Smooth endoplasmic reticulum- It has no ribosomes and used in the synthesis and storage of lipids as well as carbohydrates. The smooth ER also detoxifies harmful chemicals like ethanol and drugs

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• Cell wall- made of Cellulose microfibrils in plants, chitin in fungi and peptidoglycan in Bacteria. The cell wall maintains cell structure and provides protection due to its high tensile strength 

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• Cilia- These are hair like organelles extending from the cell surface which waft substance. They also have the role of sensing chemicals around the cell. Its made up of 9 pairs of microtubules arranged in a circle with another pair in th centre. It requires ATP for movement

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• Mitochondria- This is an oval or rod shaped double membrane organelle. The inner membrane is folded to form cristae. It contains a fluid centre called the matrix. The mitochondria is a site of aerobic respiration and ATP production

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• Chloroplast- This organelle absorbs sunlight needed for photosynthesis. It is surrounded by a double membrane which has membrane bound flattened disks called thylakoids inside. It has chlorophyll so is where light dependent reactions take place. These thylakoids are stacked onto one another which is a structure called a granum, allowing for more efficient light absorption. These granums are joined together by lamellae, allowing chemicals to pass  between them. Mitochondria have a fluid material called stroma containing enzymes for light dependent enzymes. They have a loop of DNA and ribosomes, which synthesise and make proteins needed for photosynthesis. 

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• Microvili- Microvilli are extensions of villi and are made of microfilaments, specifically actin filaments.The main function of microvilli are to increase surface (like in the case of the small intestine)

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• Plasmodesmata- These are membrane-bound tunnels, 40nm in diameter, that connect the cytosol of neighboring plant cells, which facilitates the movement of molecules and substances between cells. They have three main layers, the plasma membrane, the cytoplasmic sleeve, and the desmotubule 

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Electron micrographs:

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An electron micrograph is an image of a specimen taken using an electron microscope.

>Micrographs provide you with an indication of what organelles and components are in the cell. This allows you to analyse their structures but also compare these with micrographs of other cells. 

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To be able to gage certain structures, you must focus on:

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- Colour —> The thicker or denser the structure is, the more darker it will look as electrons can be absorbed more easily, giving you an idea of what it could be size wise

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- shape—> components of the cell have distinct physical features which are different from others in order to be most suited to carry out its function so can be used to distinguish between organelles

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- Location—> most organelles are found in a similar location so you can assume what a certain structure is based on its position in relation to others

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- Size in relation to other organelles—> you can use this as an indicator as the nucleus will always be the largest organelle, Mitochondria are the next largest, with lysosomes being much smaller

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- A photomicrograph is a micrograph prepared using an optical microscope and can be interpreted in a similar way to electron micrographs 

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 Cell drawings are constructed using thin pencil lines, keeping structures unshaded, and have labels describing the components of the drawing. They must be drawn in proportion to the real cell size, which can be measured using an eyepiece graticule and stage micrometer.

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Compare the structure of typical plant and animal cells:

• Centrosomes and lysosomes are found in animal cells, but do not exist within plant cells. The lysosomes break down worn and senile components in animal cells, while in plant cells the same function takes place in vacuoles.

• Plant cells have a cell wall, chloroplasts and a central permanent vacuole, which are not found within animal cells

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Cells use ATP from respiration for energy-requiring processes:

In a molecule of ATP, energy is stored in the bonds joining the 3 phosphate groups, so these bonds must be broken via hydrolysis in order to release energy for cellular processes.

 

The Hydrolysis of ATP forms ADP (adenosine diphosphate) and an inorganic phosphate group (Pi), catalysed by the enzyme ATP hydrolase.

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Examples of Energy requiring cellular processes are muscle contraction, proteins and lipids synthesis, active transport and cell division

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Prokaryotic cells:

A prokaryotic cell is one that lacks a distinct nucleus and other organelles due to the absence of internal membranes. 

Prokaryotic organisms are unicellular, generally 1-5 micrometers in diameter and have peptidoglycan cell walls

 

Structural features of a prokaryotic cell as found in a typical bacterium are 

• Flagella— These are long structures in the form of a whip, which assist with cell locomotion

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• Cell Wall— the outermost layer of the cell which provides protection and maintains structure

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• Cytoplasm— made up of the liquid cytosol composed of enzymes, salts amd organelles. Its a site of chemical reactions

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• Ribosomes—  involved in protein synthesis and 70s in size

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• Nucleoid Region— It’s the region in the cytoplasm where the genetic material is present.

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• Slime Capsule— a layer on the outside of the cell wall which helps in moisture retention, protection from chemical changes and is adhesive, allowing for the attachment of cells to nutrients and surfaces.

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• Cell membranes— controls the exit and entry of substances into and out of the cell

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• Plasmids— small, circular rings of double-stranded DNA which are non-chromosomal found in Bacteria and other microscopic organisms. They can self-replicate

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• Pili— These are hair-like outgrowths that attach to the surface of other bacterial cells.

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Viruses:

• All viruses are non-cellular structures

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• They have a nucleic acid core (either DNA or RNA)— It contains the virus's genetic material, holding the instructions for virus replication and the synthesis of viral proteins

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• Have a capsid made of protein— It is composed of protein subunits called capsomeres and used to protect the nucleic acid. It also plays a role in infecting host cells.

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• Some viruses have an outer envelope made of phospholipids— This is composed of portions of the host cell membranes but contains some viral glycoproteins.