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Specimen Preparation and Staining

Answer: A

  1. Cytoplasm *
  2. Chloroplast *
  3. Nuclear membrane
  4. Nucleolus/ nucleus *
  5. Endoplasmic Reticulum
  6. Ribosome
  7. Golgi bodies
  8. Vacuoles *
  9. Cell 1: Lysosome; Cell 2: Golgi vesicles
  10. Plasma membrane or cell membrane *
  11. Mitochondria
  12. Cell wall *

Answer B

Light Microscope

Electron Microscope

Cell components seen

Cell membrane, Nucleus, Nuclear membrane, Cytoplasm, Vacuoles, Cell wall, Chloroplast

Cell membrane, Nucleus, Nuclear membrane, Cytoplasm, Vacuoles, Cell wall, Chloroplast, Nucleolus, Mitochondria, Ribosomes, Endoplasmic reticulum, Lysosomes, Centrioles, Golgi body

Specimen preparation

Living organisms can be directly seen under light microscope. However, fixation and staining is used in order to increase the visibility and to accentuate the morphological features.

Fixation is a process by which the external and internal structures of the cells are fixed at a particular position. There are two types of fixation (i) heat fixation (ii) chemical fixation.

Staining is done via dyes. The main types of dyes used in staining are (i) basic dyes and (ii) acid dyes. Basic dyes include methylene blue, crystal violet, saffranin and are known as positive dyes (basic dyes) because they have positively charged groups (pentavalent nitrogen). Basic dyes bind to the negatively charged molecules of the cell. Acidic dyes include eosin, acid fuschin, they has negatively charged carboxylic group or hydroxyl group. They bind to positively charged components of the cell.

There are different types of staining

(i) Simple staining

(ii) Differential staining (Grams staining)

(iii) Acid Fast staining

(iv) Staining specific structures (negative staining)

An ideal specimen of TEM must be around 20 to 100 nm in thickness and this is about 1/50 to 1/10 of the diameter of an average bacterial cell. The importance of small specimen is, it is able to maintain its structure when bombarded with electrons under high vacuum. In order to cut the specimen into thin slice, a plastic is used as support. After the process of fixation, gultaraldehyde or osmium tetroxide is used to stabilize the structure of the cell. The specimen is then dehydrated with the help of organic solvents (ethonal or acetone). Complete dehydration is necessary, as most plastics used for embedding are not soluble in water. After this, the specimen is soaked in epoxy plastic that is unpolymerised in nature. The soaking is done until it is permeated and then the plastic is hardened to from a block of solid. Thin sections are cut out from this block with the help of diamond knife or glass via the use of a special instrument, known as ultramicrotome. The specimens are prepared for observation via mixing them with the solution of heavy metals like uranyl acetate or lead citrate. It increases the contrast of the material.

Illumination

Normal light microscope is known as bright-field microscope. It forms dark image against a brighter background. The source of illumination is light.

Electrons used in the electron microscope behave like radiation. Electrons illuminate the specimen and the resolution of microscope is enormously increased. The wavelength of the radiation is 0.005nm. This is 100,000 times shorter than the visible light. Transmission Electron Microscope (TEM) has 1000 times resolution higher than the light microscope.

Image formation

The objective lens of the light microscope forms an enlarged real image within the microscope. The eyepiece lens further magnifies the primary image. When an observer looks into a microscope, the enlarged specimen image, known as the virtual image appears to lie beyond the stage. It generally lies 25 cm away.

Modern TEM is complex and sophisticated. A heated filament made of tungsten present inside the electron gun generates a beam of electron that is focused on the specimen via the condenser. The electrons fail to pass through a glass lens and hence a doughnut-shaped electromagnets known as magnetic lens are used to focus the beam. The column containing the pair of lenses and specimen are placed under vacuum in order to obtain a clear image. The importance of creation of vacuum space lies in the fact that, when electrons collide with the air particles they get deflected. The specimen scatters the deflected electrons and the refracted beam is then focused via magnetic lenses to form a large visible image of the specimen over projected the fluorescent screen.

Magnification

The total magnification is calculated via multiplying the objective and the eyepiece magnification.

The denser portion of the specimen scatters more electrons and thus appears darken in the image as fewer electrons strike that area of the screen. On contrary, electron transparent regions are brighter.

Resolution

0.2 micro meter

0.5 nano meter

Resolution limit of human eye is 0.2 nano meter

 (Source: Reimer 2013; Wiley, Sherwood and Woolverton 2013)

 

Figure: difference between light and electron microscope

(Source: Wiley, Sherwood and Woolverton 2013)

 

Figure: The path of light of light microscope

(Source: Wiley, Sherwood and Woolverton 2013)

  1. Cell membrane
  2. Golgi body
  3. Mitochondria
  4. Nuclear membrane
  5. Nucleolus
  6. Nucleoplasm

Name of the Organelle

Size

Function

Cell membrane

7.5 to 10 nm

The main function of the cell membrane is to form a barrier from the eternal environment, protecting the internal organelles of the cell

Golgi body

20 to 200 nm

Golgi body assist in the process of post translation modification and protein folding

Mitochondria

10 micro meter

Mitochondria is the power house of the cell. It is the site of synthesis of Adenosine Triphosphate (ATP)

Nuclear membrane

40 nm

Nuclear membrane is the external layer that surrounds the nucleus. It gives the nucleus a concrete shape and protects the nuclear organelle from the exterior (the organelles of the cytoplasm)

Nucleolus

Varies with the nucleus generally 1 to 10 micro meter is diameter

Nucleolus is the site of formation of ribosomal RNA

Nucleoplasm

Varies with the size of the nucleus

Nucleoplasm is the cytoplasm of the nucleus. It contains several nuclear enzymes and other nuclear organelles

 (Source: Kierszenbaum and Tres 2015)

 

Answer a

A: Phospholipid

A1: Phospholipid polar head (hydrophilic)

A2: Fatty acid tails (non polar/ hydrophobic)

B: Carbohydrate Chain

C: Glycoprotein

  1. Glycolipid

E: Sterols

F: Inner membrane peripheral protein

G: Integral membrane protein (channel protein)

H-I: Phospholipid bilayer

Answer b

The lipid component that anchors on the exoplasmic surface of the plasma membrane is majorly Glycosylphosphatidylionositol (GPI). The phosphadityl inositol of this anchor contains a fatty acyl chain that is extended up to the bilayer. The phosphoethanolamine in the anchor links in to the protein (Opella 2013).  

Answer c

The phospholipid bilayer is semi-permeable. This selective permeability of biological membranes towards the small molecules allows cell to control and retain its internal composition. The molecules that can pass freely through the phospholipid bilayer are small uncharged molecules. O2 and CO2 are small non polar molecules and are soluble in the phospholipid bilayer and thus can readily cross the cell member. H20, an example of small uncharged polar molecules is capable of diffusing through the membrane. However, large uncharged polar molecules like glucose cannot freely pass through the member and they need support of carrier molecules along the expenditure of energy (Fragneto, Charitat and Daillant 2012).

 

Figure: Selective Permeability of Phospholipid bilayer

(Source: Fragneto, Charitat and Daillant 2012).

Answer e

1) Glucose is transported via uniporter. GLUT1, is glucose uniporter. It is found in the plasma membrane of the erythrocytes. Via uniporter, the transportation takes place down the concentration gradient, without the expenditure of energy (Deng et al. 2014). 

 

Figure: Transport of glucose via uniporter

(Source: Deng et al. 2014)

2) Water falls under the category of the small-uncharged polar molecules and is readily diffusible across the phospholipid bilayer (Stein 2012).

3) Alcohol like ethanol also falls under the category of small-uncharged polar molecules like that of water and is readily diffusible across the phospholipid bilayer (Stein 2012).

4) The size of the bacteria are many times larger than that of the viruses and thus they are too large to enter inside the cell via receptor mediated endocytosis. Bacteria in the majority of the cases enter the host cells via the process of phagocytosis. Phagocytosis of bacteria is a normal function of macrophages. Macrophages patrol the infected tissues of the body, ingest the invading bacteria and then simultaneously destroy the unwanted microbes (Allocati et al. 2015). 

Illumination and Electron Scattering

Answer A (i) 

Fick’s Law provide the simple description of diffusion.

Fick’s First Law of Diffusion

The molar flux arising out of diffusion is proportional to the concentration gradient.

Fick’s Second Law of Diffusion

The rate of change of the concentration is proportional to the second derivative of the concentration present within the same space. It is represented via a linear equation (Donev, Fai and Vanden-Eijnden 2014).

Answer A (ii)

The curve B follows the line of linear equation that is  y = mx + c. So the as the concentration of glucose increases, the molar flux also increases as demonstrated by the first law of Ficks and hence leading to the increase in the uptake of glucose inside the cell (Levkovitz et al. 2013).

Answer B (i)

 

Source: Created by author

The graph represents a rectangular hyperbola as expressed by the Michaelis-Menten equation. At the concentration less than 2mmoldm-3, the uptake of glucose inside the cell experience a shows a steep increase. However, as the concentration of the glucose rises, the rate of uptake of the glucose gradually slows down, going towards a saturation point. This is due to the fact that as the concentration of glucose increase, the glucose uptake receptors located at the cell membrane of the exterior of the cell becomes saturated with the bounded glucose leading to the gradual slow down of the glucose uptake. The reason why the graph reaches to a horizontal plateau and increases further is due to the increase in the concentration of glucose (Levkovitz et al. 2013).

Answer a

Feature

Mitosis

Meiosis

(i) Involves two successive nuclear divisions.

X

ü 

(ii) Chromosomes replicate before they can be seen in a stained cell.

X

X

(iii) Involves the formation of chiasmata.

X

ü 

(iv) Leads to random assortment of chromosomes.

X

ü 

(V) Involves the separation of sister chromatids.

ü 

ü 

(vi) Occurs during gamete formation in humans.

X

ü 

(vii) Splitting of centromere is followed by anaphase.

ü 

ü 

(viii) Daughter nuclei have identical genetic composition.

ü 

X

(ix) Occurs during vegetative growth.

ü 

X

(x) A mutation may occur during the process.

X

ü 

Answer b

Mitosis

Mitosis is the division of the nucleus, which occurs after the S-phase of the cell cycle (interphase) that is after the replication of DNA. During mitosis the chromosomes that have already duplicated, condense and then gets segregated into two different cells via the process of cytokinesis. The result of mitosis is two genetically identical daughter nuclei. Mitosis only occurs in eukaryotic cells. The basis of mitosis is, one cell divides once to from two identical cells. The major purpose of mitosis is growth. Different stages of mitosis include interphase, prophase, metaphase, anaphase and telophase (Schlegel et al. 2013).

Meiosis

Meiosis is a process where a single cell under two stages of cell divission in order to generate four cells containing half the original amount of the genetic information that was contained inside the mother cell. Meiosis generally ocuurs inside the sex cells that is sperm in makes and eggs in females(Schlegel et al. 2013).

Thus in meiosis, one cell divides twice to produce two daughter cells and these four daughter cells have only half the number of chromosomal content than that of the parent cells and hence they are termed as haploid. Meiosis has nine stages

Meiosis 1

Interphase

Prophase 1

Metaphase 1

Anaphase 1

Telophase 1 and cytokinesis

Meiosis II

Prophase II

Metaphase II

Anaphase II

Telophase II

Cytokinesis 

Meiosis is popularly known as reductional division (Schlegel et al. 2013).

References 

Allocati, N., Masulli, M., Di Ilio, C. and De Laurenzi, V., 2015. Die for the community: an overview of programmed cell death in bacteria. Cell death & disease, 6(1), p.e1609.

Deng, D., Xu, C., Sun, P., Wu, J., Yan, C., Hu, M. and Yan, N., 2014. Crystal structure of the human glucose transporter GLUT1. Nature, 510(7503), pp.121-125.

Donev, A., Fai, T.G. and Vanden-Eijnden, E., 2014. A reversible mesoscopic model of diffusion in liquids: from giant fluctuations to Fick’s law. Journal of Statistical Mechanics: Theory and Experiment, 2014(4), p.P04004.

Fragneto, G., Charitat, T. and Daillant, J., 2012. Floating lipid bilayers: models for physics and biology. European Biophysics Journal, 41(10), pp.863-874.

Kierszenbaum, A.L. and Tres, L., 2015. Histology and Cell Biology: An Introduction to Pathology ( E-Book). Elsevier Health Sciences.

Levkovitz, R., Zaretsky, U., Jaffa, A.J., Hod, M. and Elad, D., 2013. In vitro simulation of placental transport: part II. Glucose transfer across the placental barrier model. Placenta, 34(8), pp.708-715.

Opella, S.J., 2013. Structure determination of membrane proteins in their native phospholipid bilayer environment by rotationally aligned solid-state NMR spectroscopy. Accounts of chemical research, 46(9), pp.2145-2153.

Reimer, L., 2013. Transmission electron microscopy: physics of image formation and microanalysis (Vol. 36). Springer.

Schlegel, R.A., Halleck, M.S. and Rao, P.N. eds., 2013. Molecular regulation of nuclear events in mitosis and meiosis. Elsevier.

Stein, W., 2012. Transport and diffusion across cell membranes. Elsevier.

Wiley, J.M., Sherwood, L.M. and Woolverton, C.J., 2013. Prescott’s microbiology (vol. 6). The McGraw−Hill Companies

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