1Tools used in Cytology

📚
HISTOLOGICAL SLIDE PREPARATION
▢ Steps:
  • •
    Fixing
  • •
    Dehydration
  • •
    Embedding
  • •
    Sectioning
  • •
    Staining
  • •
    Mounting
▢ Flow: Fixing → Dehydration → Embedding → Sectioning → Staining → Mounting
▢ Fixing: Preservation of tissue structure
▢ Dehydration: Removal of water from tissue
▢ Embedding: Tissue support medium preparation for cutting
▢ Sectioning:
❖ Meaning: Thin section preparation
❖ Instrument: Microtome
❖ Microtome: Instrument used to prepare thin sections for histology
▢ Staining:

Table 1: Histological Stains and Uses

Stain / Test
Specific Use
Important Point
Methylene blue
Pectin substances
Middle lamella staining
Ruthenium red
Pectin substances
Middle lamella staining
Zinc-chlor-iodide
Cellulose
Cell wall specific stain
Safranin
Lignified wall
Dead thick lignified tissue; xylem
Iodine test
Starch
Endodermis / starch sheath gives iodine test
Crystal violet
Bacteria
Common bacterial stain
Haematoxylin
Nucleus + cell wall / cellulose
Nuclear stain
Acetocarmine
Chromosomes
Chromosome staining
Feulgen test
DNA / chromatin / chromosome
Specific DNA test
Basic fuchsin
DNA in Feulgen reaction
Deep red biological stain
Vital stains
Living materials
Non-toxic stains
❖ Vital Stains:
  • •
    Used for living cells / living materials
  • •
    Non-toxic
  • •
    Examples: methylene blue, Janus green
▢ Mounting: Permanent / temporary slide preparation after staining
📚
MICROSCOPE
▢ History:

Table 1: Microscopy History

Person
Contribution
Leeuwenhoek
Father of microscopy
Leeuwenhoek
Invented simple microscope
Janssen
Invented compound microscope
Knoll and Ruska
Developed electron microscope in Germany
▢ Types Based on Light Source:

Table 1: Microscope Types

Type
Light Source
Main Use
Light microscope
Visible light
Histological sections by transillumination
Electron microscope
Beam of electrons
Cell organelle ultrastructure
▢ Light Microscope:
❖ About:
  • •
    Commonly used for histological sections
  • •
    Uses transillumination
  • •
    Transillumination = light transmission through sample
❖ Special Types:

Table 1: Special Light Microscopes

Microscope
Use / Feature
Examples / Notes
Phase contrast microscope
Living cells + tissues without fixing and staining
Living chromosomes; living cell culture
Dark-field microscope
Transparent objects / objects not reflecting light
Bright object against dark background
Interference microscope
Qualitative study of cell components + cell constituents
Cell component analysis
Polarizing microscope
Spindle fibre / centriole study
Uses plane-polarized light
Fluorescent microscope
Fluorescence / phosphorescence-based study
Uses high-wavelength UV light; membrane qualities of phospholipid bilayer
▢ Electron Microscope:
❖ Key Points:
  • •
    Developed by Knoll and Ruska in Germany
  • •
    Light source = beam of electrons
  • •
    Best for ultrastructure of cell organelles
  • •
    Principle = de Broglie waves / matter waves
  • •
    Better than light microscope due to high resolving power + high magnification
  • •
    Uses high-energy / short-wavelength electrons
  • •
    Revealed ribosomes, centrioles, microbodies, etc.
  • •
    Ultra-thin specimens impregnated with heavy metals
  • •
    Scanning electron microscope → 3D image
❖ Lenses:
  • •
    Ocular / eyepiece
  • •
    Objective
  • •
    Condenser
▢ Comparison:

Table 1: Eye vs Light Microscope vs Electron Microscope

Feature
Eye
Light Microscope
Electron Microscope
Magnification
1
1,000–2,000
4,00,000–6,00,000
Resolving power
0.1 mm
0.2 μm
0.1 nm
Resolution depends on
-
Diameter of objective lens
Electron wavelength / energy
Magnification depends on
-
Eyepiece + ocular lens power
Electromagnetic lens system
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BODY EXAMINATION TECHNIQUES

Table 1: Imaging Techniques

Technique
Full Form
Key Point
CT scan
Computed Tomography
Uses X-rays; 360-degree sectional body views
X-rays
X-radiation imaging
2-D structure
MRI
Magnetic Resonance Imaging
Body structure imaging; sectional views
Ultrasound
Ultrasonography
Sound-wave based body examination
📚
SEPARATION AND ANALYTICAL TECHNIQUES

Table 1: Important Techniques and Uses

Technique
Use
High-yield Point
PAGE
Separation of proteins
Polyacrylamide Gel Electrophoresis
X-ray diffraction / crystallography
3D positions of atoms in molecules
Protein + nucleic acid structure
Autoradiography
Molecular synthesis + metabolic pathway tracing
Carbon assimilation pathway study
Radioimmunoassay
Estimation of minimum hormone + drug amount
Highly sensitive assay
Cell fractionation / centrifugation
Separation of sub-cellular components
Organelle separation
Differential centrifugation
Mitochondria separation from cell
Subcellular fractionation
Southern blotting
DNA fragment separation / detection
DNA
Northern blotting
RNA fragment separation / detection
RNA
Western blotting
Protein detection
Confirmatory / diagnostic test for HIV
ELISA
Screening test for HIV
Enzyme Linked Immunosorbent Assay
▢ X-ray Diffraction:
  • •
    Determines 3D atomic positions
  • •
    Used for proteins + nucleic acids
  • •
    First protein structure determined by X-ray diffraction = myoglobin
▢ Autoradiography:
  • •
    Used to study synthesis of molecules
  • •
    Used to trace metabolic pathways
  • •
    Used for pathway of carbon assimilation
  • •
    Hershey-Chase experiment: \(^{32}P\) + \(^{35}S\) used
  • •
    Meselson-Stahl experiment: heavy isotope \(^{15}N\) used for semiconservative DNA replication
▢ Blotting Recall:

Table 1: Blotting Techniques

Technique
Target
Southern blotting
DNA
Northern blotting
RNA
Western blotting
Protein
📚
SERENDIPITY
▢ Definition: Chance, unexpected, intuitive discovery
▢ Examples:
  • •
    Archimedes’ principle
  • •
    Discovery of penicillin by Fleming
  • •
    Newton’s law of gravitation
  • •
    Synthesis of urea by Wohler
📚
HIGH YIELD RECALL
▢ Must Remember:
  • •
    Slide preparation flow: Fixing → Dehydration → Embedding → Sectioning → Staining → Mounting
  • •
    Microtome → thin histological sections
  • •
    Methylene blue + Ruthenium red → pectin / middle lamella
  • •
    Zinc-chlor-iodide → cellulose
  • •
    Safranin → lignified wall / xylem
  • •
    Iodine test → starch
  • •
    Crystal violet → bacteria
  • •
    Haematoxylin → nucleus
  • •
    Acetocarmine → chromosome
  • •
    Feulgen test → DNA
  • •
    Feulgen stain → basic fuchsin
  • •
    Vital stains → living cells; methylene blue + Janus green
  • •
    Leeuwenhoek → father of microscopy + simple microscope
  • •
    Janssen → compound microscope
  • •
    Knoll and Ruska → electron microscope
  • •
    Phase contrast microscope → living cells without staining
  • •
    Dark-field microscope → transparent objects
  • •
    Polarizing microscope → spindle fibre / centriole
  • •
    Fluorescent microscope → phospholipid bilayer membrane quality
  • •
    Electron microscope → ultrastructure + high resolution
  • •
    Scanning electron microscope → 3D image
  • •
    Light microscope resolving power → 0.2 μm
  • •
    Electron microscope resolving power → 0.1 nm
  • •
    PAGE → protein separation
  • •
    X-ray diffraction → 3D molecular structure
  • •
    First protein by X-ray diffraction → myoglobin
  • •
    Autoradiography → metabolic pathway tracing
  • •
    Radioimmunoassay → minute hormone / drug estimation
  • •
    Differential centrifugation → mitochondria separation
  • •
    Southern → DNA
  • •
    Northern → RNA
  • •
    Western → HIV confirmatory test
  • •
    ELISA → HIV screening test
  • •
    Serendipity → chance unexpected discovery
Q1.
In an electron microscope, higher magnification is due to the use of
Q2.
Which of the following is related to genetic engineering?
Q3.
A student wants to study metaphasic behaviour chromosomes. The technique most suitable is
Q4.
Magnification of a compound microscope does not depend upon
Q5.
Dark field microscopy is used to detect bacteria. It makes use of
Q6.
Pure fractions of cellular components can be obtained by
Q7.
Biomembrane appears to be trilaminar or tripartite under
Q8.
To study the physiological function of a cell organelle, the method used is
Q9.
Feulgen reaction, a technique developed by Feulgen and Rossenbeck to study or to stain (or a Feulgen reaction is a specific test for establishing the presence of
Q10.
Which microscope would you require if asked to examine a culture of unstained living and dividing cells?
Q11.
The technique used for estimation of minor amounts of hormones and drugs is called
Q12.
Vital stains are employed in the study of
Q13.
Dye used in Feulgen's test for detection of DNA is
Q14.
If the dicot stem is stained for starch. The most intense colouration would develop in
Q15.
The process through which the amount of DNA, RNA and protein can be known at a time is called
Q16.
Dye which cannot stain chromatin is
Q17.
Electron microscope has revealed the occurrence of
Q18.
A living cell can be studied by means of
Q19.
Intact chloroplast of green cells can be isolated with the help of
Q20.
To determine the ultra structure of a cell organelle, the most likely method to be used would be
Q21.
Organelles can be separated from cell homogenate through
Q22.
The most significant drawback in electron microscope is that
Q23.
Which rays are used for making image for CT scan?
Q24.
Iodine solution is used to test the presence of
Q25.
The technique used for separating DNA fragments is
Q26.
Fluidity of bio-membranes can be shown by
Q27.
DNA synthesis can be measured by estimating incorporation of radio-labelled
Q28.
Binding of specific protein on regulatory DNA sequences can be best studied by means of
Q29.
Angstrom (A) is equal to
Q30.
The unit for measuring size of the cell is
Q31.
The technique of obtaining large number of plantlets by tissue culture method is called
Q32.
The maximum magnification produced by light microscope is about
Q33.
Cell organelles can be physically separated of each other by
Q34.
The first protein whose structure was determined by X-ray diffraction
Q35.
Feulgen reaction is
Q36.
The technique used for estimation of minimum amount of hormones and drugs is called
Q37.
Radioactive isotope P32 has been used experiment, carried out by
Q38.
If the cells are broken up and sedimented by centrifugation the new structure formed in one of fraction is
Q39.
Mitochondria was discovered with the help of
Q40.
Transmission electron microscope has high resolution of
Q41.
Phenology concerns with
Q42.
Middle lamella can be stained with
Q43.
Autoradiography technique was used to study
Q44.
The quantitative analysis of a substance can be made with the help of
Q45.
The resolution power of light microscope is
Q46.
In the experiment of Melson and Stahl to demonstrate semiconservative mode of DNA replication, the radio-isotope used was
Q47.
Electron microscope works on the principle of
Q48.
The first operational microscope was produced by
Q49.
The smallest size of cell visible to naked eye
Q50.
How many angstroms make a metre
Q51.
Electron microscope was invented by:
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📅BPKIHS - 2014
Q52.
In electron microscopy, ultra - thin specimens are impregnated with [BPKIHS - 2014]
📅BPKIHS - 2014