Download BSc/Diploma in Medical Laboratory Technology 2 BLT202

PROGRAM
BSc/Diploma in Medical Laboratory Technology
SEMESTER
2
SUBJECT
BLT202 - MICROBIOLOGY-I: PRINCIPLES AND TECHNIQUES
BOOK ID
B1821
SESSION
Winter 2015
No
Q 1
Question/Answer key
Marks Total Marks
10
Classify the various types of microscopes. Describe dark field microscopy in
detail.
( Unit 13 ; Section
13.2.2 )
A 1
Types of microscopes
• 1. Optical Microscopes
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• (a) Dark field microscope
• (b) Phase-contrast and differential interference contrast microscope
• (c) Confocal scanning microscope
• (d) Fluorescence microscope
• 2. Electron Microscopes: The types of electron microscopes are following:
• (a) Transmission Electron Microscope (TEM)
• (b) Scanning Electron Microscope (SEM)
Dark field microscopy
• Dark Field Microscopy
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• In dark field microscopy, the non-diffracted rays are removed altogether so that
the image is composed solely of diffracted wave components. This technique is
very sensitive because images based on small amounts of diffracted light from
minute phase objects are seen clearly against a black or very dark background.
Dark-field microscopy is most commonly used for minute light-diffracting
specimens such as diatoms, bacteria and bacterial flagella, isolated organelles
and polymers such as cilia, flagella, microtubules
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and actin filaments, and silver grains and gold particles in histochemically labelled
cells and tissues. An example is dark-field image of labeled neurons. The number
of scattering objects in the
specimen is an important factor, because the scattering of light from too many
objects may brighten the background and obscure fine details.
• A. Theory and Optics
• Dark-field conditions are obtained by illuminating the specimen at an oblique
angle such that direct, non-diffracted rays are not collected by the objective lens.
The effect of dark-field optics can be obtained quickly with bright-field optics by
rotating the condenser turret so that rays illuminate the specimen obliquely. Only
diffracted light from the specimen is captured by the objective, and the direct
waves pass
uncollected off to one side of the lens. The disadvantage of this technique is that
unidirectional illumination of highly refractile objects can introduce large amounts
of flare. Much better images are obtained with a special dark-field condenser
annulus, which is mounted in the condenser turret. Special oil-immersion darkfield
condensers must be used for oil immersion objectives. Dark-field microscopy
resembles phase-contrast microscopy in that the specimen is illuminated by rays
originating at a transparent annulus in the condenser.
• However, in dark-field optics only diffracted rays are collected by the objective
and contribute to the image; non-diffracted rays are pitched too steeply and do
not enter the lens. Since non-diffracted background light is absent from the
image, light-diffracting objects look bright against a dark field.
• B. Image Interpretation
• The appearance of a dark-field image is similar to one of self-luminous or
fluorescent objects on a dark background, but with the difference that edges of
extended, highly refractile objects diffract the greatest amount of light and
dominate the image, sometimes obscuring the visibility of fainter, smaller objects.
In addition, details in dark-field images are broader and less distinct compared to
other imaging modes such as phase contrast, because removal of one entire
order of light information from the diffraction plane makes edge definition less
distinct in the image. Further, if the NA of the objective selected is too restricted,
many diffracted waves are also eliminated, resulting in a loss of definition of fine
details in the specimen.
Q 2
10
Describe different blotting techniques.
( Unit 10 ; Section 10.4 )
A 2
Blotting techniques
• Blotting is a method used to detect a particular target component with a
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high level of sensitivity. Therefore, it is popular as a basic method to identify
proteins.
• Identification of a specific protein in a complex mixture of proteins can be
accomplished by a technique known as Western blotting, named for its similarity
to Southern blotting, which detects DNA fragments, and Northern blotting, which
detects mRNAs
Western Blot:
• In Western blotting, a protein mixture is electrophoretically separated on an
sodium dodecyl sulphate polyacrylamide gel (SDS–PAGE), a slab gel infused
with SDS, a dissociating agent.
Southern Blot:
• Southern blot is more formally called a DNA blot.
• Southern blot allows the visualization of one DNA fragment from a whole
genome DNA extract.
Northern Blot:
• ‘The Northern blot method reveals information about RNA identity, size and
abundance’.
• Northern blots are not used very often for diagnostic purposes; they are used
mainly in research. The techniques are fairly sophisticated and other methods
yield acceptable results (such as Southern blots or PCR).
Q 3
( Unit 12 ; Section 12.5 )
A 3
10
Diagnostic and therapeutic uses of radioisotopes
1. Enzyme and ligand-binding studies
2. Isotope dilution analysis
3. Radioimmunoassay
4. Radiodating
5. Molecular biology techniques
6. Clinical diagnosis
7. Ecological studies
8. Therapeutic radiopharmaceuticals
9. Radiotherapy
Q 4
10
Discuss the various diagnostic and therapeutic uses of radioisotopes.
10
Discuss bacterial nutrition and metabolism.
( Unit 2 ; Section 2.4 )
A 4
Bacterial nutrition
The elements essential for nutrition of a bacterium include C, H, O, N, S, P, K,
Mg, Fe, Ca, Mn, and traces of Zn, Co, Cu, and Mo.
• These elements are found in water, inorganic ions, small molecules and
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macromolecules, which serve either a structural or a functional role in the cells.
Environmental Factors Affecting Bacterial Growth:
(i) Oxygen: Based on their O2 requirements, aerobic bacteria require oxygen for
growth.
ii) Carbon dioxide: Brucella abortus requires much higher levels of carbon dioxide
(5–10%) for growth (capnophilic).
(iii) Moisture and drying: Water is an essential ingredient of bacterial protoplasm.
(iv) pH: Bacteria multiply within pH 5 (acidic) to pH 8 (basic) and have a neutral
pH 7.
(v) Light: Bacteria are sensitive to ultraviolet light.
(vi) Osmotic effect: Besides mycoplasma and cell wall-defective organisms, the
majority of the bacteria are osmotically tolerant.
(vii)Mechanical and sonic stresses: Bacteria may be ruptured by mechanical
stress or vigorous agitation employing glass beads or by exposure to ultrasonic
vibration.
Bacterial metabolism
The prokaryotes, as a group, conduct all the same types of basic metabolism and
its diversity is expressed by their great variation in modes of energy generation
and metabolism.
• Escherichia coli can produce energy for growth by fermentation or respiration,
aerobically using O2 as a final electron acceptor or respire anaerobically using
NO3 as a terminal electron acceptor.
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• Metabolism is defined as the series of changes in carbohydrate, protein or fat
within a bacterial cell. It may be aerobic or anaerobic.
Q 5
10
Discuss various methods of gene transfer in bacteria.
( Unit 6 ; Section 6.4 )
A 5
Various methods of gene transfer in bacteria
1. Transformation:
A free or naked DNA released by a donor can be lifted by a recipient cell and the
process was demonstrated for the first time in an experiment conducted by
Frederick Griffith in 1928.
• 2 Transduction Including Lysogenic Conversion:
It is a process of transfer of genetic material or DNA from one bacterium cell to
the other with the help of a virus.
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• 3 Conjugation:
Transfer of genetic material takes place between two bacterial cells through
physical contact or formation of a bridge between the two cells.
Q 6
10
Discuss radioimmunoassay under principles, methods and applications.
( Unit 10 ; Section 10.2 )
A 6
Principle
It is a competitive binding assay in which a fixed amount of antibodies and
labeled antigen, i.e. conjugated to a radioisotope (Ag*), react in the presence of
the known (standards) or the unknown (samples) amounts of the antigen for
binding site to the antibody (Ab). At equilibrium, the amount of
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radiolabelled Ag* bound to
antibody will decrease as the amount of unlabelled Ag increases.
Methods
A series of test tubes are taken in which varying amounts of pure antigen are
incubated with fixed amounts of the labelled antigen and antibody for 6 hours.
• The free and the bound antigen are separated and the radioactivity is
calculated in the bound Ag*Ab fraction.
A. Methods of separation of bound and unbound antigen
B. Separation of bound and free antigen:
(i) Solution methods
(ii) Solid-phase methods
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Applications
Used in the measurement of peptide/steroid hormones such as T3, T4,TSH,
HCG, progesterone,
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