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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 4 • (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 6 • 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 Ver : BScMLT_1308 1 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 Ver : BScMLT_1308 10 2 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 Ver : BScMLT_1308 6 3 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. 4 • 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. 10 • 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 Ver : BScMLT_1308 3 4 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 5 Applications Used in the measurement of peptide/steroid hormones such as T3, T4,TSH, HCG, progesterone, 2 Ver : BScMLT_1308 5