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Microbiology and Parasitology Reviewer on Microscope
Bachelor of Science in Biology (BSBiol)
Saint Louis University (Philippines)
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Care of the microscope: When carrying the microscope, hold it firmly by the arm with one hand and support it at the base with the other. Do not allow liquids, particularly acids and alcohols to come in contact with any part of the microscope. Always lower the stage sufficiently before either placing a slide/removing a slide from the stage. Remove immersion oil from the microscopes with lens tissue, paper. After using the microscope, always rotate and position to SO. Do not place in the edge of the laboratory table. Do not tamper with any parts of the microscope. Switch off light before disconnecting the plug from the socket. Do not let the electric cord dangle in such a way to risk foot entanglement. Use dustcovers to protect the microscope during storage.
TYPES OF MICROSCOPES
1. Brightfield Microscope Distinguish Features Uses visible light Specimen appears on a bright background. Principal uses: To observe various stained specimens To count microbes 2. Darkfield Microscope Distinguish Features Uses a special condenser with opaque disk Light reflected by specimen enters obj. lens Specimen appears light against a black background. Principal uses: To examine unstained specimens (ex. Detecting Treponema pallidum in diagnosing syphilis) To examine living microorganisms not visible in brightfield microscope
3. Phase-Contrast Microscope Distinguishing Features Special condenser containing annular diaphragm allows direct light to pass
through condenser – specimen – diffraction plate Direct and reflected or diffracted light rays – image Principal use: To facilitate detailed examination of the internal structures of living specimens 4. Differential interference contrast Microscope Distinguishing Features 2 beams of light separated by prisms Specimen appears colored as a result of prism effect Principal use: To provide 3D images 5. Fluorescence Microscope Distinguishing features UV light or near UV light Fluorescent compounds – emit light Principal use To rapidly detect and identify microbes in tissues or clinical specimens Ex: fluorescent – antibody techniques (immunofluorescence) 6. Confocal Microscope Distinguishing Feature Uses a single photon to illuminate one plane of a specimen at a time Principal use To obtain 2D or 3D images of cells for biomedical applications 7. Two photon Microscope Distinguishing feature Use 2 photons to illuminate a specimen Principal uses: To image living cells up to a depth of 1 mm To observe cell activity in real time
8. Scanning acoustic Microscope Distinguishing Feature Sound wave of specific frequency --- travels through specimen --- hits an interface ---a portion is reflected Principal use To examine living cells attached to another surface (ex. Cancer cells, artery plaque , biofilms 9. Electron microscopes Transmission Electron Microscope (TEM)
Beams of electrons – shorter wavelengths; passes through specimen – structures < 0. um can be resolved 2D image To examine viruses or internal ultrastructures in thin sections of cells
Scanning Electron microscope (SEM) Beams of electrons --- reflected from the specimen 3D image To study the surface features of cells and viruses 10. Scanned Probe Microscopes Scanning Tunneling Microscopes Uses a thin metal probe – scans specimen Image : reveals bumps and depressions of atoms on the surface of the specimen. Provides very detailed views of molecules inside cells Atomic Force Microscope Uses metal and diamond probe – gently forced down along the surface of the specimen 3D image Optimal Characteristics Magnification : The number of times the image is enlarged by the lenses Total magnification : objective lens magnification power x ocular lens magnification power. Resolution : ability of a lens to separate or distinguish between small objects that are close together Ex. 0,2 um resolving power – the microscope can distinguish 2 points if they are at least 0 um apart. Affected by: Wavelength of light source , Refractive index of medium Working Distance (WD) : distance between the top surface of cover glass and objective extremity Number of aperture (NA) : the figure corresponding to the F-number of camera Focal depth : the depth of range of a specimen, in which focusing is obtained at a time Field number (FN) : the diameter of the image observed through an eyepiece (mm) Actual field of view: diameter of the field of view, expressed as the size on the specimen surface
LABORATORY EQUIPMENTS AND GLASSWARES
1. Screw- capped tubes : used for transporting , growing , and storing cultures. 2. Centrifuge tubes : used in centrifugation 3. Durham fermentation tube : used to detect production of gas by fermenting microorganisms 4. Smith fermentation tube : used in observing microorganisms , capable of anaerobic fermentation with collection of any evolved gas. 5. Plain petri plates : used to culture cells 6. Petri plates with divisions: Used to contain several media in one plate for culturing several microorganisms. 7. Erlenmeyer Flask : for measuring large quantity of liquid , as receiver in titration and distillation, as container for liquid samples 8. Volumetric flask: used to prepare a solution of fixed volumes very accurately 9. Serological pipette: used to transfer a desired volume of solution from one container to another. 10. Volumetric pipette: measure or transfer volume of liquid 11. Beaker: used as a reaction container or to hold liquid/solid samples, also used to catch liquids from titrations and filtrates from filtering operations 12. Funnel : direct / guide liquids when pouring 13. Watch glass : for holding small samples or for covering beakers or evaporating dishes 14. Graduated cylinder : for measurement of an amount of liquid 15. Plain glass slides : used for mounting a specimen for examination under the microscope 16. Hanging drop slide : used to examine freely moving microorganisms 17. Inoculating loop : used to smear or to streak an inoculum taken from a culture of microorganisms into culture media 18. Inoculating needle : used to inoculate agar slants and agar deeps with microorganisms used to tease fungal hyphae 19. Test tube rack : used to hold test tubes 20. Slant rack : used to hold tubes in slant position to solidify agar at a slant. 21. Staining rack: used to hold slides with specimens being stained 22. Bunsen burner: used for heating, sterilization and combustion
cells Other methods of studying unstained specimens (refer to pg 1-2)
Temporary slide preparation Advantages Disadvantages Few artifacts Drying out Cellular activities can be observed (motility cell division, etc)
Difficult in focusing moving organisms
Inexpensive , easier to prepare Cannot be stored over a longer time Permanent slide preparation Advantages Disadvantages Preservation of specimen for long period of time
Color may fade over time
Specimen can be observed in more detail (stained)
Difficult to prepare, expensive, artifact formation
COMPARISON OF THE THREE DOMAINS OF LIFE Characteristic Bacteria Archaea Eukarya Nuclear envelope
Absent Absent Present
Membrane- enclosed organelles
Absent Absent Present
Peptidoglycan in cell wall
Present Absent Absent
Membrane lipids
Unbranched hydrocarbons
Some branched hydrocarbons
Unbranched hydrocarbons
RNA polymerase
One kind Several kinds Several kinds
Introns in genes
Very rare Present in some genes
Present in many genes Response to the antibiotics streptomycin and chloramphenico l
Growth usually inhibited
Growth not inhibited
Growth not inhibited
Histones associated with DNA
Absent Present in some species
Present
Circular chromosome
Present Present Absent
Growth at temperatures 100 C
No Some species No
Microbiology and Parasitology Reviewer on Microscope
Course: Bachelor of Science in Biology (BSBiol)
University: Saint Louis University (Philippines)
