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Blueboom/Microbiology

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Medical Microbiology

Study of microscopic organisms includes

  • Bacteriology Bacteria simplest smallest single celled free living organisms
  • Virology viruses - non cellular parasitic, not living organisms
  • Mycology fungi - microscopic (molds and yeast), macroscopic (mushrooms and puff balls), unicellular and multicellular
  • Protozoology - protozoa, single celled
  • Phycology/Algology - algae - simple aquatic organisms ie seaweeds

Koch's postulates

  1. The same microbe is always associated witha specific disease
  2. this microbe can be recovered and grown in pure culture
  3. The pure culture must cause disease in an experimental animal
  4. The original microbe must be recovered from t he experimental disease

Examples of Problems:

  • Mycobacterium leprae - requres animal host
  • Neisseria gonorrhoeae - only human host
  • Opportunistic pathogens - immunocompromised host

Use of Molecular Postulates

  • Criteria a for determining cause of infections using molecular techniques
  1. Virulence factor: gene or gene product must be found in pathogenic stains but not in non-pathogenic stains
  2. When a virulence factor is introduced into a non-pathogenic strain it should become a pathogenic strain
  3. Genes for virulence must be expressied during the course of disease
  4. Antibodies specific for the virulence gene products should be protective


Classifications of Microogranisms

Organisms:

  • Bacteria, Archaeabacteria and Eukaryotes.
  • Infectious Agents: Viruses, viroids, prions

Bacterial and fungal classification using taxonomy

  • Kingdom, Phylum, Class, Order, Family, Genus, Species
  • Order has suffix of ales, Family has suffix of aceae
  • Biovar - variant strain that differs physiologically or biochemically
  • Serovar - sub division of species
  • Strain - a genetic varient or subtype

Viral Taxonomy

  • Order suffix virales
  • Family suffix viridae
  • Subfamily suffix virinae
  • Genus suffix virus
  • Species individual virus

Bacterial Morphology

Size

Bacteria range in size between .2 micrometers and 10 micrometers , usually around 1-2 micrometeres in diameter (Mycoplasma species range from .15 -.3 micrometers in diameter Although bacteria have small size, the importance of this is their large surface area to volume ratio. Prokaryotes do not require complex nutrient uptake and transportation mechanisms (use passive diffusion)

Bacterial Shapes

  1. Spherical - Coccoid (Coccus (singular), cocci (plural)
  2. Cylindrical - Rod (Bacillus (singular), Bacilli (plural)
  3. Curved, Helical Twisted, Spiral Vibrio (refers to vibratory motility)
  4. Square Cuboid (not infectious)

Cell Arrangement

  • Bacteria can be arranged singularly, in pairs, in chains or in clusters
  • Specific Terminology is used for coccoidal Bacterial arrangements
    • Single Single
    • Pairs: Diplococci Streptococcus pneumoniae
    • Chains Streptococci Streptococcus pyogenes
    • Clusters Staphylococci All Staphlococcus species
    • Tetrads Example: Sarcina species
    • These terms do not exsist for bacillus
  • Coccobacillus - a very short bacillus, a term indicating an intermediate appearance of a bacilli and coccus
  • Pleomorphic - cannot retain proper shape or proper stain. Could be very old or very young bacteria

Staining Techniques

Grams staining

Invented in 1884 by Hans Christian gram. This is the most important staining technique for identifying and classifying bacteria.
Utilizes the ability of bacteria to retain crystal violet-iodine complex after exposure to organic solvent.
Note: Never carried out on a throat swab or fecal location.
Steps:

  1. Flood with Crystal violet (primary stain)
  2. Flood with Gram's iodine (mordant)
  3. Drop wise add decoloriser (acetone or alcohol) *critical step*
  4. Immediately rinse with water
  5. Flood with safranin (counter stain)

Gram Positive Bacteria stain Blue/Purple Gram Negative Bacteria stain Red

Utility of Gram Stains

  1. Determine the adequacy of the specimen for culture
  2. Make a presumptive etiologic diagnosis and early clinical decision
  3. Suggest a need for non-routine laboratory procedure
  4. Help make accurate interpretation of culture results
  5. Provide better insight into the current infection

Acid Fast Staining

Devised By Paul Ehrlich Used for bacteria resistant to Gram's stain ie: M. Tuberculosis and Nocardia species

Ziehl-Neelsen staining (hot method)

  1. Flood with hot basic carbolfuchsin; decolorize with acid-alkali
  2. Counterstain with methylene blue or malachite green
  3. Acid fast bacteria - Red/Pink; Non Acid-fast bacteria - Blue/Green

Kinyoun Stain (cold method)

Same as Ziegl-neelsen method but does not require heat

Fluorochrome stain

(auramine-rhodamine)

  • Primary stain is a fluorescent dye and the counterstain is an oxidizing agent (Potassium permanganate)
  • The bacteria would be yellowish/green against black background

Bacterial Ultrastructure

Flagellum

Flagella (plural form)

General features
  • Extends outwards from within the cell
  • Provides motility...but some bacteria are motile but lack flagella
  • May have an essential role for colonization (contributes to virulence)
  • Not essential for bacterial survival
  • All coccoidal cells lack flagella
Flagellum arrangement

Polar (at one or both ends/poles) or Lateral(situated around the entire surface)

Composition of flagellum

Composed of 3 parts

Helical filaments
  • long & thin ~20nm dia by 1-7 micrometers in length
  • Composed of protein - flagellin, antigenic termed the H (Haugch antigen). This H-antigen is useful for identifcation of bacteria
    • <math>H^+</math> possess flagella (motile)
    • <math>H^-</math> lack flagella (non-motile)
Hook
  • a short curved structure which anchors the filament into the basal body
Basal Body
  • Contains rod and 1-2 sets of double plates/rings.
  • All flagella contain two rings (S & M) for rotation located in the cytoplasmic membrane.
  • Gram negative flagella possess additional 2 rings (L & P) located in outer membrane of cell wall which stabilize the filament)
    • 2 rings Gram positive bacteria
    • 4 rings Gram negative bacteria
Movement
  • Taxis - involuntary movement of organism in response to a stimulus
  • Magnetotaxis - magnetic field
  • Phototaxis - light
  • Thermotaxis - heat
Chemotaxis
  • response to chemical stimulus (nutrients or growth inhibitors within the environment
  • Chemicals are recognized by protein receptors located in the cytoplasmic membrane
Recognition of response
  • + response- organism swims from low concentration to high concentration (up the concentration gradient)
  • - response - organism swims down the concentration gradient
  • Non response- random walk
Mechanism
  • Rotation of basal body plates in the clockwise or counterclockwise direction
  • ccw rotation - flagella sweeps around cell in common axis which results in a forward motion
  • cw rotation - Reversal of basal plates which causes the flagella to stop rotating and results in a tumbling motion to allow for a change in direction
  • random movement indicates absence of concentration gradient
  • movement during the presence of an attractant gradient resulting in a reduced tumbling frequency.

Additional outer appendages

  • Play no role in motility
  • Hollow , helical 9-10 nm diameter, thinner, shorter and more numerous than flagella.
  • Composed of PILIN protein (antigenic, can be used for classification & identification)
Pilus/Pili
  • F-Pilus (Fertility/sex)
  • A bacterium may possess a single pilus or up to 10
  • Found only in Gram negative bacteria
  • Function: transfer of genetic material between bacterial cells

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