Morphology and staining of microorganism

• Methods of identifying microorganisms include:

  • Morphology and staining of microorganisms
  • Cultural methods
  • Physiological characteristics
• General ways of observing microorganisms under a microscope:

  • Living State:
  • Wet mount (observe the shape and arrangement of the microorganism)
  • Hanging drop method (best for observing motility)
  • Depression slide
  • Fixed State:
  • Prepared slides (best for viewing)
• Ways to demonstrate motility of microorganisms:

  • Hanging Drop Methods
  • SIM (Sulfide Indole Motility Medium)
  • Staining the flagella by simple stain (Leifson Stain)
  • Serological Test (Flagellar Ag and Flagellar Ab)
  • Fluorescent Ab Test
• Classification of Bacteria based on morphology/fundamental shapes:

  • Coccus (Cocci): round/spherical organisms
  • Bacilli (Bacillus): rod-shaped
  • Spirilli (Spirillum): spiral-shaped
  • *Vibrios- straight rod w/ single rigid curve
  • *Spirilla -rigid helical
  • *Spirochaetes - flexus helical rod
  • Pleomorphic Organisms: vary in size and shapes of bacteria
• Arrangement of bacteria:

  • Grape-like Cluster: mostly Staphylococcus
  • Chain of Bacilli: Streptococci, Streptobacilli
  • Diplo arrangement: in pairs like Gonorrhea, Pneumonia
  • Tetrads: a group of 4 cells arranged in a square like Micrococcus, Peptococcus, Gaffkya
  • Cubical: arranged in cubes of 8 cells like Sarcina
  • Pallisade: tends to place themselves side by side, resembling a pack of cigarettes
  • Chinese Character
• 7. Chinese Character

  Average size of bacteria = 0.5-2.0 umum= 1/25k of an inch

  Smallest Pathogenic Organism: Haemophilus -Up to 0.2x 0.5 um Largest Pathogenic Organism: Bacillus Anthracis -Up to 1x3-10 um
• STAINING

  Process of artificially coloring

  the microorganism with the

  use of dyes or reagent

  Purpose:

  1. To observe and appreciate

  the appearance of

  microorganisms

  2. To differentiate one

  organism from another

  Organism

  3. Helps in identifying

  microorganisms and some

  special structures
• Kinds of Stains utilized in the Study

  of Bacteria;

  •Simple Stain

  •Differential Stain

  •Special stain

  •INDIRECT, RELIEF OR NEGATIVE

  STAINING METHODS
• Simple stain:

  • Uses only one particular dye to stain organisms
  • Crystal violet results in violet color
  • Safranin end results in red or pink color
• Differential stain:

  • Uses two or more dyes
  • VIAS method includes Violet, Iodine, Alcohol, and Safranin
  • Primary stain is Violet
  • Iodine acts as a mordant to enhance the substance
  • Decolorizer is used, followed by a secondary stain
  • After 1 min, air dry and observe under the microscope
• Gram stain:

  • Differentiates organisms as gram-positive (violet, all purple) or gram-negative (red or pink)
• Acid-fast stain:

  • Differentiates acid-fast from non-acid-fast organisms
  • Acid-fast organisms are hard to stain but once stained, they are hard to decolorize
  • Nocardia Asteroides is slightly acid-fast, not found in the microbacteria group
• Methods of acid-fast staining:

  • Ziehl Neelsen Method:
  • Involves a steaming process and requires heat
  • Reagents: Carbon fuchsin, Acid ROH, Methylene blue
  • Results: red for acid-fast, blue for non-acid fast
  • Kinyoun/ Cold Method:
  • Does not employ steaming methods
  • Reagents: Carbon fuchsin, Acid alcohol, Malachite green
  • Results: acid-fast shows red or green background, organisms are straight or slightly curved in groups of 3-10 bacilli
  • Non-acid fast shows no results
• Pappenheim Method:

  • Used to differentiate Mycobacterium lacticola or Mycobacterium Smegmatis from Mycobacterium TB
• • Baumgarten's Method

  • To differentiate mycobacterium leprae

  (red in color, easily stained with dilute alcoholic fuchsin) to mycobacterium TB(difficult to stain, blue)
• Special stains are used to emphasize certain special structures or features of organisms
• Types of special stains:

  • Cell wall stain using Dyar method
  • Capsular stain
  • Metachromatic granules stain
  • Spores stain
  • Flagellar stain
• Indirect, relief, or negative staining methods make microorganisms appear colorless
• Methods of indirect, relief, or negative staining:

  • India ink method
  • Nigrosine method
• 2 rapid non-staining systems:

  • LANA (alanine, nitroalamide): Microorganisms turn yellow when touched to gram-negative bacteria
  • 3% KOH
• Cultural methods involve culture media that provide nutritional and environmental requirements for bacterial growth
• Types of cultures:

  • Pure culture: one species
  • Mixed culture: made up of organisms belonging to different species
  • Stock culture: pure culture of organisms used in supplying industry, research, etc.
• Classification of culture media:

  • According to consistency or physical state
  • According to composition
  • According to how the medium is dispersed
  • According to use
• Agar, polysaccharide extracts of seaweed, is used as a base medium

  • Liquid culture medium contains no agar (e.g., Broth, Thioglycollate media)
  • Semi-solid medium contains gelatin or agar(0.5%-1% of agar)