Methods of ID

• General rules for identifying bacteria

  • All cocci are gram (+) EXCEPT Neisseria, Branhamella, Moraxella, and Veilonella
  • All bacilli are gram (-) EXCEPT Mycobacterium, Cornybacterium, Lactobacillus, Listeria, Clostridium, Bacillus, Erysipelothrix, and Nocardia
  • All spiral bacteria are gram (-) when stained

  
• Neisseria and Branhamella


  The most isolated in the laboratory, reporting these bacteria is important because they are sexually transmitted, particularly N. gonorrhea
• Moraxella


  Causes conjunctivitis
• Organisms that are acid-fast

  • Mycobacterium
  • Nocardia spp.
  • Corynebacterium spp.
• Nocardia and Corynebacterium


  Only partially acid fast - do not contain up to 60% of lipid mycolic acid in their cell wall
• Types of acid fast staining

  • Ziehl-Neelsen Method
  • Kinyoun Method
  • Pappenheim's
  • Baumgartens'
• Ziehl-Neelsen Method


  Uses heat (hot stain), red AFB, blue non-AFB
• Kinyoun Method


  Cold method using phenol as mordant, red AFB, green/blue non-AFB
• Pappenheim's


  Resolic acid in alcohol = decolorizer, differentiates M. tb from M. lacticala and M. smegmatis
• Baumgartens'


  Diluted alcoholic fuchsin = primary stain, differentiates M. tb (Blue) from M. leprae (Red)
• Types of culture

  • Pure
  • Mixed
  • Stock
• Pure culture


  Contains a single species of bacteria
• Mixed culture


  More than one type of bacteria is present
• Stock culture


  Being maintained solely for making it available for subculture
• Types of colonies

  • Mucoid
  • Smooth
  • Rough
• Mucoid colony

  Organisms are mucoid because they are encapsulated, the most typical colony in the laboratory, example: E. coli, Klebsiella pneumoniae
• Neufeld-Quellung test


  Tests if an organism has a capsule, (+) bacteria swells
• Culture media classification by physical state/consistency

  • Liquid
  • Semisolid
  • Solid
• Semisolid media

  Contains 0.5 - 1.5% agar, can detect formation of sulfide, indole, and motility
• Solid media


  Contains 2 - 3% agar, e.g. Blood Agar Plate, Triple Sugar Iron
• Culture media classification by composition

  • Synthetic or Defined Medium
  • Non Synthetic or Complex Media
  • Tissue Culture Medium
• Synthetic or Defined Medium


  All components are known, preferred for isolation of cyanobacteria and chemoorganotrophs, example: BG 11 medium
• Non Synthetic or Complex Media


  Some substances are unknown (peptones, meat and yeast extracts), used for isolation of medically important bacteria, example: TSB, MAC
• Tissue Culture Medium


  For obligate intracellular bacteria (Rickettsia and Chlamydia), example: W138 cells, HeLa 229 Cells, Mccoy cells

  
• Culture media classification by dispensing or distribution

  • Plated
  • Tubed
• Plated media

  Distributed in sterile petri dish or plate
• Tubed media

  • Liquid
  • Slant
  • Butt and Slant
  • Butt
• Slant orientation


  Streak only the top portion of the culture media
• Slant/deep orientation


  Slant area is streaked, and the butt is stabbed
• Deep orientation


  Culture is stabbed
• Broth


  Culture is mixed
• Durham tube


  Tool used to indicate if a bacteria is producing gas

  
• Autoclave


  Used for sterilization at 121 *C, 15 psi, for 15 - 30 minutes, Bacillus stearothermophilus is the biological indicator
• Triple Sugar Iron


  Contains glucose, lactose, and sucrose, tube a is sulfide positive, tube b is gas positive
• Lysine Iron Agar


  Tests for deamination, decarboxylation, and sulfide formation
• Lysine Decarboxylation (butt)


  Positive: purple slant/purple butt, Negative: purple slant/yellow butt
• Lysine Deamination (slant)


  Positive: red slant, Negative: slant remains purple
• H2S Production

  Positive: black precipitate, Negative: no black color development
• Simmons' Citrate


  Initially green, becomes blue when positive (organism can utilize citrate), uses thymol blue as pH indicator

  
• Christensens Urea Agar


  Initially yellow, becomes pink if bacteria can produce urease, complete pink = fast urease producer, incomplete pink = late urease producer