Lab Exam 3

• Coliform Characteristics


  • Gram-negative, non-spore-forming rods
  • Facultative anaerobes that ferment lactose to produce gas
  • Commonly found in the intestines of warm-blooded animals
  • Examples include Escherichia coli, which is often used as an indicator of fecal contamination
• Non-Coliform Characteristics


  • Include organisms other than coliforms, such as Enterococcus faecalis
  • Gram-positive cocci
  • Not typically used as fecal indicators
• MPN Test - Presumptive Test

  1. Inoculate lactose broth with varying amounts of water samples

  2. Incubate tubes at 35-37°C for 24 hours

  3. Observe gas production, indicating the presence of coliforms
• MPN Test - Confirmed Test

  1. Streak growth from positive tubes onto EMB and MacConkey plates

  2. Incubate plates at appropriate conditions for 24 hours

  3. Observe colony characteristics to confirm the presence of coliforms
• MPN Test - Completed Test


  1. Inoculate lactose broth and TSA slant with isolated colonies from EMB or MacConkey plates

  2. Incubate and observe for gas production in lactose broth and perform Gram stain on TSA slant
• Compact Dry


  1. Inoculate 1ml of water sample onto Compact Dry™ EC plates

  2. Ensure even spreading of sample across the plate surface

  3. Incubate plates upside down at 35-37°C for 24 hours

  4. Count blue colonies for E. coli and total blue and pink-purple colonies for coliforms
• Presumptive Test


  Detects the presence of coliforms through gas production in lactose broth
• Confirmed Test


  Confirms the presence of coliforms and differentiates them using selective media like EMB and MacConkey agar
• Completed Test


  Verifies the identity of coliforms and performs additional tests for confirmation
• MPN Method - Pros


  • Provides uniform recovery of microbial population
  • Offers a realistic representation of count
  • Allows growth of bacteria unculturable on agar
• MPN Method - Cons


  • Labor and materials intensive
  • Requires three days for completion
  • MPN estimates often have a lower order of precision
• Compact Dry Method - Pros


  • Easy to use, no media preparation is needed
  • Does not require an incubator
  • Highly portable and can be stored for up to 18 months
  • Relatively inexpensive
• Compact Dry Method - Cons


  • Limited sample size accuracy above 100 cfu/100ml
  • Not suitable for high counts
• Interpret gas production in lactose broth and colony characteristics on selective media
• Count colonies on Compact Dry™ EC plates and differentiate between E. coli and other coliforms
• MPN Table


  • Determine MPN index by comparing observed gas production to MPN chart
  • Use MPN index to estimate coliform count per 100 ml of water with 95% confidence
• Analyze results without memorizing specific data, focusing on interpreting and comparing methods
• Staphylococcus


  • Gram-positive cocci, arranged in clusters
  • Facultative anaerobes, catalase-positive, and halotolerant
  • Commonly found in skin, nasal membranes, and wounds
  • Pathogenic species include Staphylococcus aureus, known for causing abscesses, impetigo, toxic shock syndrome, and other severe infections
• Staphylococcus Differentiation


  • Coagulase Test: Detects coagulase produced by pathogenic species like S. aureus
  • Mannitol Salt Agar (MSA): Differential media with mannitol and phenol red, used to identify mannitol-fermenting S. aureus
  • Catalase Test: Distinguishes Staphylococcus from Streptococcus; Staphylococcus is catalase-positive
• Streptococcus


  • Gram-positive cocci, arranged in chains
  • Catalase-negative, often found in throat, mouth, nose, and intestine
  • Pathogenic species include Streptococcus pyogenes (Group A Streptococcus), causing strep throat, necrotizing fasciitis, and other infections
• Streptococcus Differentiation


  • Bile Esculin Test: Identifies Group D Streptococci by hydrolyzing esculin
  • Salt Broth Test: Determines salt tolerance, distinguishing Group D Streptococci
  • Hemolysis Patterns on Blood Agar: Differentiates species based on their ability to lyse blood cells
• Neisseria


  • Gram-negative diplococci
  • Oxidase-positive, catalase-positive
  • Major pathogens include Neisseria meningitidis and Neisseria gonorrhoeae, causing meningitis and gonorrhea, respectively
• Coagulase Test


  • Reagent: Rabbit plasma
  • Used to detect coagulase produced by pathogenic Staphylococcus species like S. aureus
• Mannitol Salt Agar (MSA)


  • Contains 7.5% salt, mannitol, and phenol red
  • Differentiates mannitol-fermenting Staphylococcus aureus
• Catalase Test


  Utilizes hydrogen peroxide to differentiate Staphylococcus (catalase-positive) from Streptococcus (catalase-negative)
• Bile Esculin Test


  • Media: Bile esculin agar (BEA)
  • Identifies Group D Streptococci by hydrolyzing esculin
• Salt Broth Test


  • Media: Brain Heart Infusion (BHI) broth with 6.5% NaCl
  • Differentiates Group D Streptococci by their salt tolerance
• Oxidase Test


  • Reagent: Oxidase strips or paper
  • Detects cytochrome C oxidase, positive for Neisseria species
• Coagulase Test - Positive


  Gel-like solidification
• Coagulase Test - Negative


  No gel formation
• Mannitol Salt Agar (MSA) - Positive


  Acidic byproducts turn phenol red yellow
• Mannitol Salt Agar (MSA) - Negative


  No color change
• Catalase Test - Positive


  Bubbling indicates the presence of catalase
• Catalase Test - Negative

  No bubbling
• Bile Esculin Test - Positive


  Formation of black coloration indicates esculin hydrolysis
• Bile Esculin Test - Negative


  No color change
• Salt Broth Test - Positive


  Turbidity indicates growth
• Salt Broth Test - Negative

  Clear broth with no growth
• Oxidase Test - Positive


  Purple coloration within 20 seconds
• Oxidase Test - Negative


  No color change