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Video Notes: Isolation Streaks

Overview - This note details common microbiological tests and media used for bacterial identification and characterization.

  • These processes are fundamental in diagnostic microbiology to differentiate various bacterial species based on their metabolic activities and growth characteristics.

Urease Test

  • Purpose: Detects the presence of the enzyme urease, which hydrolyzes urea into ammonia and carbon dioxide. This test is crucial for identifying rapid urease producers.
  • Principle:
    • Urea is a common substrate present in the medium.
    • Bacteria possessing the enzyme urease break down urea (NH\text{2})2CO into ammonia (NH\text{3}), carbon dioxide (CO\text{2}) and water (H\text{_2}O).
    • The ammonia produced increases the alkalinity of the medium, raising the pH.
    • A pH indicator, typically phenol red, is included in the medium.
    • At a neutral pH (6.8), phenol red is yellow. As the pH rises above 8.1 (alkaline), phenol red turns a vibrant pink/red color.
  • Medium: Urea broth or agar, containing urea (2%), a pH indicator (phenol red), and a buffer to initially maintain the pH. The concentration of urea is critical for detecting both rapid and slow urease producers.
  • Inoculation & Incubation: Inoculate with a heavy inoculum and incubate at 35^ ext{o}C for up to 24-48 hours. Rapid urease producers may show a result in as little as 4 hours.
  • Interpretation:
    • Positive result (Rapid): Intense pink/red color change throughout the medium, often visible within 4-6 hours. This indicates strong urease activity.
    • Positive result (Delayed): Pink/red color appears after 24 hours or beyond, indicating weaker urease activity.
    • Negative result: No color change (remains yellow or straw-colored) or acidic (yellow) color if the organism ferments other components in the medium, but does not produce urease. No urease activity detected.
  • Examples of urease-positive bacteria: Proteus spp. (rapid and strong producer, causing urinary tract infections), Klebsiella pneumoniae, Helicobacter pylori (associated with gastric ulcers), Cryptococcus spp. (yeast).

Bile Esculin Test

  • Purpose: Identifies bacteria capable of hydrolyzing esculin in the presence of bile. Primarily used for presumptive identification of Group D streptococci (e.g., S. gallolyticus) and enterococci (Enterococcus spp.).
  • Principle:
    • The medium contains bile salts (e.g., oxgall), which selectively inhibit the growth of many Gram-positive bacteria, except for those tolerant to bile.
    • Esculin is a glycoside composed of glucose and esculetin.
    • Bile-tolerant organisms that possess the enzyme esculinase can hydrolyze esculin into glucose and esculetin.
    • Esculetin then reacts chemically with ferric citrate (an indicator present in the medium) to form a dark brown to black precipitate of iron phenolic complexes.
  • Medium: Bile Esculin Agar (BEA), containing bile salts (oxgall, 40%), esculin, ferric citrate, and nutrients for bacterial growth.
  • Inoculation & Incubation: Streak the agar surface with the test organism and incubate at 35^ ext{o}C for 18-24 hours. Some slow reactors may require up to 48 hours.
  • Interpretation:
    • Positive result: Blackening of the agar surrounding the growth (at least half of the slant if in a tube) due to the formation of iron phenolic complexes. This indicates both bile tolerance and esculin hydrolysis. Read within 18-24 hours for optimal results.
    • Negative result: No blackening of the agar or only a slight discoloration with no more than 1/4 of the slant turning black. Indicates either inability to grow in bile or inability to hydrolyze esculin.
  • Examples of positive bacteria: Enterococcus faecalis (strong positive), Enterococcus faecium, Streptococcus gallolyticus (formerly S. bovis), Listeria monocytogenes (weak positive, but useful differentiation).

Triple Sugar Iron (TSI) Agar

  • Purpose: Differentiates Gram-negative enteric bacilli based on their ability to ferment glucose, lactose, sucrose, and produce hydrogen sulfide (H\text{_2}S) and gas (CO\text{2} and H\text{2}).
  • Principle: TSI agar is a differential medium in a slant/butt format, allowing for the assessment of both aerobic (slant) and anaerobic (butt) metabolism.
    • Carbohydrates: Contains three sugars:
    • Glucose (dextrose) at a low concentration (0.1%): ensures that any organism capable of fermenting glucose will exhaust it quickly, leading to specific reactions.
    • Lactose (1.0%) and Sucrose (1.0%): present at higher concentrations. Organisms fermenting these will produce more acid.
    • pH Indicator: Phenol red, which is yellow below pH 6.8 (acidic) and red above pH 8.1 (alkaline).
    • H\text{2}S Indicator: Ferrous sulfate (and sodium thiosulfate), which reacts with H\text{2}S gas to form a black precipitate (ferrous sulfide).
    • Inoculation: The butt (anaerobic) is inoculated by stabbing, and the slant (aerobic) is streaked. This creates distinct oxygen environments.
  • Interpretation (Read after 18-24 hours):
    1. Fermentation of Glucose only (K/A - Alkaline slant/Acid butt):
      • The organism ferments the small amount of glucose in both the aerobic slant and anaerobic butt, producing acid (yellow). Due to the low glucose concentration, the aerobic slant quickly exhausts glucose, switches to peptone utilization (via oxidative deamination), producing alkaline byproducts (ammonia), which turns the slant red. The anaerobic butt, however, remains acidic (yellow) because acid production from glucose fermentation continues under anaerobic conditions and is not overcome by alkaline byproducts.
      • Appearance: Red slant, yellow butt.
      • Examples: Shigella spp., Salmonella spp. (most, but many produce H\text{_2}S), Proteus spp.
    2. Fermentation of Lactose and/or Sucrose (A/A - Acid slant/Acid butt):
      • The organism ferments lactose and/or sucrose (which are in high concentrations) in addition to glucose. This produces a large amount of acid that overwhelms any alkaline production from peptone deamination in both the slant and the butt.
      • Appearance: Yellow slant, yellow butt. Often accompanied by gas bubbles or cracks in the agar.
      • Examples: Escherichia coli, Klebsiella pneumoniae, Enterobacter spp.
    3. No Carbohydrate Fermentation (K/K or K/NC - Alkaline slant/Alkaline butt or No Change):
      • The organism does not ferment any of the sugars. It primarily utilizes peptones (proteins) in the medium, producing alkaline byproducts both aerobically (slant) and anaerobically (butt).
      • Appearance: Red slant, red butt (K/K) or red slant, no change in butt (K/NC). (No change typically means initial red color remains, or is slightly orange if weak peptone utilization)
      • Examples: Pseudomonas aeruginosa (obligate aerobe, will show K/NC or K/K mostly on slant only), Alcaligenes spp.
    4. H\text{_2}S Production:
      • If the organism produces hydrogen sulfide (H\text{_2}S), usually from the breakdown of sulfur-containing amino acids (cysteine or methionine) or reduction of thiosulfate.
      • The H\text{_2}S gas reacts with ferrous sulfate (an iron salt) in the medium to form black ferrous sulfide.
      • Appearance: Black precipitate, usually observed in the butt (anaerobic conditions favoring H\text{_2}S production).
      • Examples: Salmonella spp., Proteus spp., Citrobacter spp.
    5. Gas Production:
      • Fermentation of carbohydrates can produce gas (CO\text{2} and H\text{2}).
      • Appearance: Bubbles, cracks, or displacement of the agar in the butt.
      • Examples: E. coli, Klebsiella spp., many Enterobacter spp.
  • Combined Interpretations:
    • K/A: Red slant/Yellow butt (glucose fermenter only)
    • A/A: Yellow slant/Yellow butt (lactose and/or sucrose fermenter)
    • K/K or K/NC: Red slant/Red butt or Red slant/No change (non-fermenter)
    • K/A, H\text{_2}S: Red slant/Yellow butt with black precipitate (e.g., Salmonella typhi)
    • A/A, Gas: Yellow slant/Yellow butt with gas bubbles (e.g., E. coli)

Blood Agar Plate (BAP)

  • Purpose: A general-purpose enriched medium used for the isolation and cultivation of fastidious microorganisms and, most importantly, for the detection and differentiation of hemolytic activity (the lysis of red blood cells).
  • Principle:
    • BAP consists of a nutritious base (e.g., Tryptic Soy Agar, Columbia Agar) supplemented with 5-10% sterile sheep red blood cells (or other animal blood).
    • The blood provides essential growth factors (hemin, NAD) for fastidious organisms that cannot synthesize them.
    • Hemolytic reactions are observed as characteristic zones of clearing or discoloration around bacterial colonies, which are caused by bacterial exotoxins called hemolysins.
  • Inoculation & Incubation: Streak for isolation, typically incubated at 35^ ext{o}C with 5-10% CO\text{2} (capnophilic conditions) for 18-24 hours to enhance growth of streptococci.
  • Types of Hemolysis:
    1. Alpha (\alpha)-hemolysis (Partial Hemolysis):
      • Mechanism: Incomplete lysis of red blood cells around the colonies. Hemoglobin is oxidized to methemoglobin.
      • Appearance: A green or brownish discoloration/zone around the colonies. The zone is typically opaque, not completely clear.
      • Examples: Streptococcus pneumoniae (causes alpha-hemolysis and is sensitive to optochin), viridans streptococci (a group of streptococci that often cause alpha-hemolysis and are resistant to optochin).
    2. Beta (\beta)-hemolysis (Complete Hemolysis):
      • Mechanism: Complete lysis of red blood cells, resulting from the production of potent hemolysins (e.g., streptolysin O, streptolysin S by S. pyogenes; alpha-toxin by S. aureus).
      • Appearance: A clear, transparent zone around the colonies through which the underlying agar plate can be easily seen. This zone indicates complete destruction of red blood cells.
      • Examples: Streptococcus pyogenes (Group A Strep), Streptococcus agalactiae (Group B Strep), Staphylococcus aureus, Clostridium perfringens.
    3. Gamma (\gamma)-hemolysis (Non-hemolytic):
      • Mechanism: No lysis of red blood cells.
      • Appearance: No change in the agar around the colonies; the agar remains red and opaque.
      • Examples: Enterococcus faecalis, Staphylococcus epidermidis, many non-pathogenic Bacillus species.
  • Applications:
    • Primary Isolation: Used to grow a wide range of bacteria, including fastidious ones.
    • Differentiation: Key for differentiating streptococci and staphylococci based on their distinct hemolytic patterns.
    • Susceptibility Testing: Can be used in some antimicrobial susceptibility tests.
    • Virulence Factor Detection: Hemolysis is an important virulence factor for many pathogens.

Summary Takeaways

- Microbiological tests like Urease, Bile Esculin, TSI, and BAP provide essential information for identifying bacteria based on their metabolic and enzymatic capabilities and growth characteristics.