A Comprehensive Guide to Bacterial Culture Media and Identification Techniques

Posted on Jul 30, 2024 in Biology

Bacterial Culture Media

Agar is a solid culture medium that contains agarose, a gelatinous substance derived from seaweed, which allows it to solidify. Broth is a liquid culture medium used for growing bacteria in a liquid state.

Types of Media

  • Selective: Media that favor the growth of particular microorganisms and inhibit the growth of others. Example: Mannitol Salt Agar (MSA).
  • Differential: Media that distinguish between different groups of bacteria based on their biological characteristics. Example: Eosin Methylene Blue (EMB) agar.
  • All-purpose/Supportive: Media that support the growth of a wide variety of microorganisms. Example: Tryptic Soy Agar (TSA).
  • Enrichment: Media that contain special nutrients to enhance the growth of particular bacteria. Example: Blood agar.

Common Culture Media

Eosin Methylene Blue (EMB) Agar

  • Selective for: Gram-negative bacteria.
  • Differentiation: Lactose fermentation. Lactose fermenters produce acid, leading to a color change.
  • Color of E. coli on EMB plate: Metallic green sheen.
  • Selective Ingredients: Eosin Y and methylene blue dyes (select for Gram-negative bacteria).
  • Differential Ingredient: Lactose (differentiates lactose fermenters, resulting in color change).
  • Lactose Fermenters: Dark colonies with metallic green sheen (e.g., E. coli).
  • Non-Lactose Fermenters: Colorless or light pink colonies (e.g., Salmonella).

Mannitol Salt Agar (MSA)

  • Selective Ingredient: High salt concentration (selects for Staphylococcus species).
  • Differential Ingredient: Mannitol and phenol red (differentiates mannitol fermenters, turning yellow).
  • Mannitol Fermenters: Yellow colonies and medium (e.g., Staphylococcus aureus).
  • Non-Mannitol Fermenters: Red or pink colonies (e.g., Staphylococcus epidermidis).
  • pH > 8.4 = pink, pH 6.9 – 8.4 = red, pH < 6.9 = yellow

Other Media

  • TSA plate: Used to culture Staphylococcus epidermidis, Micrococcus luteus, and Serratia marcescens.
  • EMB plate: Used to culture Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae.

Bacterial Plating Techniques

  • Spread Plate: Original culture is serially diluted, then the final dilution is spread on the surface of a plate. Surface colonies grow.
  • Pour Plate: A diluted microbial sample is mixed with melted agar and poured into a petri dish to solidify, allowing colonies to grow within and on the surface of the agar.
  • Streak Plate: Original culture directly diluted across a new plate with a loop.

Hemolysis on Blood Agar

  • Beta hemolysis: Complete lysis of red blood cells around the colony. Example: Streptococcus pyogenes.
  • Alpha hemolysis: Partial lysis of red blood cells, resulting in a greenish discoloration around the colony. Example: Streptococcus pneumoniae.
  • Gamma hemolysis: No lysis of red blood cells, no change in the blood agar around the colony. Example: Enterococcus faecalis.

Antibiotic Resistance

  • MRSA: Methicillin-Resistant Staphylococcus aureus.
  • VRE: Vancomycin-Resistant Enterococci.
  • ESBL: Extended-Spectrum Beta-Lactamases.
  • PRSP: Penicillin-Resistant Streptococcus pneumoniae.
  • CRE: Carbapenem-Resistant Enterobacteriaceae. CRE is often considered the most severe due to its high level of resistance and limited treatment options.

Biochemical Testing

Simmons Citrate Agar

  • Differential medium used to determine if an organism can utilize citrate as a sole carbon source.
  • Organisms with the enzyme citrate lyase can utilize citrate, turning the medium from green to blue due to the pH indicator bromothymol blue.
  • Positive reaction: Blue agar slant (e.g., Enterobacter aerogenes).
  • Negative reaction: Green agar slant (e.g., Escherichia coli).

Urea Agar

  • Differential medium used to identify bacteria that can hydrolyze urea into ammonia and carbon dioxide using the enzyme urease.
  • The release of ammonia increases pH, changing the medium from yellow to fuchsia (hot pink) due to the pH indicator phenol red.
  • Positive reaction: Fuchsia agar slant (e.g., Proteus vulgaris).
  • Negative reaction: Yellow agar slant (e.g., Escherichia coli).

Bacterial Morphology and Identification

Staphylococcus

  • Gram-positive cocci (round) that appear in grape-like clusters.
  • Catalase positive.
  • S. aureus: Coagulase positive, Staphyloside test positive.
  • S. epidermidis: Coagulase negative, Staphyloside test negative.
  • Commonly found: Nasal passages (nares), skin (particularly in moist areas like armpits and groin).

Streptococcus

  • Gram-positive cocci (round) that appear in chains or pairs.
  • Catalase negative.
  • Lancefield classification and hemolytic activity are used for identification.
  • Group A: Contains Streptococcus pyogenes (beta-hemolytic). Example: S. pyogenes, known for causing strep throat and skin infections.
  • Group B: Contains Streptococcus agalactiae (beta-hemolytic). Example: S. agalactiae, known for causing neonatal infections and sepsis.
  • Group D: Contains Enterococci and non-enterococci species (gamma-hemolytic). Example: Enterococcus faecalis (part of the normal gut flora).