M BACTE: CULTURE MEDIA

• Culture Media


  Media containing the required nutrients for bacterial growth
• Culture Media


  • Used for: Isolation and identification of micro-organisms
  • Performing anti-microbial sensitivity tests
• Common Ingredients of Culture Media

  • Free amino acids
  • Peptides
  • Proteoses (large sized peptides)
  • Carbohydrates
  • Nucleic acid fractions
  • Minerals
  • Vitamins
• Peptone


  Hydrolyzed product of animal and plant proteins
• Meat extract


  Supplies amino acids, vitamins and mineral salts
• Yeast extract


  Bacterial growth stimulant
• Agar


  • Inert polysaccharide of seaweed
  • Has high gelling strength
  • High melting temperature (90-95°C)
  • Low gelling temperature
  • Forms firm gel at 1.5% W/V concentration
  • Forms semisolid gel at 0.4-0.5% W/V concentration
• Agar


  • Solidifies culture media
  • May provide calcium and organic ions to inoculated bacteria
• Water


  Deionized or distilled water must be used in the preparation of culture media
• Factors that determine selection of culture media

  • The major pathogens to be isolated, their growth requirements and the features by which they are recognized
  • Whether the specimens being cultured are from sterile sites or from sites having normal microbial flora
  • The cost, availability and stability of media
  • The training and experience of laboratory staff in preparing, using and controlling culture media
• Liquid (Broth) Media


  Nutrients are dissolved in water, and bacterial growth is indicated by a change in broth's appearance from clear to turbid (i.e. cloudy)
• Liquid (Broth) Media


  • Used as: Enrichment media
  • Biochemical testing media
  • Blood culture media
• Solid (Agar) Media


  • Made by adding a solidifying agent (usually agarose) to the nutrients and water
  • The Petri dish containing the agar is referred to as agar
• Solid Agar Media


  • Plate cultures in petri dishes
  • Stab/slope cultures in tubes and bottles
• Characteristics of bacterial colonies on solid agar media

  • Size: diameter in mm
  • Outline: circular, entire, wavy, indented
  • Elevation: flat, raised, low convex and dome shaped
  • Transparency: transparent, opaque, and translucent
  • Surface: smooth (mucoid) and shiny, rough and dull
  • Color: colorless, white, pink, and pigmented
  • Changes in medium: e.g. Hemolysis in Blood Agar / Blackening of medium due to hydrogen sulfide production
• Basic /Simple / All Purpose Media


  Contain only basic nutrients required for the growth of ordinary organisms, and used as a general purpose media
• Basic /Simple / All Purpose Media

  • Used to: Prepare enriched media
  • Maintain stock cultures of control bacterial strains
  • Subculture pathogenic bacteria from selective/differential medium prior to performing biochemical or serological tests
• Basic /Simple / All Purpose Media

  • Nutrient Broth / Nutrient Agar
• Complex media


  Media that contain some ingredients of unknown chemical composition
• Nutrient broth


  Commonly used complex medium, a simple basal liquid medium that supports growth of many organisms
• Types of Nutrient Broth

  • Meat infusion broth
  • Meat extract broth
  • Digest broth
• Nutrient agar


  • Prepared by adding agar at a concentration of 2% to the nutrient broth
  • Simplest and most common medium used routinely in microbiology laboratories, to grow nonfastidious bacteria
• Semisolid agar


  If the concentration of agar is reduced to 0.2–0.5%, enables motile organisms to spread
• Firm agar


  If the concentration of agar is increased to 6%
• Synthetic or Chemically Defined Media


  Prepared exclusively from pure chemical substances and their exact composition is known
• Enriched Media


  Prepared to meet the nutritional requirements of more bacteria by the addition of substances such as blood, serum or egg to a basal medium
• Blood agar


  • Used for isolation of streptococci, pneumococci, Haemophilus
• Chocolate agar


  • Used to culture even more fastidious bacteria
  • Used for isolation of Neisseria (Meningococci and Gonococci) and Haemophilus
• Loeffeler's serum


  • Serum is added for enriching the medium
  • Used for the isolation of Corynebacterium diphtheriae
• Enrichment Media


  Substance added to a liquid medium which inhibits the growth of unwanted bacteria and favors the growth of wanted bacteria
• Enrichment Media


  • Tetrathionate broth: inhibits coliforms while allowing typhoid-paratyphoid bacilli to grow freely in fecal sample
  • Selenite F (F for feces) broth: used for dysentery bacilli
  • Alkaline peptone water: used for Vibrio cholerae from feces
• Selective Media


  Substance is added to a solid medium which inhibits the growth of unwanted bacteria but favors the growth of wanted bacteria
• Selective Media


  • Deoxycholate citrate agar (DCA): acts as a selective agent for dysentery bacilli (isolation of Shigellae)
  • Lowenstein-Jensen medium: used for Mycobacterium tuberculosis
  • Bile salt agar (BSA): a selective agent, allows the growth of only Vibrio cholerae and inhibits the growth of intestinal organisms
• Indicator Media


  Contain an indicator which changes color when a bacterium grows in them
• Indicator Media


  • Wilson and Blair medium: incorporation of sulfite, S. typhi reduces sulfite to sulfide in the presence of glucose and the colonies of S. Typhi have a black metallic sheen
  • Macconkey agar: indicates lactose fermenting property, lactose fermenter (LF) produces pink colonies and non-lactose fermenter (NLF) produces colorless colonies due to a neutral red indicator
• Differential Media


  Has substances incorporated in it, enabling it to bring out differing characteristics of bacteria and thus helping to distinguish between them
• Differential Media


  • Macconkey agar is both differential and selective, contains peptone, meat extract, NaCl, bile salt, lactose and neutral red indicator
• Sugar Media


  Used to test carbohydrate fermentation, which is important for characterization and identification of bacteria, particularly Gram negative bacilli
• Sugar Media


  • Usual sugar media consist of 1% of the sugar in peptone water along with an appropriate indicator (Andrade's indicator—0.005% acid fuchsin in NaOH)
  • Color of the medium is light yellow
  • Gas accumulates in the inner Durham's tube
  • Acid production is indicated by the development of pink color
• Sugar Media


  • Glucose, lactose, sucrose and mannitol