culture media

• Culture Media


  Liquid or solid substance - contains nutrients to support the growth, and survival of microorganisms
• Constituents of Culture Media


  • Water and Electrolytes
  • Peptone: Mixture of partially digested proteins - obtained from various sources - heart muscle, casein or fibrin, or soya
  • Agar: Used for solidifying the culture media - Solid Media: 1-2% Agar, Semi solid Media: 0.5%
  • Meat extract
  • Yeast extract
  • Blood and serum: Important components of enriched media; provide extra nutrition to fastidious bacteria
• Types of Culture Media (based on consistency)

  • Liquid (or broth) media
  • Semisolid media
  • Solid media
• Types of Culture Media (conventional)

  • Prepared from nutrients - aqueous extract of meat, peptone
  • Simple/basal media
  • Enriched media
  • Enrichment broth
  • Selective media
  • Differential media
  • Transport media
  • Anaerobic media
• Types of Culture Media (automated)

  • Mainly available for blood and sterile body fluid culture
  • Based on the growth detection
• Simple/Basal Media


  Contain minimum ingredients that support the growth of non-fastidious bacteria
• Simple/Basal Media


  • Peptone water: Contains peptone (1%) + NaCl (0.5%) + water
  • Nutrient broth: Peptone water + meat extract (1%)
  • Nutrient agar: Nutrient broth + 2% agar
  • Semisolid medium: Concentration of agar - reduced to 0.2–0.5 %
• Uses of Basal Media


  • Testing the non-fastidiousness of bacteria
  • Serve as base for the preparation of many other media
  • Performing the biochemical tests, such as oxidase, catalase, etc.
  • Study the colony morphology
  • Pigment demonstration
• Enriched Media


  • To Basal Media Substances such as blood, serum or Egg are added
  • Used to grow bacteria which require extra nutrients
• Enriched Media


  • Blood Agar
  • Chocolate Agar
• Blood agar


  • Prepared by adding 5-10% of sheep blood to the molten nutrient agar at 45°C
  • Tests the hemolytic property of the bacteria, which may be either partial or α (green) hemolysis and complete or β hemolysis
• Chocolate agar


  • Heated blood agar, prepared by adding 5 -10% of sheep blood to the molten nutrient agar at 70°C
  • Supports certain highly fastidious bacteria - Haemophilus influenzae that does not grow on blood agar
• Enrichment Broth


  • Liquid media added with inhibitory agents which selectively allow certain organism to grow and inhibit others
  • Important for isolation of pathogens from clinical specimens which also contain normal flora (e.g. stool and sputum specimen)
• Enrichment Broth


  • Tetrathionate broth—Used for Salmonella Typhi
  • Selenite F broth—Used for Shigella
• Selective Media


  Solid media containing inhibitory substances - inhibit the normal flora present in the specimen and allow the pathogens to grow
• Selective Media


  • Lowenstein Jensen (LJ) medium : Mycobacterium tuberculosis
  • DCA (Deoxycholate Citrate Agar) : Salmonella and Shigella from stool
• Lowenstein–Jensen medium


  • Selective media for Mycobacterium tuberculosis
• Transport Media


  • Used for the transport of the clinical specimens suspected to contain delicate organism or when delay is expected while transporting the specimens from the site of collection to the laboratory
  • Bacteria do not multiply in the transport media - they only remain viable
• Transport Media

  • Alkaline peptone water for Vibrio cholerae
  • Amies medium, Stuart's medium for Neisseria
• Differential Media


  Differentiate between two groups of bacteria - by using an indicator
• Differential Media


  • MacConkey agar : Differentiates organisms into LF (pink colonies, e.g. Escherichia coli) and NLF or (colorless colonies, e.g. Shigella)
• Anaerobic Culture Media


  Contain reducing substances which take up oxygen and create lower redox potential - permit the growth of obligate anaerobes, such as Clostridium
• Anaerobic Culture Media


  • Robertson's cooked meat (RCM) broth
• Blood Culture Media


  • Recovery of bacteria from blood is difficult - they are usually present in lesser quantity and many of the blood pathogens are fastidious
  • Enriched media - used for isolating microorganisms from blood
• Conventional Blood Culture Media

  • Monophasic medium: Contains brain–heart infusion (BHI) broth
  • Biphasic medium: Liquid phase containing BHI broth and a solid agar slope made up of BHI agar
• Automated Blood Culture Techniques


  • Continuous automated monitoring: Blood culture bottles - periodically monitored for the microbial growth - every 10 minutes by the instrument
  • BacT/ALERT 3D: When bacteria multiply - produce CO2 - increases the pH - changes the color of a blue-green sensor present at the bottom of the bottle to yellow
• Culture Methods


  • Involve inoculating the specimen on to appropriate culture media, followed by incubating the culture plates in appropriate conditions
• Culture Methods


  • Bacteriological loop and straight wire
  • Flaming the loop (red hot)
• Biosafety Cabinet (BSC)


  Enclosed, ventilated laboratory work station, used to protect the laboratory personnel while working with potential infectious clinical specimens
• Streak Culture


  • Used for culture of specimens - blood or body fluids - inoculated by directly adding the specimen in to the liquid medium or with the help of a syringe or pipette
  • Bacterial growth - detected by observing turbidity in the medium. Some aerobic bacteria form surface pellicles
• Lawn or Carpet Culture


  • Uniform lawn of bacterial growth is obtained by either swabbing or flooding with a bacterial broth onto the culture plate
  • Useful to carry out antimicrobial susceptibility testing (AST) by disk diffusion method
• Stroke Culture


  • Carried out on agar slopes or slants by streaking the straight wire in a zigzag fashion
  • Used for biochemical test such as urease test
• Stab Culture


  • Made by stabbing the semisolid agar butt by a straight wire
  • Used for motility testing using mannitol motility medium
• Incubatory Conditions


  • Most pathogenic bacteria - aerobes or facultative anaerobes - grow best at 37°C
  • Inoculated culture plates - incubated at 37°C aerobically for overnight in an incubator
• Incubatory Conditions for Capnophilic Bacteria

  • Candle jar is used
  • Inoculated media - placed inside a jar with a lighted candle - burning candle reduces oxygen to a point where the flame goes off
  • Provides an atmosphere of approximately 3–5% CO2
  • Useful for capnophilic bacteria - Brucella, Streptococcus, pneumococcus and gonococcus
• Anaerobic Culture Methods


  • Evacuation and Replacement
  • Absorption of Oxygen by Chemical Methods
  • Anaerobic Glove box and Anaerobic Work Station
  • Reducing Agents
• Evacuation and Replacement (Manual Method)


  McIntosh and Filde's anaerobic jar - most popular method for creating anaerobiosis in the past, now not in use
• Evacuation and Replacement (Automated System)

  • Anoxomat: Automatically evacuates air - replaces by hydrogen gas from a cylinder
  • Catalyst - sachet containing aluminum pellets coated with palladium
• Absorption of Oxygen by Chemical Methods

  GasPak system: Traces of oxygen - removed by same catalyst used for Anoxomat
• Reducing Agents


  • Oxygen in culture media - reduced by various reducing agents - glucose, thioglycollate, cooked meat pieces, cysteine and ascorbic acid
  • Robertson cooked meat broth - most widely employed anaerobic culture medium - uses chopped meat particles (beef heart) as reducing agent