Culture Media

• Bacteria have to be grown (cultured) for them to be identified
• Agar

  • Used for preparing solid medium
  • Obtained from seaweeds
  • No nutritive value
  • Not affected by the growth of the bacteria
  • Melts at 98°C & sets at 42°C
  • 2% agar is employed in solid medium
• Solid medium


  • Discrete colonies
• Special media based on Oxygen requirement

  • Aerobic media
  • Anaerobic media
• Enriched media

  • Substances like blood, serum, egg are added to the basal medium
  • Used to grow bacteria that are exacting in their nutritional needs
  • Examples: Blood agar, Chocolate agar
• Liquid medium


  • Diffuse growth
• Indicator media contain an indicator which changes its colour when a bacterium grows in them
• Gelatin is not satisfactory as a solid medium as it liquefies at 24°C
• Synthetic or defined media

  • Media prepared from pure chemical substances and its exact composition is known
  • Example: peptone water – 1% peptone + 0.5% NaCl in water
• Special media

  • Enriched media
  • Enrichment media
  • Selective media
  • Indicator media
  • Differential media
  • Sugar media
  • Transport media
  • Media for biochemical reactions
• Anaerobic media are used to grow anaerobic organisms
• Transport media are used for transporting samples
• Selective media have inhibitory substances added to a solid media
• The original media used by Louis Pasteur were urine or meat broth
• Complex media

  • Media other than basal media
  • They have added ingredients
  • Provide special nutrients
• Sugar media contain fermentable substances like glucose, arabinose, lactose, starch, etc.
• Enrichment media are liquid media used to isolate pathogens from a mixed culture
• Types of culture media based on the constituents/ingredients

  • Simple medium
  • Complex medium
  • Synthetic or defined medium
  • Special media
• Biochemical tests

  • Oxidase test
  • Triple sugar iron agar (TSI)
  • Indole test
• Robert Koch used cooked cut potato as the earliest solid medium
• Colony

  Macroscopically visible collection of millions of bacteria originating from a single bacterial cell
• Simple media / basal media

  • Examples: NB, NA
  • NB consists of peptone, meat extract, NaCl
  • NB + 2% agar = Nutrient agar
• Procedures for growing bacteria

  They have to be grown separately (isolated) on culture media and obtained as pure for study
• Blood agar and Chocolate agar are examples of enriched media
• Differential media are able to distinguish between bacteria
• Biochemical tests and reactions provide additional information for the identification of the bacterium
• Types of culture media based on their consistency

  • Solid medium
  • Liquid medium
  • Semi solid medium
• Transport media for delicate organisms

  • Stuart’s medium - non-nutrient soft agar gel containing a reducing agent
  • Buffered glycerol saline - enteric bacilli
• Indole test

  1. Used to detect indole production by the organism

  2. Positive test indicated by a pink ring

  3. Indole positive - E.coli; Indole negative - Klebsiella, Salmonella
• Culture methods

  • Streak culture
  • Lawn culture
  • Stroke culture
  • Stab culture
  • Pour plate method
  • Liquid culture
  • Anaerobic culture methods
• Triple sugar iron agar (TSI)

  1. Composite media used to study different properties of a bacterium - sugar fermentation, gas production, and H2S production

  2. Contains Glucose, Lactose, Sucrose, Iron salt (Ferric citrate), and Phenol red as the indicator

  3. Shows yellow (acid) or pink (alkaline) reactions

  4. Differentiates between Lactose fermenters (A/A), Non-lactose fermenters (K/A), and Non-fermenters (K/K)

  5. Indicates H2S production with black color and gas production with gas bubbles or cracks

  6. Lactose fermenters - E.coli, Klebsiella; Non-lactose fermenters - Salmonella, Shigella; H2S production - Proteus
• Anaerobic media

  • Robertson’s cooked meat medium
  • Thioglycolate medium
• Indications for culture methods

  • To isolate bacteria in pure cultures
  • To demonstrate their properties
  • To obtain sufficient growth for the preparation of antigens and for other tests
  • For bacteriophage & bacteriocin susceptibility
  • To determine sensitivity to antibiotics
  • To estimate viable counts
  • Maintain stock cultures
• Citrate utilization

  1. Done in Simmon’s Citrate medium

  2. Detects the ability of certain bacteria to utilize citrate as the sole source of carbon

  3. Citrate positive - blue color; Citrate negative - green color

  4. Positive - Klebsiella; Negative - E.coli
• Biochemical tests & reactions

  • Provide additional information for the identification of the bacterium
  • Include Oxidase test, Triple sugar iron agar (TSI), Indole test, Citrate utilization, Urease test
• Streak culture

  1. Used for the isolation of bacteria in pure culture from clinical specimens

  2. Inoculum is distributed thinly over the plate by streaking it with a loop in a series of parallel lines in different segments of the plate
• Urease test

  1. Done in Christensen’s urease medium

  2. Used to detect organisms that produce urease

  3. Urease positive - pink color; Urease negative - yellow color

  4. Positive - Proteus, Klebsiella; Negative - E.coli, Salmonella
• Oxidase test

  1. Detects the presence of an enzyme “oxidase” produced by certain bacteria which will reduce the dye – tetramethyl-p-phenylene diamine dihydrochloride

  2. Positive test is indicated by the development of a purple colour

  3. Oxidase positive - Pseudomonas, Vibrio, Neisseriae

  4. Oxidase negative - Salmonella, Shigella
• Pour plate culture preparation

  Agar medium is melted (15 ml) and cooled to 45°C. 1 ml of the inoculum is added to the molten agar. Mix well and pour to a sterile petri dish. Allow it to set. Incubate at 37°C, colonies will be distributed throughout the depth of the medium
• Chemical method for anaerobic culture

  Alkaline pyrogallol absorbs oxygen