Principle:
Triple sugar iron agar test is used to determine whether gram negative bacilli utilize glucose and lactose or sucrose fermentatively and produce hydrogen sulfide (H2S).  It contains 10 parts of lactose: 10 parts of sucrose: 1 part of glucose and peptone.  Phenol red and ferrous sulphate serves as an indicator for acidification of medium and H2S production respectively.
Glucose is utilized first by a fermentative organism and the entire medium becomes acidic (yellow) in 8 to 12 hours. Butt remains acidic even after 18 to 24 hours incubation peroid because of the presence of organic acids resulting from the fermentation of glucose under anaerobic conditions in the butt of the tube.The slant reverts to alkaline state that is indicated by red color as the fermentation products gets oxidised to carbon dioxide (CO2) and water (H2O) and peptone in aerobic condition the slant undergoes oxidation releasing alkaline amines (Phenol red in alkaline pH turns red while in acidid pH turns yellow).
  • If the organism ferments glucose but does not ferment lactose and/or sucrose, then the slant becomes red and butt remains yellow (K/A) within 18 to 24 hrs.
  • If the organism in addition to glucose ferments lactose and/or sucrose, the fermentation product formed on the slant will more than neutralize the alkaline amines rendering the slant acidic (yellow) (A/A) provided the reaction is read within 18 to 24 hours.
  • If the organism is non fermenter, instead of sugars, peptone is utilised as an alternate source of energy under aerobic condition on the slant which makes it alkaline indicated by the red color while there is no change in the color of the butt. K/NC

Reactions in TSI should not be read after 24 hours of incubation, because aerobic oxidation of fermentation products form lactose and/or sucrose will occur and the slant will eventually revert to alkaline state.
The formation of CO2 and H2 is indicated by the presence of bubbles or cracks in the medium or by the separation of the agar from sides or bottom of the tube. The production of H2S rqires an acidic condition and is indicated by blackening of the butt of the medium in the tube.

Procedure:
  1. Touch a well isolated colony with a sterile straight wire.
  2. Inoculate TSI by first stabbing through the centre of the medium to the bottom of the tube and then streak the surface of the slant.
  3. Leave the cap loose and incubate the tube at 35 in ambient air for 18 to 24 hours.
  4. Observe the reaction

Expected results:

 
Alkaline slant/no change in butt (K/NC) or Alkaline slant/Alkaline butt (K/K) = glucose, lactose and sucrose nonfermenter.(red/red)
Alkaline slant/acidic butt (K/A)- glucose fermentation only. (red/yellow)

Acidic slant/acidic butt (A/A)- glucose, lactose and/or sucrose fermenter. (yellow/yellow)
A black precipitate in the butt indicates production of H2S . H2S produced reacts with ferric salt to produce black precipitate of ferrous sulfide.
Bubbles or cracks in the tube indicate the production of CO2 or H2. Drawing of circle around butt indicates that gas is produced by glucose and sucrose or glucose and lactose and glucose, sucrose and lactose fermenter.
 

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