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Originally described by FF. Russel, the (TSIA) triple Sugar. Iron Agar test is used to identify enteric bacteria. It is based on the ability to catalyse glucose, lactose or sugar and to liberate sulphur from ferrous ammonium sulphate or sodium thiosulfate. Triple Sugar Iron Agar slants contain all concentration of lactase and sucrose, and a 0.1 % glucose concentration. The PH indicator, phenol red, is also incorporated into the medium to detect acid production from carbohydrate fermentation.

Materials Required:

i) Sample

ii) straight Inoculating wire

iii) Test organism

iv) Bunsen Burner

v) Incubator.


i) Inoculate TSIA agar slants with sample organisms. Inoculation is done by stabbing in the butt by straight wire and streaking in the slant with the same wire.

ii) Incubate only for 18 to 24 hours at 37°c.

iii) Examine TSIA tube for the colour change in slant and butt and for the presence of blackening of the medium and for gas formation. Note that the initial colour of the medium is pink. 

iv) Tally your result with the table to identify the micro-organism (for table refer theory portion, chapter 6).

Observation Table:

S.NMedia UsedInitial colourColour of slant Colour of buttGas productionRemarks 
1TripleSugar IronAgar(TSIA)RedYellowYellowGas was produceGlucose was fermented with acid tgas.lactose and sucrose was fermented

Result and Discussion:

From the observation, we found that the test organism is a member of Enterobacteriae family which metabolises glucose from media anaerobically in butt and aerobically in the slant. The acid in the media course, PH indicator phenol red to assume a yellow colour. Then the organism also utilize sucrose from the media and the media will remain yellow. There is the production of gas which breaks up the media and is pushed up in the tube.


Hence the ISIA test proceeded to test the organism by fermentation of nutrients present in media.


i) The presence of O₂ is necessary for proper reaction so the capps may be kept loose.

II) Careless stabbing may form a channel in the agar inside the tube’s wall, through which gas may escape preventing its detection. 

iii) In TSIA test incubation period is critical, i.e. 37°c for 18 hours. Otherwise, we may get false results within 18 hours, all sugars will be used up, reaching the degradation stage.


Shah Ple, Dahal, Amatya J (2013) “Techniques in Microbiology” Heritage Publisher and Distributor Bhotahity, KTM Nepal Pg no. 139.

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