Triple Sugar Iron Agar (TSI): Principle, Procedure and Interpretation

TSI Agar Test results
Triple Sugar Iron Agar Test Results Image source: Clark College

Whenever you see the name of this test i.e. Triple Sugar Iron Agar ,you have to remember that it’s a test which has three sugar (Lactose, Sucrose, and Glucose) and also iron; and it contains Agar Agar as solidifying agent (TSI is a semi solid media having slant and butt).

CLUE: You might have (or not) realized the rationale behind the use of three different sugar and adding iron. Lets start with very basic information and we will proceed towards principle and interpretations.

Composition of Triple Sugar Iron Agar (TSI)
Lactose, Sucrose and Glucose in the concentration of 10:10:1 (i.e. 10 part Lactose (1%), 10 part Sucrose (1%) and 1 part Glucose (0.1%)). TSI is similar to Kligler’s iron agar (KIA), except that Kligler’s iron agar contains only two carbohydrates: glucose (0.1%) and lactose (1%).

  • 0.1% Glucose: If only glucose is fermented, only enough acid is produced to turn the butt yellow.  The slant will remain red
  • 1.0 % lactose/1.0% sucrose:  a large amount of acid turns both butt and slant yellow, thus indicating the ability of the culture to ferment either lactose or sucrose.
  • Iron: Ferrous sulfate: Indicator of H2S formation
  • Phenol red: Indicator of acidification (It is yellow in acidic condition and red under alkaline conditions).
  • It also contains Peptone which acts as source of nitrogen. (Remember that when ever peptone is utilized under aerobic condition ammonia is produced)

Why Sucorse is added in TSI? 

Inoculation in TSI Agar
Inoculation in TSI Agar

Addition of sucrose in TSI Agar  permits  earlier detection of coliform bacteria that ferment sucrose more rapidly than lactose. Adding sucrose also aids the identification of certain gram-negative bacteria that could ferment sucrose but not lactose. Other basic understanding is TSI Tube contains butt (poorly oxygenated area on the bottom) slant (angled well oxygenated area on the top).

Procedure for Triple Sugar Iron Agar (TSI) Test

  1. With a sterilized straight inoculation needle touch the top of a well-isolated colony
  2. Inoculate TSI Agar by first stabbing through the center of the medium to the bottom of the tube and then streaking on the surface of the agar slant. 
  3. Leave the cap on loosely and incubate the tube at 35°C in ambient air for 18 to 24 hours.

Interpretation of Triple Sugar Iron Agar Test

  1. If lactose (or sucrose) is fermented, a large amount of acid is produced, which turns the phenol red indicator yellow both in butt and in the slant. Some organisms generate gases, which produces bubbles/cracks on the medium.
  2. If lactose is not fermented but the small amount of glucose is, the oxygen deficient butt will be yellow (remember that butt comparatively have more glucose compared to slant i.e. more media more glucose), but on the slant the acid (less acid as media in slant is very less) will be oxidized to carbondioxide and water by the organism and the slant will be red (alkaline or neutral pH).
  3. If neither lactose/sucrose nor glucose is fermented, both the butt and the slant will be red. The slant can become a deeper red-purple (more alkaline) as a result of production of ammonia from the oxidative deamination of amino acids (remember peoptone is a major constitutents of TSI Agar) .
  4. if H2S is produced, the black color of ferrous sulfide is seen.

So the expected results of TSI Agar test are:

TSI Agar Test results
Triple Sugar Iron Agar Test Results
Image source: Clark College
  1. Alkaline slant/no change in butt (K/NC) i.e Red/Red = glucose, lactose and sucrose non-fermenter
  2. Alkaline slant/Alkaline butt (K/K) i.e Red/Red = glucose, lactose and sucrose non-fermenter
  3. Alkaline slant/acidic butt (K/A); Red/Yellow = glucose fermentation only, gas (+ or -), H2s (+ or -)
  4. Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or sucrose fermenter gas (+ or -), H2s (+ or -).

Some example of Triple Sugar Iron (TSI) Agar Reactions: 


Name of the organisms Slant Butt Gas H2S
Escherichia, Klebsiella, Enterobacter Acid (A) Acid (A) Pos (+) Neg (-)
Shigella, Serratia Alkaline (K) Acid (A) Neg (-) Neg (- )
Salmonella, Proteus Alkaline (K) Acid (A) Pos (+) Pos (+)
Pseudomonas Alkaline (K) Alkaline (K) Neg (-) Neg (-)


About Tankeshwar Acharya 250 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion, I am working as a Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


    • In my opinion, that result is not posible to achieve. There is only one way to get a yellow slant: with lactose/sucrose fermenters. In that case, even the butt will be yellow a cause of the quantity of lactose/sucrose.

    • Hello Sir, i have isolate a pathogen from food sample that gives some positive reaction for Salmonella, but it gives red slant and yellow butt with gas production on TSI agar. Is it the positive reaction for Salmonella ???

    • H2S is produced only under anaerobic respiration. Anaerobic respiration is occurred only in the butt

  1. Komal, in the slant we streak the colony but in the butt we stab the colony. When the H2S is produced in the slant, it get escaped but in the slant, it get trapped and react with Iron, resulting the black pigmentation..

    • but in case of my isolates there is h2s production in slants not in butt. why this is so? and also my results dont come in 24 hours it takes more time almost more then 30 hours. is this possible? i have islotaes of salmonella

  2. Ayesha, Yellow comes because of Acid production, for slant to be acidic, the organism has to ferment glucose, but the concentration of glucose is less, so after exhaustion of glucose source, the organism may use sucrose/lactose or peptone. If organism is fermenter, it will ferment at the butt also, if organism is non-fermenter, it will oxidize the peoptone and alkali condition will come. So as per my knowledge and understanding such condition is unlikely to occur.

    • HI ! referring to her question, I finished working on an unknown project and my bacteria turned out to be E. coli. I know E.coli is supposed to turn the TSI media yellow completely, however, my TSI had a yellow slant and a red butt. Could it have been that there was not enough bacteria inoculated? or contamination? or incubated for too long?

      • Hello Alianis Hernandez, yellow slant and red butt reaction can not be justified in any circumstances, the reverse can happen in case of NLF and no change in both slant and butt can be seen in case non fermenter and in case of oxidative organisms, the slant become red but butt may not change. I suggest you to repeat the inoculation of TSI again and incubate for 18-24 hours and also please use known strain (organism) to check the TSI agar is giving actual result.

  3. sir, i have performed TSI test with an unknown isolate.. i observed that after 48 hr incubation slant was yellow but butt was red. what could be interpretation for it? if the organism catabolize glucose i should observe yellow colour in butt only due to its low concentration and if the organism is catabolizing lactose/sucrose both slant and butt should be yellow coloured due to high concentration. in either case yellow colour on the slant with red colour butt is unexpected..

    • Such type of result is unexpected. Did you sub-culture the isolate in MacConkey Agar? What was the colony characteristics? What about Catalase and Oxidase test results?
      Please repeat the procedure (following all the steps and precautions as mentioned) with a single isolated colony; and observe the result within 18-24 hours.

  4. Can you tell me which bacteriums has these apperances: for the Phenol Red Broth all slants turned yellow (glucose, sucrose & lactose). I know that Phenol red is a pH indicator which turns yellow below a pH of 6.8. Also air bubbles were observed inside all 3 Durham tubes. And the Kligler Iron Agar was completely yellow with one or two small bubbles at the bottom.

    • Dear Olivia, thank you so much for your query. Based on the information you have provided here, the isolate can be one of them; Escherichia coli, Klebsiella spp (pneumoniae or oxytoca), Enterobacter spp or Citrobacter spp. These are the most common lactose fermenter. You need other information to identify the isolate such as production of Hydrogen sulphide gas, indole production, citrate utilization, urease production, motility etc.

  5. Sir I have a question in which mechanism bacteria that produce gas for example Proteus produce copious of carbon dioxide which causing gap at the but and separate at the slant I need to know the mechanism behind which leading the occurrence of that gaps

  6. Thanks Tankeshwar Acharya for directing me to the Lab diagnosis of vibrio i found out that there are other biochemical test for example oxidase test and others including TSI for identifying Vibrio and thats is clear.also i have a question ,is CHOLERA RED TEST Part of diagnosing cholera

    • Dear Anderson koech,
      Thank you for your comment. In our settings; we do not perform Cholera Red Reaction as a part of routine test to identify Vibrio cholerae. We perform simple test from Hanging drop (for rice watery stool with mucus flecks), followed by plating in TCBS agar. Observation of colony morphology and performing other biochemical test is done after that. Finally we perform serological test to differentiate the serotypes, O1 and Non O1. I have listed the tests we perform in our laboratory here:

    • The rationale is, MTB concentrate in the sputum at night and sensitivity increases if early morning sample is taken.

  7. Sir I have a question, I have an unknown sample and on the TSI, it showed K/A but on the MacConkey agar, it showed red colonies which indicates that it is a Lactose fermenter. Why is the organism positive for the agar and negative on the TSI? thanks for answering.

  8. Sir I have a question. I have an unknown sample and it showed K/A on my TSI which indicates that my organism is a non lactose fermenter/glucose fermenter. But on my MacConkey agar, it showed red colonies that indicates that it is a lactose fermenter. Why is that possible?

    • Dear Diego
      Thank you for the query. This is very unlikely as results are contradictory. Sometime this may happen because of mixed culture, I suggest you to do full plate subculture and perform TSI from single isolated colony. To find out if the organism is oxidative or fermentative, you can also perform OF Test, if you wish.

  9. Hello sir..i have a question. Is there any other biochemical test that can be done to differentiate the Enterobacteriaceae such as E.coli and salmonella typhimurium more accurate than TSI test?

    • According to the information you have provided, the organism (if its a pure culture) must be lactose fermenter which produces H2S gas. Four most common lactose fermenter are Escherichia coli, Klebsiella pneumoniae, Enterobacter spp, and Citrobacter spp. Out of these only few strains of Citrobacter spp may produce H2S gas. So you have to perform other biochemical test to find out, if these isolates also share similar properties like that of Citrobacter. Other possibility can be mixed culture of Lactose fermenter with H2S producing NLF such as Proteus, Salmonella.

    • I just conduct the TSI test on Vibrio parahaemolyticus and the result is A/A.. For specific isolation of Vibrio parahaemolyticus, you should culture on TCBS, if growth with dark green colony at center, oxidase positive and turbid grow with NACL at 8% and 10% can consider as Vibrio parahaemolyticus.

  10. One thing please, why we talke about glucose fermentation in the slant where we have aerobic condition ?! in fact, in the same place the AAs are oxidized !
    All my thanks

  11. Dear Acharya
    This kheiri from Iran. I have isolated a bacteria with small colonies and metal sheen on EMB agar. TSI result is A/A, citrate negative, Mug negative, indole negative, XGAL negative.
    I can’t distinguish the genus. Can you help me?

    • Dear Roohollah
      Thank you for your question and the information.
      Four possible organisms based on TSI result is Escherichia, Klebsiella, Enterobacter and Citrobacter. Out of them, only Escherichia is citrate negative. But you said that particular isolate is MUG and Indole negative too, that made the result confusing, as now it’s hard to say that the isolate is E.coli. So my recommendation is please perform panel of tests, MR/VP, Indole, Citrate, Motility, TSI, Urease again using pure isolate or perform API 20E test system if you have. Please share your identification result once you identify the isolate.

  12. Thank you Tankeshwar Acharya for such a good explanations and all the discussion here.. but to my knowledge, the principle behind turning red color of the slant when organism ferments only glucose is because of the aerobic condition and oxidation of the whatever little amount of acid that is formed over the slant. I have seen some literature explaining the way you have explained here but some professors prefer the oxidative wash out mechanism only but not the glucose concentration differences between slant and butt.. Hope you would share a positive comment on this.. Thanks and regards!!!

    • Dear Amit
      We want to differentiate among organism who ferments lactose and which can not. If you use 10% of Glucose, sufficient amount of acid will produce in the slant also and the reaction will be yellow/yellow. In such case we wont able to differentiate between lactose fermenter and those who can not ferment lactose.

  13. Dear sir, is it possible for a bacteria eg f. columnare to give H2S before storage, and later after cryopreservation grow without giving H2S on TSI?

  14. Dear sir, is it true that H2S can only grow in acidic condition? If so, why is it that my experiment of E.Coli and P.A did not have blackening of agar eventhough the medium is all yellow?

  15. I did a TSI slant test and the slant was slight red and yellow and the butt yellow with lots of gas. Having trouble debating between ecoli or enterobacter. Can u help?

  16. I thought H2S production only occur under anaerobic and alkaline environment? Or does H2S production also happen under acidic environment? Thank you

  17. May I have a question? If the presented result from TSI is K/N, for me my understand is that this organism can not ferment any sugar and my question is Does this organism use glucose in aerobic condition before peptone? /DOES this organism use only peptone but not glucose? Could anyone explain that?

  18. Sir, if H2S masks the color of the slant and the butt and all you can see is black, how can you say or why would you assume that it is K/A when you cannot see the colors of the slant/butt?

  19. Dear sir.Thanks a lot for your informative articles but still have not been able to understand why is it that we group M.tuberculosis as gram negative bacilli and yet they stain poorly with gram stain instead we use auramine or Zien nelson stain. Nicholas from Kenya.

  20. Sir,
    I have a question.
    I’m working with e. coli.after storing positive samples on i m doing chemical test by re-culturing them & in tsi test i found y/black butt in many samples. Can you please tell me about this result?
    Thank you in advance!

    • Sorry, I did not understand your query. Once you isolated Gram negative bacilli, you can run couple of biochemical tests, and TSI is one of them.

        • Before jumping here, please use the food sample and try to isolate Salmonella (or any other bacteria) if present there. If you isolated NLF colonies in MacConkey Agar, you can perform series of biochemical test to know, if this isolate is Salmonella or not.

          • im working on a research sir about boiled egg using its color, i can give probabilities that there are presence of any bacteria.. there are research that if a boiled egg is overcook there would be a chemical reaction which is the hydrogen sulfide and the color of these is greyish or greenish.. can you give me any possible test that i can use?


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