Triple Sugar Iron Test – Procedure, Interpretation And Uses

One of the defining abilities of bacterias is their ability to ferment carbohydrates. Sugars are particularly great at being fermented by bacteria but not all bacterias are made equally.

While some types of bacteria are great for fermenting sugars, others cannot ferment them at all – and this lack of fermentation abilities is key to defining and identifying bacterias. 

Therefore, the Triple Sugar Iron test is an important part of microbiology because it is the best test to use to seperate the microorganisms that can ferment sugars from the ones that can’t.

Triple Sugar Iron Test - Procedure, Interpretation And Uses

Sometimes called the TSI test for short, the Triple Sugar Iron test puts each bacteria to the test to see if they can ferment sugar and helps classify them – so it’s easy to see why every microbiologist needs to be familiar with this test. 

Here, we are going to go through the Triple Sugar Iron test so you can understand everything you need to know about this procedure including how to interpret your results.

So, check out the information below so you have everything you need to carry out a Triple Sugar Iron test of your own. 

What Is The Triple Sugar Iron Test?

As we said earlier, the Triple Sugar Iron test is an important method that microbiologists use to identify which microorganisms can ferment sugars and which cannot.

This helps to identify different sugars, classify them and understand more about their fermentation patterns.

It is used mainly to separate members of the Enterobacteriaceae family based on their fermentation patterns, helping microbiologists to seperate them from other gram negative rods. 

It involves exposing certain bacterias to fermentative sugars and oxygen, then waiting to see the results.

Indication that the bacteria have indeed fermented the sugars includes seeing the production of gas and a pH indicator coloring from red to yellow.

These changes occur when hydrogen sulfide is produced and when carbohydrate fermentation is underway. 

There are a range of ingredients used in the TSI test including an agar slant of multiple special sugars with a pH sensitive dye.

These fermentative sugars are lactose, sucrose and glucose – the ‘triple’ sugars of which the test gets its name. Lactose and glucose are both present in 1% concentrations while glucose is present at 0.1%.

The reason for this is because some organisms are only able to ferment glucose but cannot ferment other sugars like lactose or sucrose.

In order to separate these two types of organism apart, a lower concentration of glucose is used. 

If the fermentation process starts, then the pH will fall as acid will build up during this process. So, including a pH indicator for acids, such as phenol red, will help indicate if the fermentation process has worked.

If it has, then the phenol red in the solution will shift from its usual red or red/orange color to yellow.

This is how microbiologists know if there are acids in the solution and if acids are present, that means that fermentation has taken place.

If the organism can only ferment glucose, it will use that small amount of glucose first. Then, it will revert to the oxygen containing surface (known as the aerobic area) of the slant and turn to alkaline.

The bottom of the slant which has not been exposed to oxygen will remain acidic.

The difference in color between the aerobic and anaerobic areas will indicate if the organism can only ferment glucose. 

Another important result to look for during the TSI test is to see if ferrous ammonium and sodium thiosulfate have been produced in the medium.

Their presence is able to detect if hydrogen sulfide has been produced during the fermentation process – hence why it is known as the Triple Sugar Iron test.

This is indicated by the presence of a black color at the very butt (or bottom) of the test tube. 


Here, we are going to go through the method of the Triple Sugar Iron test.

There are not that many steps to follow once you have all of your ingredients and media together, so let’s go through them one by one so you can understand the exact process of the test. 

The first step is to take your inoculation needs and use it to touch the top of your colony.

This colony must have been isolated beforehand and the inoculation needle should be straight so it can enter the tube with ease. 

Then, you need to inoculate your colony by piercing the center of the colony until you reach the bottom of the tube and then streak the surface of the agar slant. Once this is done, remove the inoculation needle.

Place the cap on the tube loosely so it has a free exchange of air – this is important so the glucose can ferment and react if the colony is capable of fermenting. 

Now, you must incubate the tube between 95 and 98.60 degrees Fahrenheit (or 35 to 37 degrees Celsius) in ambient air. Leave the tube to incubate for at least 18 to 24 hours before examining your results. 


Now that you know how to carry out the procedure for the Triple Sugar Iron test, it’s time to go through the results and what they mean.

This way, you can understand what results to expect and how to interpret the results you actually get (if there is a difference at all!). 

If your results show a red slant but a yellow butt of the tube, then this is an indication that the organism has only fermented the glucose present in the agar.

This result is also known as an alkaline/acid reaction and means that the organism can only ferment glucose but not other sugars like glucose or sucrose. 

If your results show a yellow slant and a yellow butt of the tube, then this is an indication that the organism has fermented all three sugars in the solution.

This means that your organism can ferment carbohydrates. This is known as an acid/acid reaction. 

And finally, if your results show a red slant and a red butt of the tube, then this is an indication that no fermentation has taken place so your organism cannot ferment carbohydrates.

This is known as an alkaline/alkaline reaction. 

Other important results you may notice includes blackening anywhere in the solution but specifically at the butt of the tube. As we mentioned above, this is an indication of the presence of hydrogen sulfide.

Any bubbles or cracks indicate the production of gas during the fermentation process.

They are mostly a side production and their presence does not indicate if the organism has fermented all three sugars or just the glucose. 


So, that is everything you need to know about the Triple Sugar Iron test! 

We have gone through what the test is and why it is performed by microbiologists so you can understand its uses, described the procedure and each step of the method in detail.

Plus, we have interpreted the results so you can understand what each reaction means.

Now, you can use this information to understand your own results when you try out this vital microbiological test in your own lab time! 

We hope that this has helped you understand all you need to know about the Triple Sugar Iron test!

Jennifer Dawkins