Triple Sugar Iron (TSI) Agar test Principle, Procedure, Result

• Triple Sugar Iron Agar is used to identify Enterobacteriaceae based on the fermentation of glucose, lactose, and sucrose and the formation of gas and H2S.
• Additionally, gas from glucose metabolism can be detected. Bacteria can either aerobically (with oxygen) or fermentatively (without oxygen) consume carbohydrates (without oxygen).
• Triple Sugar Iron Agar consists of three sugars (glucose, lactose and sucrose).
• The differential medium triple sugar iron agar (TSI) comprises lactose, sucrose, a little amount of glucose (dextrose), ferrous sulphate, and the pH indicator phenol red. The ability to decrease sulphur and ferment carbohydrates is utilised to differentiate enterics.
• Similar to the phenol red fermentation broths, if any of the three sugars present in the medium can be fermented, the medium will turn yellow.
• If an organism can only ferment dextrose, it will consume the minimal amount of dextrose in the medium within the first ten hours of incubation.
• After this period, the reaction that produced acid reverses in the aerobic regions of the slant, and the medium in these regions turns red, indicating alkaline conditions.
• The anaerobic regions of the slant, including the butt, will not return to an alkaline state and will remain yellow. This is a characteristic of Salmonella and Shigella.
• Russell described the use of an agar medium containing two sugars in 1911 as a means of identifying intestinal gram-negative bacilli.
• Other researchers added lead or iron salts to Russell’s Double Sugar medium after recognising that the ability of bacteria to create hydrogen sulphide was a valuable trait.
• To produce Kigler’s Iron Agar, Kliger added lead acetate and iron salts to detect hydrogen sulphide formation and utilised phenol red as a pH indicator.
• In 1917, Krunweide and Kohn added a third sugar, sucrose, to Russell’s Double Sugar agar.
• Sucrose allowed for the quicker discovery of coliform bacteria, which ferment sucrose more rapidly than lactose.
• The addition of sucrose also assisted in identifying gram-negative bacteria that could ferment sucrose but not lactose.
• 1940 saw the introduction of a comparable triple sugar ferrous sulphate medium for the identification of enteric bacilli by Difco Laboratories, Sulkin and Willet, and Hajna.
• The present recipe of triple sugar iron medium is nearly identical to that of Sulkin and Willet, with the exception of the use of phenol red as the pH indicator instead of brom thymol blue, the substitution of Bacto Peptone and Proteose Peptone for Tryptone, and the addition of yeast extract.

Purpose of Triple Sugar Iron (TSI) Agar test

• To differentiate gram-negative enteric bacilli on the basis of carbohydrate fermentation and hydrogen sulphide generation.
• To recognise the Enterobacteriaceae family.

Principle of Triple Sugar Iron (TSI) Agar test

• Three carbohydrates are included in TSI: glucose (0.1%), sucrose (1%), and lactose (1%). TSI is comparable to Kligler’s iron agar, with the exception that Kligler’s iron agar only contains two carbohydrates: glucose (0.1%) and lactose (1%).
• The medium also contains beef extract, yeast extract, and peptones, which are the sources of nitrogen, vitamins, and minerals, in addition to the specified carbs.
• The pH indicator is phenol red, and agar is employed to harden the medium.
• During preparation, molten agar-filled tubes are tilted.
• The medium slope is aerobic, whereas the medium depth (or butt) is anaerobic.
• When any of the carbs are fermented, the pH decrease causes the medium’s colour to change from reddish-orange to yellow.
• A rich crimson hue shows that the peptones have been alkalized. Some microorganisms in the media convert sodium thiosulfate to colourless hydrogen sulphide (H2S).
• Hydrogen sulphide reacts with ferric ions in the medium to form iron sulphide, a dark, insoluble precipitate.
• Based on carbohydrate consumption and hydrogen sulphide generation, there are multiple ways to interpret a TSI slant:

Glucose Fermenter

• The alkaline over acid (K/A) tube reaction indicates that only glucose is processed.
• In a few hours, the bacteria swiftly digested the glucose, producing an acid slant and an acid butt (acid over acid; A/A).
• ATP and pyruvate were produced via the Emben-Meyerhof-Parnas route both aerobically (on the slant) and anaerobically (in the butt).
• On the slope, pyruvate was converted into carbon dioxide, water, and energy.
• After an additional 18 hours of incubation, the glucose was depleted, and because the bacteria could not utilise lactose or sucrose, the peptones (amino acids) were used as an aerobic energy source on the slant.
• Utilization of peptones results in the release of ammonia (NH3), which raises the pH, as indicated by the colour change of phenol red from yellow to red.
• In the anaerobic butt, the bacteria utilize the Embden-Meyerhof-Parnas route to metabolize glucose, generating ATP and pyruvate, which are transformed into stable acid end products, hence maintaining the acidity of the butt.
• The outcomes would be recorded as alkaline versus acidic (K/A).
• Citrobacter freundii*, Citrobacter koseri*, and Morganella morganii* are bacteria that produce a K/A reaction with or without gas. * = variable reactions

Glucose, Lactose and/or Sucrose Fermenter

• The tube response is acid over acid (A/A), showing the metabolism of glucose, lactose, and/or sucrose.
• In a few hours, the bacteria rapidly digested the glucose, producing an acid slant and an acid butt.
• The Emben Meyerhof-Parnas route is utilised aerobically (on the slope) and anaerobically (in the butt) to generate ATP and pyruvate.
• On the slope, pyruvate is converted to carbon dioxide, water, and energy.
• After an additional 18 hours of incubation, the bacteria metabolised lactose and/or sucrose while maintaining an acidic slant.
• The outcomes are shown as acid over acid (A/A).
• If the medium were to be incubated for more than 48 hours, the lactose and sucrose would be depleted and the slant’s pH would become alkaline due to peptone metabolism.
• In the anaerobic butt, bacteria convert pyruvate to stable acid endproducts, thus maintaining the acidity of the butt.
• The bacteria that typically cause an A/A reaction with or without gas include Enterobacter aerogenes, E. coli, and E. coli phage. cloacae, Escherichia coli, Klebsiella oxytoca, and K. pneumoniae.

Glucose, Lactose and Sucrose Nonfermenters

• Either alkaline over alkaline (K/K) or alkaline over no change (K/NC) indicates that none of the three sugars have been digested.
• The distinction between K/K and K/NC) is minimal. Certain nonenteric bacteria, including pseudomonads, are incapable of fermenting glucose, lactose, or sucrose.
• These bacteria obtain energy from peptones either aerobically or anaerobically.
• Utilizing peptones results in the release of ammonia (NH3), which causes the pH indicator phenol red to change colour from pink to red.
• Non-glucose fermenters are capable of two distinct processes.
• If bacteria can both aerobically and anaerobically metabolise peptones, the slant and butt will be red (alkaline over alkaline; K/K).
• If peptones can be digested purely aerobically, the slant will be red and the butt will remain unchanged (K/NC). Among the bacteria that produce K/K or K/NC are the Acinetobacter spp. together with Pseudomonas spp.

Gas Production

• The detection of gas generation (CO2 and O2) is achieved by dividing the agar.
• In certain instances, the amount of gas created is sufficient to push the agar to the top of the tube. These bacteria typically produce an A/A reaction with gas: Enterobacter aerogenes, E. coli, and E. coli. cloacae, Escherichia coli, Klebsiella oxytoca, and K. pneumoniae. Nonetheless, several strains do not produce gas.

Glucose Fermenter and Hydrogen Sulfide Production.

• The reaction in the test tube is alkaline over acid (K/A) with a black precipitate.
• In a few hours, the bacteria swiftly digested the glucose, producing an acid slant and an acid butt (acid over acid; A/A).
• ATP and pyruvate are produced via the Emben-Meyerhof-Parnas route both aerobically (on the slant) and anaerobically (in the butt).
• On the slope, pyruvate is converted to carbon dioxide, water, and energy. After an additional 18 hours of incubation, the glucose was depleted, and because the bacteria could not utilise lactose or sucrose, the peptones (amino acids) were used as an aerobic energy source on the slant.
• Utilizing peptones results in the release of ammonia (NH3), which causes the pH indicator phenol red to change colour from yellow to red.
• In the anaerobic butt, the bacteria utilise the Embden-Meyerhof-Parnas route to metabolise glucose, creating ATP and pyruvate, which are then transformed into stable acid endproducts, hence maintaining the acidity of the butt.
• The dark precipitate suggests that the bacteria were able to convert sodium thiosulfate into hydrogen sulphide (H2S).
• Due to the absence of colour in H2S, ferric ammonium citrate is utilised as an indicator, leading in the creation of insoluble ferrous sulphide.
• Formation of H2S requires an acidic environment; despite the fact that a yellow butt cannot be seen due to the black precipitate, the butt is acidic.
• The results will be recorded as alkaline over acid (K/A), positive for H2S. Citrobacter freundii*, Edwardsiella tardi*, Proteus mirabilis*, and Salmonellaspp* are examples of bacteria that produce a K/A using H2S. Citrobacter freundii*, Proteus mirabilis*, and P. vulgaris* are examples of bacteria that frequently produce an A/A with H2S.

Glucose, Lactose and/or Sucrose Fermenter and Hydrogen Sulfide Producer

• The reaction in the test tube is acid over acid (A/A) with a dark precipitate. In a matter of hours, the bacteria rapidly metabolised the glucose, producing an acid slant and an acid butt (acid over acid; A/A).
• ATP and pyruvate are produced via the Emben-Meyerhof-Parnas route both aerobically (on the slant) and anaerobically (in the butt).
• On the slope, pyruvate is converted to carbon dioxide, water, and energy.
• After an additional 18 hours of incubation, the bacteria metabolised lactose and/or sucrose while maintaining an acidic slant.
• The outcomes are shown as acid over acid (A/A).
• If the medium were to be incubated for more than 48 hours, the lactose and sucrose would be depleted and the slant’s pH would become alkaline due to peptone metabolism.
• In the anaerobic butt, bacteria convert pyruvate to stable acid endproducts, thus maintaining the acidity of the butt.
• The dark precipitate suggests that the bacteria were able to convert sodium thiosulfate into hydrogen sulphide (H2S).
• Due to the absence of colour in H2S, ferric ammonium citrate is utilised as an indicator, leading in the creation of insoluble ferrous sulphide.
• Formation of H2S requires an acidic environment; despite the fact that a yellow butt cannot be seen due to the black precipitate, the butt is acidic.
• The results will be recorded as acid over acid (A/A) and positive for H2S.
• Citrobacter freundii*, Proteus mirabilis*, and P. vulgaris* are examples of bacteria that typically produce A/A with H2S. * = varying responses

Glucose Non Fermenter Hydrogen Sulfide Producer

• The tube is alkaline above no change (K/NC) and contains a black precipitate (H2S).
• Thiosulfate reduction in KIA and TSIA requires H+.
• The fermentation of carbohydrates cannot produce an acidic environment in nonfermenters.
• Cysteine and maybe other organic sulphate compounds are converted to pyruvic acid, ammonia, and H2S during metabolism.
• Positive non-fermentative H2S response is very predictive of Shewenella species.

Composition of TSI (Triple Sugar Iron) agar

Ingredients	Grams/liter
Beef extract	3.0 g
Yeast extract	3.0 g
Peptone	20.0 g
Glucose	1.0 g
Lactose	10.0 g
Sucrose	10.0 g
Ferrous sulfate or ferrous ammonium sulfate	0.2 g
NaCl	5.0 g
Sodium thiosulfate	0.3 g
Phenol red	0.024 g
Agar	13.0 g
Distilled water	1,000 mL

Note: The following combination of ingredients can substitute for the first two components listed: Pancreatic digest of casein USP 10.0 g, Peptic digest of animal tissue USP 10.0g,

Preparation of TSI (Triple Sugar Iron) agar

1. Mix 65 grammes of the powder with 1 litre of filtered water.
2. Blend thoroughly
3. The powder must be heated with constant stirring and boiled for one minute to completely dissolve.
4. Dispense into tubes and autoclave for 15 minutes at 121 degrees Celsius.
5. Cool in a tilted angle so as to develop deep butts.
6. Examine the performance of samples of the final product using stable, typical control cultures.

Procedure of Triple Sugar Iron (TSI) Agar test

1. To inoculate, touch only the centre of an isolated colony on an enteric-plated media with a cool, sterile needle, insert the needle into the medium at the base of the tube, and then streak back and forth across the surface of the slant.
2. Multiple colonies from each primary plate should be analysed independently, as mixed infections are possible.
3. After 18 to 24 hours of incubation at 35 degrees Celsius with the caps removed, look for carbohydrate fermentation, gas production, and hydrogen sulphide creation.
4. Any combination of the following reactions may occur.
5. The acid reaction in the slant of lactose and sucrose fermenters may return to an alkaline reaction if incubated for longer than 24 hours.

Expected Results of Triple Sugar Iron (TSI) Agar test

1. If the bacteria only use glucose: the bottom turns yellow and the slope red (Acid butt, alkaline slant – yellow butt, red slant)

• The bacterium rapidly metabolises glucose, initially giving an acid slope and an acid bottom (acid on acid A / A) with the production of pyruvate. After a further incubation (18 hours), the glucose will be consumed, and since the bacteria cannot use lactose and sucrose, peptones (amino acids) will be used as an aerobic energy source (slope), resulting in the release of ammonia (NH 3), which raises the pH, changing the

2. If the bacteria use glucose, sucrose and / or lactose: the bottom and the slope will turn yellow (Acid butt, acid slant – yellow butt, yellow slant)

• After consuming glucose, the bacterium consumes sucrose and/or lactose, resulting in a yellow slope and bottom (acid on acid (A / A)).
• If the medium is incubated for longer than 48 hours, the lactose and sucrose will be depleted and the pH will return to an alkaline state as a result of peptone metabolism.

3. Bacteria does not use any of the sugars: In this case the bacteria will consume the peptones (Alkaline butt, alkaline slant – red butt, red slant)

• If bacteria can both aerobically and anaerobically metabolise peptones, the slope and pellet will be red (alkaline on alkaline; K / K).
• If peptones can be digested purely aerobically, the slope will be red and the base will remain constant (K / NC).

4. Gas production

• Indicated by the presence of bubbles in the butt.
• With substantial quantities of gas, the agar can be shattered or propelled upward.
• The formation of gas (CO2 and O2) is identified through the separation of the agar.

5. H2s production

• The creation of hydrogen sulphide from thiosulfate is shown by the formation of black ferrous sulphide from the interaction of hydrogen sulphide with ferrous ammonium sulphate. The dark precipitate suggests that the bacteria were able to convert sodium thiosulfate into hydrogen sulphide (H2S).
• Due to the absence of colour in H2S, ferric ammonium citrate is utilised as an indicator, leading in the creation of insoluble ferrous sulphide.
• Formation of H2S requires an acidic environment; despite the fact that a yellow butt cannot be seen due to the black precipitate, the butt is acidic.
• The results will be recorded as acid over acid (A/A) and positive for H2S.

Results (slant/butt)	Symbol	Interpretation
Red/yellow	K/A	Glucose fermentation only; Peptone catabolized
Yellow/yellow	A/A	Glucose and lactose and/or sucrose fermentation
Red/red	K/K	No fermentation; Peptone catabolized
Red/no color change	K/NC	No fermentation; Peptone used aerobically
Yellow/yellow with bubbles	A/A,G	Glucose and lactose and/or sucrose fermentation; Gas produced
Red/yellow with bubbles	K/A,G	Glucose fermentation only; Gas produced
Red/yellow with bubbles and black precipitate	K/A,G, H2S	Glucose fermentation only; Gas produced; H2S produced
Red/yellow with black precipitate	K/A, H2S	Glucose fermentation only; H2S produced
Yellow/yellow with black precipitate	A/A, H2S	Glucose and lactose and/or sucrose fermentation; H2S produced
No change/no change	NC/NC	No fermentation

A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction

A/A , G	A/A, G, H2S+	ALK/A	ALK/A, G	ALK/A, G, H2S+	ALK/A, H2S(w)
Citrobacter spp. Cronobacter Enterobacter Escherichia coli Klebsiella spp. Pantoea Yersinia spp.	Citrobacter spp. Proteus vulgaris	Escherichia coli Morganella Proteus penneri Providencia spp. Serratia spp. Shigella spp. Yersinia spp.	Escherichia coli Citrobacter spp. Enterobacter spp. Hafnia Klebsiella spp. Proteus myxofaciens Providencia alcalifaciens Salmonella enterica serovar Paratyphi Serratia spp. Yersinia kristensenii	Citrobacter spp. Edwardsiella tarda Proteus mirabilis Salmonella serovars other than S. enterica serovar Typhi and Paratyphi	Salmonella enterica serotype Typhi

A, acid; ALK, alkaline; G, gas; +, positive; w, weak.

Organisms	Slant	Butt	Gas	H2S
Escherichia coli	Acid (A)	Acid (A)	Pos (+)	Neg (-)
Shigella flexneri	Alkaline (K)	Acid (A)	Neg (-)	Neg (-)
Salmonella enterica subsp. enterica serotype Enteritidis	Alkaline (K)	Acid (A)	Pos (+)	Pos (+)
Pseudomonas	Alkaline (K)	Alkaline (K)	Neg (-)	Neg (-)
Klebsiella pneumoniae	Acidic reaction (A)	Acidic reaction (A)	Positive	Negative
Salmonella Typhimurium	Alkaline (K)	Acid (A)	Positive	Blackening of medium
Proteus mirabilis	Alkaline (K)	Acid (A)	Neg (-)	Pos (+)
Salmonella Typhi ATCC 6539	Alkaline (K)	Acid (A)	Neg (-)	Blackening of medium
Enterobacter aerogenes	Acid (A)	Acid (A)	Positive	No blackening of medium
Citrobacter freundii	Acid (A)	Acid (A)	Positive	Blackening of medium
Salmonella Paratyphi AATCC 9150	Alkaline (K)	Acid (A)	Positive	No blackening of medium
Proteus vulgaris	Alkaline (K)	Acid (A)	Neg (-)	Blackening of medium
Shigella sonnei	Alkaline (K)	Acid (A)	Neg (-)	Neg (-)

Limitations of Triple Sugar Iron (TSI) Agar test

• On Kligler Iron Agar, hydrogen sulphide generation may be noticeable, but not on Triple Sugar Iron Agar. Bulmash and Fulton’s research demonstrated that sucrose consumption can inhibit the enzymes responsible for H2S generation. Padron and Dockstade discovered that not all H2S-positive Salmonella is also TSI-positive.
• Sucrose is added to TSI in order to eradicate certain sucrose-fermenting lactose-nonfermenters, including Proteus and Citrobacter spp.
• To definitively identify and confirm organisms, additional biochemical testing and serological typing must be undertaken.
• Avoid using an inoculating loop while inoculating a Triple Sugar Iron Agar tube. During the process of stabbing the butt, mechanical splitting of the medium occurs, resulting in a false-positive gas production result.
• When inoculating Triple Sugar Iron Agar, a pure culture is needed. If inoculated with a mixed culture, there may be erratic observations.
• Reactions in TSI should not be measured beyond 24 hours of incubation, as aerobic oxidation of the fermentation products from lactose and/or sucrose continues and the slant reverts to an alkaline condition.
• The formation of hydrogen sulphide by an organism may disguise acid production in the bottom of the medium. However, the creation of hydrogen sulphide requires an acidic environment, thus the butt should be considered acidic.
• Incubate tubes with their caps loosened. This permits a free circulation of air, which is required to improve the alkaline condition on the slope.
• Compared to other iron-containing mediums, such as Sulfide Indole Motility (SIM) Medium, TSI is less sensitive to the detection of hydrogen sulphide. Thus, organisms with modest hydrogen sulphide production may exhibit negligible or no hydrogen sulphide activity.
• Certain species or strains may exhibit delayed reactions or fail to ferment the carbohydrate in accordance with the instructions. If the bacterium fails to ferment glucose within 48 hours, it is most likely not a member of the family Enterobacteriaceae.

Quality Control

References

• https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/226540.pdf
• https://www.neogen.com/categories/microbiology/triple-sugar-iron-tsi-agar/
• https://www.neogen.com/globalassets/pim/assets/original/10007/ncm0144_ts_en-us.pdf
• https://asm.org/ASM/media/Protocol-Images/Triple-Sugar-Iron-Agar-Protocols.pdf?ext=.pdf
• https://microbe-canvas.com/tests.php?p=2089
• https://www.himedialabs.com/TD/MM021.pdf
• https://www.austincc.edu/microbugz/triple_sugar_iron_agar.php
• https://microbeonline.com/triple-sugar-iron-agar-tsi-principle-procedure-and-interpretation/
• https://microbiologie-clinique.com/triple-sugar-iron-agar.html
• https://microbenotes.com/triple-sugar-iron-agar-tsia-test/