Triple Sugar Iron Agar (TSI)

The TSI is a multiple test medium. Its a slanted medium with a deep butt that is used to further investigate Gram-negative microorganisms. It differentiates the microbes by their ability to ferment glucose, lactose and/or sucrose with or without the production of gas and production of hydrogen sulfide.

The TSI medium contains three carbohydrates; 1.0% lactose, 1.0% sucrose and 0.1% glucose. Phenol red is added as a pH indicator. Ferric ammonium citrate and sodium thiosulfate are added as indicators for the production hydrogen sulfide.

There are multiple reactions that can be observed;

A reaction of alkaline/no change is denoted K/NC and it means that the organism can only catabolize peptones aerobically, hence only the slant exhibited a color change, usually a red/orange color. This means that no carbohydrates were utilized.

When the slant is alkaline after 18-24 hours of incubation it means that there was rapid depletion of glucose and there is a subsequent reliance on peptides for nutrients. This occurs because the concentration glucose is so low and therefore it is consumed quickly. Catabolism of peptones results in the release of ammonia (NH3) which yields an alkaline pH. The butt of the medium remains acid because the degradation of peptones occurs aerobically (i.e. in the slant).

Some organisms have the ability to ferment lactose and/or sucrose with glucose for their nutrients. This results in an acid slant and acid butt reaction denoted A/A and a color change of yellow/yellow will appear. Because the concentrations of lactose and sucrose are 10x the amount of glucose, therefore a large amount of acid is produced.

A TSI medium also is used to determine whether or not a microorganism can produce carbon dioxide and hydrogen gases from the fermentation of the carbohydrates present. Gas production is seen when a bubble forms, which splits the medium. A clear disc shaped area is seen within the medium.

Ferric ammonium citrate and sodium thiosulfate are both indicators that are added to view the presence of H2S hydrogen sulfide. A microorganism in an acidic environment acts on the sodium thiosulfate to produce H2S gas. H2S reacts with ferric ions to produce ions that produce ferrous sulfide which is an insoluble black precipitate.

It should be noted that the black precipitate of ferrous sulfide that indicates H2S production may mask an acidic condition in the butt of the tube. Since H2S is only produced under acidic conditions, when the butt of the tube is black, an acid butt exists as well even without the presence of the yellow color.

TSI Results K/K K/A A/A +g A/A +g,+H2S A/K +g, +H2S



The Gram Stain

Gram staining is the first step and common technique used to differentiate Gram-positive bacteria and Gram-negative bacteria. Bacterial cell walls contain the constituent peptidoglycan.


In the photo above you’ll see Staphylococcus aureus gram positive stained purple and Escherichia coli gram-negative stained pink.

Bacterial cell walls lack membranes around their organelles. A major component of a prokaryotic cell wall is the structure of peptidoglycan. The peptidoglycan gives the cell shape and surrounds and surrounds the cytoplasmic membrane. Peptidoglycan consists of a polymer of disaccharides (glycan) which is cross-linked by short chains of amino acid monomers. The backbone of the peptidoglycan molecule consists of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) that are connected via peptide bridges. The NAM molecule varies slightly between bacterial species. The peptidoglycan molecules are transported across the cytoplasmic membrane by bactoprenol; a carrier molecule. The peptidoglycan provides receptor sites for viruses and antibiotics.

In Gram-Negative bacteria the cell wall is composed of a single layer of peptidoglycan surrounded by a membrane structure called an outer membrane. The outer membrane contains a unique lipopolysaccharide (LPS) component. The LPS is an endotoxin that generates a strong immune response when come into contact with. The gram-negative bacteria do not retain the crystal violet which stains the peptidoglycan, but are able to retain he safranin which is a counterstain added after the crystal violet. The safranin is responsible for the red or pink color that gram-negative bacteria appear under the microscope.

Gram-positive bacteria are stained dark blue or purple due to the thick layer of peptidoglycan which retains the crystal violet. Gram-positive bacteria lack the LPS that gram-negative have, but contain a group of molecules called teichoic acids. Teichoic acids give the bacteria an overall negative charge due to the presence of phosphodiester bonds between the monomers. The primary function is unknown, but they are believed to serve as a means of adherence for the bacteria.

The process of gram staining is quite simple;

First make a slide of cell sample that needs to be stained. Heat fix the slide by carefully passing it through a Bunsen burner a few times. Then add the primary stain crystal violet and let it sit for one minute. After one minute rinse the remaining stain off the slide. The next step is to grams iodine for one minute. The Gram’s iodine fixes the crystal violet to the bacterial cell wall. After the Gram’s iodine rinse the sample with acetone or alcohol and rinse with water. The alcohol acts to decolorize the sample in the case of a Gram-negative bacteria, however its important to be careful because if the alcohol remains on the sample for too long, it may decolorize Gram-positive cells. The last step is to add the secondary stain called the safranin and let sit for one minute. After one minute, wash the stain off and let dry.

Gram staining is an important step in identify cultures and often the first step in the process in differentiating whether the pathogen is gram-negative or gram-positive.