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.


No Organisms Were Recovered On Buffered Charcoal Yeast Extract Medium After 2 Days of Incubation

Serum samples collected from a patient with pneumonia demonstrate a rising antibody titer to Legionella. A bronchoalveolar lavage (BAL) specimen from this patient had a positive antigen test for Legionella but no organisms were recovered on buffered charcoal yeast extract medium after 2 days of incubation. What is the best explanation?

Legionella BCYE

First of all, Legionella is a pathogenic group of gram-negative bacteria with the chief species being Legionella pneumophilia which is the causative agent of legionnaires disease. Legionella lives within amoeba in the natural environment. Transmission occurs via inhalation of water droplets from a contaminated source. Upon inhalation the bacteria infects alveolar macrophages where it begins to replicate. Legionella pneumophilia is a non-encapsulated aerobic bacillus with a single flagellum. It is unable to hydrolase gelatin or produce urease. Legionella is a non-pigment producer and does not auto fluoresce. It requires cysteine and iron to survive, thus media used to inoculate legionella must contain those two nutrients to grow.

The pathogenesis of legionella begins after an incubation period of up to two weeks. The initial prodromal symptoms are flu-like that include fever, chills and dry-cough. Through advancement of the disease the patient will experience gastrointestinal tract and the nervous system involvement. Pneumonia is also a common occurrence. The most at risk for infection with legionella are those that are immunocompromised, and the elderly. Legionella is also a prevalent nosocomial infection that has a fatality rate of 28%.

The medium of choice for when legionella is suspected is a buffered charcoal yeast extract agar (BCYE). The BCYE is a modified medium from the pre-existing F-G agar. Yeast serves as the protein source instead of casein. Beef extractives and starch are not added in the BCYE like they are in the F-G agar. Macroscopic colonies of legionella pneumophilia are visible on the BCYE after 3 days, but media should be incubated at 35-37 degrees Celsius for at least 7 days for a definitive answer. This is compared to the F-G agar where visible colonies were present after 4 days of incubation.

To answer the original question, the best explanation as to why colonies were present on the BCYE after 2 days is because the medium was simply not incubated long enough. It should be incubated for 7 days to make a definitive diagnosis even though colonies should start to appear after 3 days. The colonies on the BCYE should appear gray-white with a textured, cut glass appearance or opal.


Presence of Thermally Dimorphic Organism with Microconidia/Macroconidia in Immunocompromised Patient

A bone marrow sample obtained from an immunocompromised patient revealed small intracellular cells using a Wright’s stain preparation. Growth on Sabouraud-dextrose agar plates of a mold phase at 25 degrees Celsius and a yeast phase at 37 degrees Celsius designates the organism as dimorphic. The mold phase produced thick, spherical tuberculated macroconidia. What is the most likely identification?


Initial clues point towards some sort of systemic organism of fungi origin. Growth at both 25 and 37 degrees confirms dimorphism which is important for the differential diagnosis.

Microscopy examination revealed macroconidia indicative of Histoplasma capsulatum. When all other information is taken into consideration, a diagnosis H. capsulatum seems most plausible.

Histoplasma capsulatum is mostly found in its natural setting in the soil. It is endemic in the Ohio and Mississippi river valleys, and in some parts of South America as well. Transmission of the mycosis occurs through inhalation of aerosolized microconidia with activity that disturbs the natural environment. The severity of the illness depends on the host immunity status coupled with the intensity of the exposure. Only 1% of infections are symptomatic.

In this case the patient was immunocompromised. There are three distinct courses the disease can take depending on the immune status of the host. Acute primary histoplasmosis is typically a self-limiting disease. Localized to the lung and the patient may experience fever, cough, chest pain, and malaise (general unwell). Chronic cavitary histoplasmosis is characterized by atypical pulmonary lesions that resemble tuberculosis. Progression can lead to disabling respiratory dysfunction. Progressive disseminated histoplasmosis is generalized involvement of the RES with hepatosplenomegaly, lymphadenopathy, bone marrow involvement, with sometimes GI involvement.

The yeast form is able to survive within circulating monocytes and tissue macrophages. This is the primary way that the fungi disseminates. When H. capsulatum converts to the yeast form, it expresses proteins that interact with the phagocytic receptors on the macrophage surface. These interactions allow entry into the macrophage without activating it to undergo phagocytosis.

Treatment for the patient mentioned above should include amphotericin B including combinations of both liposomal and lipid formulations as well as itraconazole. Amphotericin B binds to ergosterol which is a component of fungal cell membranes. This causes pores to form in the membrane causing leakage of the monovalent ions K+, Na+, H+ and Cl- which subsequently induces fungal cell death.



Gram-negative diplococci failed to grow at 48 hours in 35 degree Celsius.

An urethral swab was obtained from a man with an urethral exudate was plated directly on chocolate agar and modified Thayer-Martin agar and a gram stain was made. The culture plates were incubated at 35 degrees celsius, but showed no growth at 48 hours. The gram stain showed gram-negative diplococci.

What is a Thayer-Martin agar? A Thayer-Martin agar is a Mueller-Hinton agar with 5% chocolate sheep blood and a combination of antibiotics. The antibiotics include vancomycin, colistin, nystatin, and trimethoprim. Vancomycin kills most gram-positive organisms, except Lactobacillus and Pediococcus species are inherently resistant. Colistin is added to kill most gram-negative organisms except Neisseria. Nystatin is added to kill most fungi. Trimethoprim inhibits gram-negative organisms especially Proteus species.

A Thayer-Martin agar is used to isolate suspected pathogenic Neisseria gonorrhoeae and meningitidis.

A chocolate blood agar (CBA, CHOC) is a nonselective enriched growth media used for isolation of pathogenic bacteria. The chocolate agar is used for growing fastidious respiratory bacteria such as Haemophilus influenzae or Neisseria meningitidis. Some of the fastidious bacteria need NAD and hemin which are inside RBCs, which are lysed in the chocolate agar.

Typically when a pathogenic Haemophilus or Neisseria is suspected, the swab will first be plated onto a chocolate agar, and then a more selective media, either being the chocolate agar with the addition of bacitracin for selection of Haemophilus, or the Thayer-Martin agar for the selection of Neisseria species.

Because of the source of the swab, being from a urethral exudate, this makes Neisseria more likely so sample was restreaked onto a Thayer-Martin agar.

Neisseria gonorrhoeae is a gram-negative diplococci (which was observed on upon gram stain). It is oxidase positive, aerobic, and non-mobile. It survives within PMNs within the host and is the causative agent of gonorrhea.

The reason that it failed to grow at 35 degrees celsius after 48 hours is because Neisseria gonorrhoeae requires an environment enriched with 3-7% carbon dioxide.  This species is capnophilic meaning that it strives in higher levels of carbon dioxide.

A way to differentiate Neisseria gonorrhoeae from Neisseria meningitidis is from a carbohydrate utilization test. N. gonorrhoeae ONLY oxidizes glucose while N. meningitidis is able to ferment glucose and maltose.


BOC Online Study Guide; Microbiology