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.



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.