Gram staining
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Gram stain (Gram staining or Gram's method), is a method of
staining Staining is a technique used to enhance contrast in samples, generally at the Microscope, microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissue (biology), tissues), in cytology (microscopic ...
used to classify
bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
l species into two large groups:
gram-positive bacteria In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. The Gram stain ...
and
gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...
. It may also be used to diagnose a
fungal infection Fungal infection, also known as mycosis, is a disease caused by fungi. Different types are traditionally divided according to the part of the body affected: superficial, subcutaneous, and systemic. Superficial fungal infections include common ...
. The name comes from the Danish
bacteriologist A bacteriologist is a microbiologist, or similarly trained professional, in bacteriology— a subdivision of microbiology that studies bacteria, typically Pathogenic bacteria, pathogenic ones. Bacteriologists are interested in studying and learnin ...
Hans Christian Gram Hans Christian Joachim Gram (13 September 1853 – 14 November 1938) was a Danish bacteriologist noted for his development of the Gram stain, still a standard technique to classify bacteria and make them more visible under a microscope. Earl ...
, who developed the technique in 1884. Gram staining differentiates bacteria by the chemical and physical properties of their
cell wall A cell wall is a structural layer that surrounds some Cell type, cell types, found immediately outside the cell membrane. It can be tough, flexible, and sometimes rigid. Primarily, it provides the cell with structural support, shape, protection, ...
s. Gram-positive cells have a thick layer of
peptidoglycan Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like layer (sacculus) that surrounds the bacterial cytoplasmic membrane. The sugar component consists of alternating ...
in the cell wall that retains the primary stain, crystal violet. Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out on addition of
ethanol Ethanol (also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound with the chemical formula . It is an Alcohol (chemistry), alcohol, with its formula also written as , or EtOH, where Et is the ps ...
. They are stained pink or red by the counterstain, commonly
safranin Safranin (Safranin O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endo ...
or fuchsine. Lugol's iodine solution is always added after addition of crystal violet to form a stable complex with crystal violet that strengthens the bonds of the stain with the
cell wall A cell wall is a structural layer that surrounds some Cell type, cell types, found immediately outside the cell membrane. It can be tough, flexible, and sometimes rigid. Primarily, it provides the cell with structural support, shape, protection, ...
. Gram staining is almost always the first step in the identification of a bacterial group. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to ''gram-variable'' and ''gram-indeterminate'' groups.


History

The method is named after its inventor, the Danish scientist
Hans Christian Gram Hans Christian Joachim Gram (13 September 1853 – 14 November 1938) was a Danish bacteriologist noted for his development of the Gram stain, still a standard technique to classify bacteria and make them more visible under a microscope. Earl ...
(1853–1938), who developed the technique while working with Carl Friedländer in the morgue of the city hospital in
Berlin Berlin ( ; ) is the Capital of Germany, capital and largest city of Germany, by both area and List of cities in Germany by population, population. With 3.7 million inhabitants, it has the List of cities in the European Union by population withi ...
in 1884. Gram devised his technique not for the purpose of distinguishing one type of bacterium from another but to make bacteria more visible in stained sections of lung tissue. Gram noticed that some bacterial cells possessed noticeable resistance to decolorization. Based on these observations, Gram developed the initial gram staining procedure, initially making use of Ehrlich's aniline-gentian violet, Lugol's iodine, absolute alcohol for decolorization, and Bismarck brown for counterstain. He published his method in 1884, and included in his short report the observation that the
typhus Typhus, also known as typhus fever, is a group of infectious diseases that include epidemic typhus, scrub typhus, and murine typhus. Common symptoms include fever, headache, and a rash. Typically these begin one to two weeks after exposu ...
bacillus ''Bacillus'', from Latin "bacillus", meaning "little staff, wand", is a genus of Gram-positive, rod-shaped bacteria, a member of the phylum ''Bacillota'', with 266 named species. The term is also used to describe the shape (rod) of other so-sh ...
did not retain the stain..
English translation in:
Translation is also at:
Gram did not initially make the distinction between Gram-negative and Gram-positive bacteria using his procedure.


Uses

Gram staining is a bacteriological
laboratory A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratories are found in a variety of settings such as schools ...
technique used to differentiate bacterial species into two large groups (
gram-positive In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. The Gram stain is ...
and
gram-negative Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelope consists ...
) based on the physical properties of their
cell wall A cell wall is a structural layer that surrounds some Cell type, cell types, found immediately outside the cell membrane. It can be tough, flexible, and sometimes rigid. Primarily, it provides the cell with structural support, shape, protection, ...
s. Gram staining can also be used to diagnose a
fungal infection Fungal infection, also known as mycosis, is a disease caused by fungi. Different types are traditionally divided according to the part of the body affected: superficial, subcutaneous, and systemic. Superficial fungal infections include common ...
. Gram staining is not used to classify
archaea Archaea ( ) is a Domain (biology), domain of organisms. Traditionally, Archaea only included its Prokaryote, prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even thou ...
, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups. Gram stains are performed on
body fluid Body fluids, bodily fluids, or biofluids, sometimes body liquids, are liquids within the Body (biology), body of an organism. In lean healthy adult men, the total body water is about 60% (60–67%) of the total Human body weight, body weight; it ...
or
biopsy A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, an interventional radiologist, or an interventional cardiology, interventional cardiologist. The process involves the extraction of sampling (medicine), sample ...
when
infection An infection is the invasion of tissue (biology), tissues by pathogens, their multiplication, and the reaction of host (biology), host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmis ...
is suspected. Gram stains yield results much more quickly than culturing, and are especially important when infection would make an important difference in the patient's treatment and prognosis; examples are
cerebrospinal fluid Cerebrospinal fluid (CSF) is a clear, colorless Extracellular fluid#Transcellular fluid, transcellular body fluid found within the meninges, meningeal tissue that surrounds the vertebrate brain and spinal cord, and in the ventricular system, ven ...
for
meningitis Meningitis is acute or chronic inflammation of the protective membranes covering the brain and spinal cord, collectively called the meninges. The most common symptoms are fever, intense headache, vomiting and neck stiffness and occasion ...
and
synovial fluid Synovial fluid, also called synovia, elp 1/sup> is a viscous, non-Newtonian fluid found in the cavities of synovial joints. With its egg white–like consistency, the principal role of synovial fluid is to reduce friction between the articul ...
for
septic arthritis Acute septic arthritis, infectious arthritis, suppurative arthritis, pyogenic arthritis, osteomyelitis, or joint infection is the invasion of a joint by an infectious agent resulting in joint inflammation. Generally speaking, symptoms typica ...
.


Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of
peptidoglycan Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like layer (sacculus) that surrounds the bacterial cytoplasmic membrane. The sugar component consists of alternating ...
(50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin. There are four basic steps of the Gram stain: # Applying a primary stain ( crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they do not rinse out during the staining procedure. # The addition of
iodine Iodine is a chemical element; it has symbol I and atomic number 53. The heaviest of the stable halogens, it exists at standard conditions as a semi-lustrous, non-metallic solid that melts to form a deep violet liquid at , and boils to a vi ...
, which binds to crystal violet and traps it in the cell # Rapid decolorization with
ethanol Ethanol (also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound with the chemical formula . It is an Alcohol (chemistry), alcohol, with its formula also written as , or EtOH, where Et is the ps ...
or
acetone Acetone (2-propanone or dimethyl ketone) is an organic compound with the chemical formula, formula . It is the simplest and smallest ketone (). It is a colorless, highly Volatile organic compound, volatile, and flammable liquid with a charact ...
# Counterstaining with
safranin Safranin (Safranin O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endo ...
.
Carbol fuchsin Carbol fuchsin, carbol-fuchsin, carbolfuchsin, or Castellani's paint is a mixture of phenol and basic fuchsin that is used in bacterial staining procedures. It is commonly used in the staining of mycobacteria because it has an affinity for the my ...
is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain. Crystal violet (CV) dissociates in aqueous solutions into and chloride () ions. These ions penetrate the cell wall of both gram-positive and gram-negative cells. The ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodide ( or ) interacts with and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV–I complex and, therefore, colors the cell. When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-negative cell loses its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain is removed from both gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsine, is applied last to give decolorized gram-negative bacteria a pink or red color. Both gram-positive bacteria and gram-negative bacteria pick up the counterstain. The counterstain, however, is unseen on gram-positive bacteria because of the darker crystal violet stain.


Examples


Gram-positive bacteria

Gram-positive bacteria generally have a single membrane (''monoderm'') surrounded by a thick peptidoglycan. This rule is followed by two phyla:
Bacillota The Bacillota (synonym Firmicutes) are a phylum of bacteria, most of which have Gram-positive cell wall structure. They have round cells, called cocci (singular coccus), or rod-like forms (bacillus). A few Bacillota, such as '' Megasphaera'', ...
(except for the classes
Mollicutes Mollicutes is a class of bacteria distinguished by the absence of a cell wall and its peptidoglycan. The word "Mollicutes" is derived from the Latin ' (meaning "soft" or "pliable"), and ' (meaning "skin"). Individuals are very small, typically on ...
and Negativicutes) and the
Actinomycetota The Actinomycetota (or Actinobacteria) are a diverse phylum of Gram-positive bacteria with high GC content. They can be terrestrial or aquatic. They are of great importance to land flora because of their contributions to soil systems. In soil t ...
. In contrast, members of the
Chloroflexota The Chloroflexota are a phylum of bacteria containing isolates with a diversity of phenotypes, including members that are aerobic thermophiles, which use oxygen and grow well in high temperatures; anoxygenic phototrophs, which use light for ph ...
(green non-sulfur bacteria) are monoderms but possess a thin or absent (class Dehalococcoidetes) peptidoglycan and can stain negative, positive or indeterminate; members of the Deinococcota stain positive but are diderms with a thick peptidoglycan. The cell wall's strength is enhanced by teichoic acids, glycopolymeric substances embedded within the peptidoglycan. Teichoic acids play multiple roles, such as generating the cell's net negative charge, contributing to cell wall rigidity and shape maintenance, and aiding in cell division and resistance to various stressors, including heat and salt. Despite the density of the peptidoglycan layer, it remains relatively porous, allowing most substances to permeate. For larger nutrients, Gram-positive bacteria utilize exoenzymes, secreted extracellularly to break down macromolecules outside the cell. Historically, the gram-positive forms made up the
phylum In biology, a phylum (; : phyla) is a level of classification, or taxonomic rank, that is below Kingdom (biology), kingdom and above Class (biology), class. Traditionally, in botany the term division (taxonomy), division has been used instead ...
Firmicutes, a name now used for the largest group. It includes many well-known genera such as '' Lactobacillus, Bacillus'', ''
Listeria ''Listeria'' is a genus of bacteria that acts as an intracellular parasite in mammals. As of 2024, 28 species have been identified. The genus is named in honour of the British pioneer of sterile surgery Joseph Lister. ''Listeria'' species ...
'', ''
Staphylococcus ''Staphylococcus'', from Ancient Greek σταφυλή (''staphulḗ''), meaning "bunch of grapes", and (''kókkos''), meaning "kernel" or " Kermes", is a genus of Gram-positive bacteria in the family Staphylococcaceae from the order Bacillale ...
'', ''
Streptococcus ''Streptococcus'' is a genus of gram-positive spherical bacteria that belongs to the family Streptococcaceae, within the order Lactobacillales (lactic acid bacteria), in the phylum Bacillota. Cell division in streptococci occurs along a sing ...
'', ''
Enterococcus ''Enterococcus'' is a large genus of lactic acid bacteria of the phylum Bacillota. Enterococci are Gram-positive cocci that often occur in pairs ( diplococci) or short chains, and are difficult to distinguish from streptococci on physical ch ...
'', and ''
Clostridium ''Clostridium'' is a genus of anaerobic, Gram-positive bacteria. Species of ''Clostridium'' inhabit soils and the intestinal tracts of animals, including humans. This genus includes several significant human pathogens, including the causative ...
''. It has also been expanded to include the Mollicutes, bacteria such as ''
Mycoplasma ''Mycoplasma'' is a genus of bacteria that, like the other members of the class ''Mollicutes'', lack a cell wall, and its peptidoglycan, around their cell membrane. The absence of peptidoglycan makes them naturally resistant to antibiotics ...
and Thermoplasma'' that lack cell walls and so cannot be Gram-stained, but are derived from such forms. Some bacteria have cell walls which are particularly adept at retaining stains. These will appear positive by Gram stain even though they are not closely related to other gram-positive bacteria. These are called acid-fast bacteria, and can only be differentiated from other gram-positive bacteria by special staining procedures.


Gram-negative bacteria

Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (''diderm'').
Lipopolysaccharide Lipopolysaccharide (LPS), now more commonly known as endotoxin, is a collective term for components of the outermost membrane of the cell envelope of gram-negative bacteria, such as '' E. coli'' and ''Salmonella'' with a common structural archit ...
(LPS) is the most abundant
antigen In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
on the cell surface of most gram-negative bacteria, contributing up to 80% of the outer membrane of ''E. coli'' and ''Salmonella''. These LPS molecules, consisting of the O-antigen or O-polysaccharide, core polysaccharide, and lipid A, serve multiple functions including contributing to the cell's negative charge and protecting against certain chemicals. LPS's role is critical in host-pathogen interactions, with the O-antigen eliciting an immune response and lipid A acting as an endotoxin. Additionally, the outer membrane acts as a selective barrier, regulated by porins, transmembrane proteins forming pores that allow specific molecules to pass. The space between the cell membrane and the outer membrane, known as the periplasm, contains periplasmic enzymes for nutrient processing. A significant structural component linking the peptidoglycan layer and the outer membrane is Braun's lipoprotein, which provides additional stability and strength to the bacterial cell wall. Most
bacterial phyla Bacterial phyla constitute the major lineages of the domain Bacteria. While the exact definition of a bacterial phylum is debated, a popular definition is that a bacterial phylum is a monophyletic lineage of bacteria whose 16S rRNA genes share ...
are gram-negative, including the
cyanobacteria Cyanobacteria ( ) are a group of autotrophic gram-negative bacteria that can obtain biological energy via oxygenic photosynthesis. The name "cyanobacteria" () refers to their bluish green (cyan) color, which forms the basis of cyanobacteri ...
, green sulfur bacteria, and most
Pseudomonadota Pseudomonadota (synonym "Proteobacteria") is a major phylum of gram-negative bacteria. Currently, they are considered the predominant phylum within the domain of bacteria. They are naturally found as pathogenic and free-living (non- parasitic) ...
(exceptions being some members of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).


Gram-variable and gram-indeterminate bacteria

Some bacteria, after staining with the Gram stain, yield a gram-variable pattern: a mix of pink and purple cells are seen. In cultures of ''Bacillus, Butyrivibrio'', and ''Clostridium'', a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain gram-negative. In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain. Gram-indeterminate bacteria do not respond predictably to Gram staining and, therefore, cannot be determined as either gram-positive or gram-negative. Examples include many species of ''
Mycobacterium ''Mycobacterium'' is a genus of over 190 species in the phylum Actinomycetota, assigned its own family, Mycobacteriaceae. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis (''Mycobacterium tuberculo ...
'', including ''Mycobacterium bovis'', ''
Mycobacterium leprae ''Mycobacterium leprae'' (also known as the leprosy bacillus or Hansen's bacillus) is one of the two species of bacteria that cause Hansen's disease (leprosy), a chronic but curable infectious disease that damages the peripheral nerves and ta ...
'' and ''
Mycobacterium tuberculosis ''Mycobacterium tuberculosis'' (M. tb), also known as Koch's bacillus, is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, ''M. tuberculosis'' ha ...
'', the latter two of which are the causative agents of leprosy and tuberculosis, respectively. Bacteria of the genus ''
Mycoplasma ''Mycoplasma'' is a genus of bacteria that, like the other members of the class ''Mollicutes'', lack a cell wall, and its peptidoglycan, around their cell membrane. The absence of peptidoglycan makes them naturally resistant to antibiotics ...
'' lack a
cell wall A cell wall is a structural layer that surrounds some Cell type, cell types, found immediately outside the cell membrane. It can be tough, flexible, and sometimes rigid. Primarily, it provides the cell with structural support, shape, protection, ...
around their
cell membrane The cell membrane (also known as the plasma membrane or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of a cell from the outside environment (the extr ...
s, which means they do not stain by Gram's method and are resistant to the antibiotics that target cell wall synthesis.


Orthographic note

The term ''Gram staining'' is derived from the surname of
Hans Christian Gram Hans Christian Joachim Gram (13 September 1853 – 14 November 1938) was a Danish bacteriologist noted for his development of the Gram stain, still a standard technique to classify bacteria and make them more visible under a microscope. Earl ...
; the
eponym An eponym is a noun after which or for which someone or something is, or is believed to be, named. Adjectives derived from the word ''eponym'' include ''eponymous'' and ''eponymic''. Eponyms are commonly used for time periods, places, innovati ...
(Gram) is therefore capitalized but not the common noun (stain) as is usual for scientific terms. The initial letters of ''gram-positive'' and ''gram-negative'', which are eponymous adjectives, can be either capital ''G'' or lowercase ''g'', depending on what
style guide A style guide is a set of standards for the writing, formatting, and design of documents. A book-length style guide is often called a style manual or a manual of style. A short style guide, typically ranging from several to several dozen page ...
(if any) governs the document being written. Lowercase style is used by the US
Centers for Disease Control and Prevention The Centers for Disease Control and Prevention (CDC) is the National public health institutes, national public health agency of the United States. It is a Federal agencies of the United States, United States federal agency under the United S ...
and other style regimens such as the AMA style. Dictionaries may use lowercase, Use search terms such as . uppercase, or both. Uppercase ''Gram-positive'' or ''Gram-negative'' usage is also common in many
scientific journal In academic publishing, a scientific journal is a periodical publication designed to further the progress of science by disseminating new research findings to the scientific community. These journals serve as a platform for researchers, schola ...
articles and publications. When articles are submitted to journals, each journal may or may not apply house style to the
postprint A postprint is a digital draft of a research journal article ''after'' it has been peer reviewed and accepted for publication, but ''before'' it has been typeset and formatted by the journal. Related terminology A digital draft before peer re ...
version.
Preprint In academic publishing, a preprint is a version of a scholarly or scientific paper that precedes formal peer review and publication in a peer-reviewed scholarly or scientific journal. The preprint may be available, often as a non-typeset versi ...
versions contain whichever style the author happened to use. Even style regimens that use lowercase for the adjectives ''gram-positive'' and ''gram-negative'' still typically use capital for ''Gram stain''.


See also

*
Bacterial cell structure A bacterium, despite its simplicity, contains a well-developed cell structure which is responsible for some of its unique biological structures and pathogenicity. Many structural features are unique to bacteria, and are not found among archaea ...
* Ziehl–Neelsen stain


References


External links


Gram staining technique video
{{Clinical microbiology techniques Bacteriology Staining Microscopy Danish inventions 1884 in biology