Gram staining
Encyclopedia
Gram staining is a method of differentiating bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
l species into two large groups (Gram-positive and Gram-negative).
It is based on the chemical and physical properties of their cell wall
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...
s. Primarily, it detects peptidoglycan
Peptidoglycan
Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria , forming the cell wall. The sugar component consists of alternating residues of β- linked N-acetylglucosamine and N-acetylmuramic acid...
, which is present in a thick layer in Gram positive bacteria. A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color.
The Gram stain is almost always the first step in the identification of a bacterial organism, and is the default stain performed by laboratories over a sample when no specific culture is referred.
While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram-variable and Gram-indeterminate groups as well.
The word Gram is always spelled with a capital, referring to Hans Christian Gram
Hans Christian Gram
Hans Christian Joachim Gram was a Danish bacteriologist.He was the son of Frederik Terkel Julius Gram, a professor of jurisprudence, and Louise Christiane Roulund....
, the inventor of Gram staining.
History
The method is named after its inventor, the DanishDenmark
Denmark is a Scandinavian country in Northern Europe. The countries of Denmark and Greenland, as well as the Faroe Islands, constitute the Kingdom of Denmark . It is the southernmost of the Nordic countries, southwest of Sweden and south of Norway, and bordered to the south by Germany. Denmark...
scientist Hans Christian Gram
Hans Christian Gram
Hans Christian Joachim Gram was a Danish bacteriologist.He was the son of Frederik Terkel Julius Gram, a professor of jurisprudence, and Louise Christiane Roulund....
(1853–1938), who developed the technique while working with Carl Friedländer
Carl Friedländer
Carl Friedländer was a German pathologist and microbiologist who helped discover the bacterial cause of pneumonia in 1882...
in the morgue of the city hospital in Berlin
Berlin
Berlin is the capital city of Germany and is one of the 16 states of Germany. With a population of 3.45 million people, Berlin is Germany's largest city. It is the second most populous city proper and the seventh most populous urban area in the European Union...
. Gram devised his technique not for the purpose of distinguishing one group of bacteria from another but to enable bacteria to be seen more readily in stained sections of lung tissue. He published his method in 1884, and included in his short report the observation that the Typhus
Typhus
Epidemic typhus is a form of typhus so named because the disease often causes epidemics following wars and natural disasters...
bacillus
Bacillus (shape)
The word bacillus may be used to describe any rod-shaped bacterium, and such bacilli are found in many different taxonomic groups of bacteria. However, the name Bacillus, capitalized and italicized, refers to a specific genus of bacteria...
did not retain the stain.
Uses
Gram staining is a bacteriologicalBacteriology
Bacteriology is the study of bacteria. This subdivision of microbiology involves the identification, classification, and characterization of bacterial species...
laboratory
Laboratory
A laboratory is a facility that provides controlled conditions in which scientific research, experiments, and measurement may be performed. The title of laboratory is also used for certain other facilities where the processes or equipment used are similar to those in scientific laboratories...
technique used to differentiate bacterial species into two large groups (Gram-positive
Gram-positive
Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain and appearing red or pink...
and Gram-negative
Gram-negative
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...
) based on the physical properties of their cell wall
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...
s. Gram staining is not used to classify archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
, formally archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups
Phylogenetics
In biology, phylogenetics is the study of evolutionary relatedness among groups of organisms , which is discovered through molecular sequencing data and morphological data matrices...
.
The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, and it is of extremely limited use in environmental microbiology
Environmental microbiology
Environmental microbiology is the study of the composition and physiology of microbial communities in the environment. The environment in this case means the soil, water, air and sediments covering the planet and can also include the animals and plants that inhabit these areas...
. It has been largely superseded by molecular techniques even in the medical microbiology lab. Some organisms are Gram-variable (that means, they may stain either negative or positive); some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and information-rich than differential staining.
Medical
Gram stains are performed on body fluid or biopsyBiopsy
A biopsy is a medical test involving sampling of cells or tissues for examination. It is the medical removal of tissue from a living subject to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically...
when infection
Infection
An infection is the colonization of a host organism by parasite species. Infecting parasites seek to use the host's resources to reproduce, often resulting in disease...
is suspected. Gram stains yield results much more quickly than culture
Microbiological culture
A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested,...
, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid
Cerebrospinal fluid
Cerebrospinal fluid , Liquor cerebrospinalis, is a clear, colorless, bodily fluid, that occupies the subarachnoid space and the ventricular system around and inside the brain and spinal cord...
for meningitis
Meningitis
Meningitis is inflammation of the protective membranes covering the brain and spinal cord, known collectively as the meninges. The inflammation may be caused by infection with viruses, bacteria, or other microorganisms, and less commonly by certain drugs...
and synovial fluid
Synovial fluid
Synovial fluid is a viscous, non-Newtonian fluid found in the cavities of synovial joints. With its yolk-like consistency , the principal role of synovial fluid is to reduce friction between the articular cartilage of synovial joints during movement.-Overview:The inner membrane of synovial joints...
for septic arthritis
Septic arthritis
Septic arthritis is the purulent invasion of a joint by an infectious agent which produces arthritis. People with artificial joints are more at risk than the general population but have slightly different symptoms, are infected with different organisms and require different treatment. Septic...
.
.
Staining mechanism
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycanPeptidoglycan
Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria , forming the cell wall. The sugar component consists of alternating residues of β- linked N-acetylglucosamine and N-acetylmuramic acid...
(50-90% of cell wall), which are stained purple by crystal violet, whereas Gram-negative bacteria have a thinner layer (10% of cell wall), which are stained pink by the counter-stain. There are four basic steps of the Gram stain:
- applying a primary stain (crystal violet) to a heat-fixed (death by heat) smear of a bacterial culture
- the addition of a trapping agent (Gram's iodineIodineIodine is a chemical element with the symbol I and atomic number 53. The name is pronounced , , or . The name is from the , meaning violet or purple, due to the color of elemental iodine vapor....
) - rapid decolorization with alcoholEthanolEthanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
or acetoneAcetoneAcetone is the organic compound with the formula 2CO, a colorless, mobile, flammable liquid, the simplest example of the ketones.Acetone is miscible with water and serves as an important solvent in its own right, typically as the solvent of choice for cleaning purposes in the laboratory...
, and - counterstaining with safraninSafraninSafranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in a Gram stain...
. carbol fuchsinCarbol fuchsinCarbol fuchsin, carbol-fuchsin, or carbolfuchsin, is a mixture of phenol and basic fuchsin, used in bacterial staining procedures. It is commonly used in the staining of mycobacteria as it has an affinity for the mycolic acids found in their cell walls.It is a component of Ziehl-Neelsen...
is sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain.
Crystal violet
Crystal violet
Crystal violet or Gentian violet is a triarylmethane dye. The dye is used as a histological stain and in Gram’s method of classifying bacteria. Crystal violet has antibacterial, antifungal, and anthelmintic properties and was formerly important as a topical antiseptic...
(CV) dissociates in aqueous solutions into and chloride ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The ion interacts with negatively charged components of bacterial cells and stains the cells purple.
Iodine ( 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
Mordant
A mordant is a substance used to set dyes on fabrics or tissue sections by forming a coordination complex with the dye which then attaches to the fabric or tissue. It may be used for dyeing fabrics, or for intensifying stains in cell or tissue preparations. The term mordant comes from the Latin...
, but is a trapping agent that prevents the removal of the CV–I complex and, therefore, color 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 will lose 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 will be 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 fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color.
Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces
Actinomyces
Actinomyces from Greek "actino" that means mucus and fungus, is a genus of the actinobacteria class of bacteria. They are all Gram-positive and are characterized by contiguous spread, suppurative and granulomatous inflammation, and formation of multiple abscesses and sinus tracts that may...
, Arthobacter, Corynebacterium
Corynebacterium
Corynebacterium is a genus of Gram-positive rod-shaped bacteria. They are widely distributed in nature and are mostly innocuous. Some are useful in industrial settings such as C. glutamicum. Others can cause human disease. C...
, Mycobacterium
Mycobacterium
Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy...
, and Propionibacterium
Propionibacterium
Propionibacterium is a genus of bacteria named for their unique metabolism: They are able to synthesize propionic acid by using unusual transcarboxylase enzymes....
have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. In cultures of Bacillus
Bacillus
Bacillus is a genus of Gram-positive, rod-shaped bacteria and a member of the division Firmicutes. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species...
, Butyrivibrio, and Clostridium
Clostridium
Clostridium is a genus of Gram-positive bacteria, belonging to the Firmicutes. They are obligate anaerobes capable of producing endospores. Individual cells are rod-shaped, which gives them their name, from the Greek kloster or spindle...
, 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-positive bacteria
Gram-positive bacteria have generally a single membrane (monoderm) surrounded by a thick peptidoglycan.This rule is followed by two phyla — the Firmicutes
Firmicutes
The Firmicutes are a phylum of bacteria, most of which have Gram-positive cell wall structure. A few, however, such as Megasphaera, Pectinatus, Selenomonas and Zymophilus, have a porous pseudo-outer-membrane that causes them to stain Gram-negative...
(except for the classes Mollicutes
Mollicutes
The Mollicutes are a class of bacteria distinguished by the absence of a cell wall. The word "Mollicutes" is derived from the Latin mollis , and cutis . They are parasites of various animals and plants, living on or in the host's cells. Individuals are very small, typically only 0.2–0.3 μm in size...
and Negativicutes
Negativicutes
The Negativicutes is a class of firmicute bacteria, whose members have a peculiar cell wall composition which stains Gram negative, unlike most other members of the Firmicutes. The family Veillonellaceae, formerly known as Acidaminococcaceae, we once considered members of the clsss Clostridia. The...
) and the Actinobacteria
Actinobacteria
Actinobacteria are a group of Gram-positive bacteria with high guanine and cytosine content. They can be terrestrial or aquatic. Actinobacteria is one of the dominant phyla of the bacteria....
. In contrast, members of the Chloroflexi
Chloroflexi
Chloroflexi is one of four classes of bacteria in the phylum Chloroflexi, known as filamentous green non-sulfur bacteria. They produce energy from light and are named for their green pigment, usually found in photosynthetic bodies called chlorosomes....
(green non-sulfur bacteria) are monoderms but possess a thin or absent (class Dehalococcoidetes
Dehalococcoidetes
"Dehalococcoides" is a class of "Chloroflexi", a phylum of Bacteria. It is also known as the DHC group.The name "Dehalococcoidetes" is a placeholder name given by Hugenholtz and Stackebrandt, 2004, after "Dehalococcoides ethenogenes" a partially described species in 1997, whereas the first species...
) peptidoglycan and can stain negative, positive or indeterminate. Members of the Deinococcus-Thermus group
Deinococcus-Thermus
The Deinococcus-Thermus are a small group of bacteria composed of cocci highly resistant to environmental hazards.There are two main groups.* The Deinococcales include two families, with three genera, Deinococcus and Truepera, the former with several species that are resistant to radiation; they...
, stain positive but are diderms with a thick peptidoglycan.
Historically
Bacterial taxonomy
Bacterial taxonomy is the taxonomy, i.e. the rank-based classification, of bacteria.In the scientific classification established by Carl von Linné, each species has to be assigned to a genus , which in turn is a lower level of a hierarchy of ranks .In the currently accepted classification...
, the Gram-positive forms made up the phylum Firmicutes
Firmicutes
The Firmicutes are a phylum of bacteria, most of which have Gram-positive cell wall structure. A few, however, such as Megasphaera, Pectinatus, Selenomonas and Zymophilus, have a porous pseudo-outer-membrane that causes them to stain Gram-negative...
, a name now used for the largest group. It includes many well-known genera such as Bacillus
Bacillus
Bacillus is a genus of Gram-positive, rod-shaped bacteria and a member of the division Firmicutes. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species...
, Listeria, Staphylococcus
Staphylococcus
Staphylococcus is a genus of Gram-positive bacteria. Under the microscope they appear round , and form in grape-like clusters....
, Streptococcus
Streptococcus
Streptococcus is a genus of spherical Gram-positive bacteria belonging to the phylum Firmicutes and the lactic acid bacteria group. Cellular division occurs along a single axis in these bacteria, and thus they grow in chains or pairs, hence the name — from Greek στρεπτος streptos, meaning...
, Enterococcus
Enterococcus
Enterococcus is a genus of lactic acid bacteria of the phylum Firmicutes. Enterococci are Gram-positive cocci that often occur in pairs or short chains, and are difficult to distinguish from streptococci on physical characteristics alone. Two species are common commensal organisms in the...
, and Clostridium
Clostridium
Clostridium is a genus of Gram-positive bacteria, belonging to the Firmicutes. They are obligate anaerobes capable of producing endospores. Individual cells are rod-shaped, which gives them their name, from the Greek kloster or spindle...
. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma
Mycoplasma
Mycoplasma refers to a genus of bacteria that lack a cell wall. Without a cell wall, they are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. They can be parasitic or saprotrophic. Several species are pathogenic in humans,...
that lack cell walls and so cannot be stained by Gram, but are derived from such forms.
Gram-negative bacteria
Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderms). Most bacterial phylaBacterial phyla
The bacterial phyla are the major lineages of the domain Bacteria.In the scientific classification established by Carl von Linné, each bacterial strain has to be assigned to a species , which is a lower level of a hierarchy of ranks...
are Gram-negative, including the cyanobacteria, spirochaete
Spirochaete
Spirochaetes belong to a phylum of distinctive Gram-negative bacteria, which have long, helically coiled cells...
s and green sulfur
Green sulfur bacteria
The green sulfur bacteria are a family of obligately anaerobic photoautotrophic bacteria. Most closely related to the distant Bacteroidetes, they are accordingly assigned their own phylum....
, and most Proteobacteria
Proteobacteria
The Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter, and many other notable genera....
(exceptions being some members of the Rickettsiales
Rickettsiales
The Rickettsiales, also called rickettsias, are an order of small proteobacteria. Most of those described survive only as endosymbionts of other cells. Some are notable pathogens, including Rickettsia, which causes a variety of diseases in humans...
and the insect-endosymbionts of the Enterobacteriales).