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Gram stain

Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan 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. They are stained pink or red by the counterstain, commonly safranin or fuchsine. Lugol's iodine solution is always added after addition of crystal violet to strengthen the bonds of the stain with the cell membrane.

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 (1853–1938), who developed the technique while working with Carl Friedländer in the morgue of the city hospital in Berlin 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 bacillus did not retain the stain. Gram did not initially make the distinction between Gram-negative and Gram-positive bacteria using his procedure.

Uses
Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large groups (gram-positive and gram-negative) based on the physical properties of their cell walls. Gram staining can also be used to diagnose a fungal infection. Gram staining is not used to classify archaea, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.

Some organisms are gram-variable (meaning they may stain either negative or positive); some are not stained with either dye used in the Gram technique and are not seen.

Medical
Gram stains are performed on body fluid or biopsy when infection 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 for meningitis and synovial fluid for septic arthritis.

Staining mechanism
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (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:
 * 1) 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.
 * 2) The addition of iodine, which binds to crystal violet and traps it in the cell
 * 3) Rapid decolorization with ethanol or acetone
 * 4) Counterstaining with safranin. Carbol fuchsin 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.

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 (except for the classes Mollicutes and Negativicutes) and the Actinomycetota. In contrast, members of the Chloroflexota (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 Firmicutes, a name now used for the largest group. It includes many well-known genera such as Lactobacillus, Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria such as Mycoplasma 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 (LPS) is the most abundant antigen 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 are gram-negative, including the cyanobacteria, green sulfur bacteria, and most Pseudomonadota (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, including Mycobacterium bovis, Mycobacterium leprae and Mycobacterium tuberculosis, the latter two of which are the causative agents of leprosy and tuberculosis, respectively. Bacteria of the genus Mycoplasma lack a cell wall around their cell membranes, 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; the eponym (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 (if any) governs the document being written. Lowercase style is used by the US Centers for Disease Control and Prevention and other style regimens such as the AMA style. Dictionaries may use lowercase, uppercase,    or both. Uppercase Gram-positive or Gram-negative usage is also common in many scientific journal articles and publications. When articles are submitted to journals, each journal may or may not apply house style to the postprint version. Preprint 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.

title: Gram stain


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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 is used by microbiologists to place bacteria into two main categories, Gram-positive (+) and Gram-negative (-). Gram-positive bacteria have a thick layer of peptidoglycan within the cell wall, and Gram-negative bacteria have a thin layer of peptidoglycan.

Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-coloured when seen through an optical microscope. This is because the thick layer of peptidoglycan in the bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test.

Conversely, gram-negative bacteria cannot retain the violet stain after the decolorization step; alcohol used in this stage degrades the outer membrane of gram-negative cells, making the cell wall more porous and incapable of retaining the crystal violet stain. Their peptidoglycan layer is much thinner and sandwiched between an inner cell membrane and a bacterial outer membrane, causing them to take up the counterstain (safranin or fuchsine) and appear red or pink.

Despite their thicker peptidoglycan layer, gram-positive bacteria are more receptive to certain cell wall–targeting antibiotics than gram-negative bacteria, due to the absence of the outer membrane.

Characteristics
In general, the following characteristics are present in gram-positive bacteria:

 * 1) Cytoplasmic lipid membrane
 * 2) Thick peptidoglycan layer
 * 3) Teichoic acids and lipoids are present, forming lipoteichoic acids, which serve as chelating agents, and also for certain types of adherence.
 * 4) Peptidoglycan chains are cross-linked to form rigid cell walls by a bacterial enzyme DD-transpeptidase.
 * 5) A much smaller volume of periplasm than that in gram-negative bacteria.

Only some species have a capsule, usually consisting of polysaccharides. Also, only some species are flagellates, and when they do have flagella, have only two basal body rings to support them, whereas gram-negative have four. Both gram-positive and gram-negative bacteria commonly have a surface layer called an S-layer. In gram-positive bacteria, the S-layer is attached to the peptidoglycan layer. Gram-negative bacteria's S-layer is attached directly to the outer membrane. Specific to gram-positive bacteria is the presence of teichoic acids in the cell wall. Some of these are lipoteichoic acids, which have a lipid component in the cell membrane that can assist in anchoring the peptidoglycan.

Classification
Along with cell shape, Gram staining is a rapid method used to differentiate bacterial species. Such staining, together with growth requirement and antibiotic susceptibility testing, and other macroscopic and physiologic tests, forms a basis for practical classification and subdivision of the bacteria (e.g., see figure and pre-1990 versions of Bergey's Manual of Systematic Bacteriology).

Historically, the kingdom Monera was divided into four divisions based primarily on Gram staining: Bacillota (positive in staining), Gracilicutes (negative in staining), Mollicutes (neutral in staining) and Mendocutes (variable in staining). Based on 16S ribosomal RNA phylogenetic studies of the late microbiologist Carl Woese and collaborators and colleagues at the University of Illinois, the monophyly of the gram-positive bacteria was challenged, with major implications for the therapeutic and general study of these organisms. Based on molecular studies of the 16S sequences, Woese recognised twelve bacterial phyla. Two of these were gram-positive and were divided on the proportion of the guanine and cytosine content in their DNA. The high G + C phylum was made up of the Actinobacteria, and the low G + C phylum contained the Firmicutes. The Actinomycetota include the Corynebacterium, Mycobacterium, Nocardia and Streptomyces genera. The (low G + C) Bacillota, have a 45–60% GC content, but this is lower than that of the Actinomycetota.

Importance of the outer cell membrane in bacterial classification
Although bacteria are traditionally divided into two main groups, gram-positive and gram-negative, based on their Gram stain retention property, this classification system is ambiguous as it refers to three distinct aspects (staining result, envelope organization, taxonomic group), which do not necessarily coalesce for some bacterial species. The gram-positive and gram-negative staining response is also not a reliable characteristic as these two kinds of bacteria do not form phylogenetic coherent groups. However, although Gram staining response is an empirical criterion, its basis lies in the marked differences in the ultrastructure and chemical composition of the bacterial cell wall, marked by the absence or presence of an outer lipid membrane.

All gram-positive bacteria are bounded by a single-unit lipid membrane, and, in general, they contain a thick layer (20–80 nm) of peptidoglycan responsible for retaining the Gram stain. A number of other bacteria—that are bounded by a single membrane, but stain gram-negative due to either lack of the peptidoglycan layer, as in the mycoplasmas, or their inability to retain the Gram stain because of their cell wall composition—also show close relationship to the gram-positive bacteria. For the bacterial cells bounded by a single cell membrane, the term monoderm bacteria has been proposed.

In contrast to gram-positive bacteria, all typical gram-negative bacteria are bounded by a cytoplasmic membrane and an outer cell membrane; they contain only a thin layer of peptidoglycan (2–3 nm) between these membranes. The presence of inner and outer cell membranes defines a new compartment in these cells: the periplasmic space or the periplasmic compartment. These bacteria have been designated as diderm bacteria. The distinction between the monoderm and diderm bacteria is supported by conserved signature indels in a number of important proteins (viz. DnaK, GroEL). Of these two structurally distinct groups of bacteria, monoderms are indicated to be ancestral. Based upon a number of observations including that the gram-positive bacteria are the major producers of antibiotics and that, in general, gram-negative bacteria are resistant to them, it has been proposed that the outer cell membrane in gram-negative bacteria (diderms) has evolved as a protective mechanism against antibiotic selection pressure. Some bacteria, such as Deinococcus, which stain gram-positive due to the presence of a thick peptidoglycan layer and also possess an outer cell membrane are suggested as intermediates in the transition between monoderm (gram-positive) and diderm (gram-negative) bacteria. The diderm bacteria can also be further differentiated between simple diderms lacking lipopolysaccharide, the archetypical diderm bacteria where the outer cell membrane contains lipopolysaccharide, and the diderm bacteria where outer cell membrane is made up of mycolic acid.

Exceptions
In general, gram-positive bacteria are monoderms and have a single lipid bilayer whereas gram-negative bacteria are diderms and have two bilayers. Exceptions include:
 * Some taxa lack peptidoglycan (such as the class Mollicutes, some members of the Rickettsiales, and the insect-endosymbionts of the Enterobacteriales) and are gram-indeterminate.
 * The Deinococcota have gram-positive stains, although they are structurally similar to gram-negative bacteria with two layers.
 * The Chloroflexota have a single layer, yet (with some exceptions ) stain negative. Two related phyla to the Chloroflexi, the TM7 clade and the Ktedonobacteria, are also monoderms.

Some Bacillota species are not gram-positive. The class Negativicutes, which includes Selenomonas, are diderm and stain gram-negative. Additionally, a number of bacterial taxa (viz. Negativicutes, Fusobacteriota, Synergistota, and Elusimicrobiota) that are either part of the phylum Bacillota or branch in its proximity are found to possess a diderm cell structure. However, a conserved signature indel (CSI) in the HSP60 (GroEL) protein distinguishes all traditional phyla of gram-negative bacteria (e.g., Pseudomonadota, Aquificota, Chlamydiota, Bacteroidota, Chlorobiota, "Cyanobacteria", Fibrobacterota, Verrucomicrobiota, Planctomycetota, Spirochaetota, Acidobacteriota, etc.) from these other atypical diderm bacteria, as well as other phyla of monoderm bacteria (e.g., Actinomycetota, Bacillota, Thermotogota, Chloroflexota, etc.). The presence of this CSI in all sequenced species of conventional LPS (lipopolysaccharide)-containing gram-negative bacterial phyla provides evidence that these phyla of bacteria form a monophyletic clade and that no loss of the outer membrane from any species from this group has occurred.

Pathogenicity
In the classical sense, six gram-positive genera are typically pathogenic in humans. Two of these, Streptococcus and Staphylococcus, are cocci (sphere-shaped). The remaining organisms are bacilli (rod-shaped) and can be subdivided based on their ability to form spores. The non-spore formers are Corynebacterium and Listeria (a coccobacillus), whereas Bacillus and Clostridium produce spores. The spore-forming bacteria can again be divided based on their respiration: Bacillus is a facultative anaerobe, while Clostridium is an obligate anaerobe. Also, Rathybacter, Leifsonia, and Clavibacter are three gram-positive genera that cause plant disease. Gram-positive bacteria are capable of causing serious and sometimes fatal infections in newborn infants. Novel species of clinically relevant gram-positive bacteria also include Catabacter hongkongensis, which is an emerging pathogen belonging to Bacillota.

Bacterial transformation
Transformation is one of three processes for horizontal gene transfer, in which exogenous genetic material passes from a donor bacterium to a recipient bacterium, the other two processes being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of donor bacterial DNA by a bacteriophage virus into a recipient host bacterium). In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.

As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between gram-positive and gram-negative bacteria; the number might be an overestimate since several of the reports are supported by single papers. Transformation among gram-positive bacteria has been studied in medically important species such as Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus and Streptococcus sanguinis and in gram-positive soil bacterium Bacillus subtilis, Bacillus cereus.

Orthographic note
The adjectives gram-positive and gram-negative derive from the surname of Hans Christian Gram; as eponymous adjectives, their initial letter can be either capital G or lower-case g, depending on which style guide (e.g., that of the CDC), if any, governs the document being written. This is further explained at Gram staining § Orthographic note.

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/

title: Gram-positive bacteria


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Gram-negative bacteria

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 their cell envelope, which consists of a thin peptidoglycan cell wall sandwiched between an inner (cytoplasmic) membrane and an outer membrane. These bacteria are found in all environments that support life on Earth.

Within this category, notable species include the model organism Escherichia coli, along with various pathogenic bacteria, such as Pseudomonas aeruginosa, Chlamydia trachomatis, and Yersinia pestis. They pose significant challenges in the medical field due to their outer membrane, which acts as a protective barrier against numerous antibiotics (including penicillin), detergents that would normally damage the inner cell membrane, and the antimicrobial enzyme lysozyme produced by animals as part of their innate immune system. Furthermore, the outer leaflet of this membrane contains a complex lipopolysaccharide (LPS) whose lipid A component can trigger a toxic reaction when the bacteria are lysed by immune cells. This reaction may lead to septic shock, resulting in low blood pressure, respiratory failure, reduced oxygen delivery, and lactic acidosis.

Several classes of antibiotics have been developed to target gram-negative bacteria, including aminopenicillins, ureidopenicillins, cephalosporins, beta-lactam-betalactamase inhibitor combinations (such as piperacillin-tazobactam), folate antagonists, quinolones, and carbapenems. Many of these antibiotics also cover gram-positive bacteria. The antibiotics that specifically target gram-negative organisms include aminoglycosides, monobactams (such as aztreonam), and ciprofloxacin.

Characteristics

Conventional gram-negative (LPS-diderm) bacteria display the following characteristics:
 * An inner cell membrane is present (cytoplasmic)
 * A thin peptidoglycan layer is present (this is much thicker in gram-positive bacteria)
 * Has outer membrane containing lipopolysaccharides (LPS, which consists of lipid A, core polysaccharide, and O antigen) in its outer leaflet and phospholipids in the inner leaflet
 * Porins exist in the outer membrane, which act like pores for particular molecules
 * Between the outer membrane and the cytoplasmic membrane there is a space filled with a concentrated gel-like substance called periplasm
 * The S-layer is directly attached to the outer membrane rather than to the peptidoglycan
 * If present, flagella have four supporting rings instead of two
 * Teichoic acids or lipoteichoic acids are absent
 * Lipoproteins are attached to the polysaccharide backbone
 * Some contain Braun's lipoprotein, which serves as a link between the outer membrane and the peptidoglycan chain by a covalent bond
 * Most, with few exceptions, do not form spores

Classification
Along with cell shape, Gram staining is a rapid diagnostic tool and once was used to group species at the subdivision of Bacteria. Historically, the kingdom Monera was divided into four divisions based on Gram staining: Firmacutes (+), Gracillicutes (−), Mollicutes (0) and Mendocutes (var.). Since 1987, the monophyly of the gram-negative bacteria has been disproven with molecular studies. However some authors, such as Cavalier-Smith still treat them as a monophyletic taxon (though not a clade; his definition of monophyly requires a single common ancestor but does not require holophyly, the property that all descendants be encompassed by the taxon) and refer to the group as a subkingdom "Negibacteria".

Taxonomy
Bacteria are traditionally classified based on their Gram-staining response into the gram-positive and gram-negative bacteria. Having just one membrane, the gram-positive bacteria are also known as monoderm bacteria, while gram-negative bacteria, having two membranes, are also known as diderm bacteria. It was traditionally thought that the groups represent lineages, i.e., the extra membrane only evolved once, such that gram-negative bacteria are more closely related to one another than to any gram-positive bacteria. While this is often true, the classification system breaks down in some cases, with lineage groupings not matching the staining result. Thus, Gram staining cannot be reliably used to assess familial relationships of bacteria. Nevertheless, staining often gives reliable information about the composition of the cell membrane, distinguishing between the presence or absence of an outer lipid membrane.

Of these two structurally distinct groups of prokaryotic organisms, monoderm prokaryotes are thought to be ancestral. Based upon a number of different observations, including that the gram-positive bacteria are the most sensitive to antibiotics and that the gram-negative bacteria are, in general, resistant to antibiotics, it has been proposed that the outer cell membrane in gram-negative bacteria (diderms) evolved as a protective mechanism against antibiotic selection pressure. Some bacteria such as Deinococcus, which stain gram-positive due to the presence of a thick peptidoglycan layer, but also possess an outer cell membrane are suggested as intermediates in the transition between monoderm (gram-positive) and diderm (gram-negative) bacteria. The diderm bacteria can also be further differentiated between simple diderms lacking lipopolysaccharide (LPS); the archetypical diderm bacteria, in which the outer cell membrane contains lipopolysaccharide; and the diderm bacteria, in which the outer cell membrane is made up of mycolic acid (e. g. Mycobacterium).

The conventional LPS-diderm group of gram-negative bacteria (e.g., Pseudomonadota, Aquificota, Chlamydiota, Bacteroidota, Chlorobiota, "Cyanobacteria", Fibrobacterota, Verrucomicrobiota, Planctomycetota, Spirochaetota, Acidobacteriota; "Hydrobacteria") are uniquely identified by a few conserved signature indel (CSI) in the HSP60 (GroEL) protein. In addition, a number of bacterial taxa (including Negativicutes, Fusobacteriota, Synergistota, and Elusimicrobiota) that are either part of the phylum Bacillota (a monoderm group) or branches in its proximity are also found to possess a diderm cell structure. They lack the GroEL signature. The presence of this CSI in all sequenced species of conventional lipopolysaccharide-containing gram-negative bacterial phyla provides evidence that these phyla of bacteria form a monophyletic clade and that no loss of the outer membrane from any species from this group has occurred.

Example species
The proteobacteria are a major superphylum of gram-negative bacteria, including E. coli, Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc. Other notable groups of gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria.

Medically-relevant gram-negative diplococci include the four types that cause a sexually transmitted disease (Neisseria gonorrhoeae ), a meningitis (Neisseria meningitidis ), and respiratory symptoms (Moraxella catarrhalis, A coccobacillus Haemophilus influenzae is another medically relevant coccal type.

Medically relevant gram-negative bacilli include a multitude of species. Some of them cause primarily respiratory problems (Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis, Salmonella typhi).

Gram-negative bacteria associated with hospital-acquired infections include Acinetobacter baumannii, which cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in hospital intensive-care units.

Bacterial transformation
Transformation is one of three processes for horizontal gene transfer, in which exogenous genetic material passes from one bacterium to another, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium). In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.

As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between gram-positive and gram-negative bacteria; the number might be an overestimate since several of the reports are supported by single papers. Transformation has been studied in medically important gram-negative bacteria species such as Helicobacter pylori, Legionella pneumophila, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae and Vibrio cholerae. It has also been studied in gram-negative species found in soil such as Pseudomonas stutzeri, Acinetobacter baylyi, and gram-negative plant pathogens such as Ralstonia solanacearum and Xylella fastidiosa.

Role in disease
One of the several unique characteristics of gram-negative bacteria is the structure of the bacterial outer membrane. The outer leaflet of this membrane contains lipopolysaccharide (LPS), whose lipid A portion acts as an endotoxin. If gram-negative bacteria enter the circulatory system, LPS can trigger an innate immune response, activating the immune system and producing cytokines (hormonal regulators). This leads to inflammation and can cause a toxic reaction, resulting in fever, an increased respiratory rate, and low blood pressure. That is why some infections with gram-negative bacteria can lead to life-threatening septic shock.

The outer membrane protects the bacteria from several antibiotics, dyes, and detergents that would normally damage either the inner membrane or the cell wall (made of peptidoglycan). The outer membrane provides these bacteria with resistance to lysozyme and penicillin. The periplasmic space (space between the two cell membranes) also contains enzymes which break down or modify antibiotics. Drugs commonly used to treat gram negative infections include amino, carboxy and ureido penicillins (ampicillin, amoxicillin, pipercillin, ticarcillin). These drugs may be combined with beta-lactamase inhibitors to combat the presence of enzymes that can digest these drugs (known as beta-lactamases) in the peri-plasmic space. Other classes of drugs that have gram negative spectrum include cephalosporins, monobactams (aztreonam), aminoglycosides, quinolones, macrolides, chloramphenicol, folate antagonists, and carbapenems.

Orthographic note
The adjectives gram-positive and gram-negative derive from the surname of Hans Christian Gram, a Danish bacteriologist; as eponymous adjectives, their initial letter can be either capital G or lower-case g, depending on which style guide (e.g., that of the CDC), if any, governs the document being written. This is further explained at Gram staining § Orthographic note.

title: Gram-negative bacteria


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Greyhound

The English Greyhound, or simply the Greyhound, is a breed of dog, a sighthound which has been bred for coursing, greyhound racing and hunting. Since the rise in large-scale adoption of retired racing Greyhounds, the breed has seen a resurgence in popularity as a family pet.

Greyhounds are defined as a tall, muscular, smooth-coated, "S-shaped" type of sighthound with a long tail and tough feet. Greyhounds are a separate breed from other related sighthounds, such as the Italian greyhound.

The Greyhound's combination of long, powerful legs, deep chest, flexible spine, and slim build allows it to reach average race speeds exceeding 64 km/h. A racing greyhound can reach a full speed of at least 21.3 m/s 47 mph.

Appearance
Males are usually 71 to 76 cm tall at the withers, and weigh on average 27 to 40 kg. Females tend to be smaller, with shoulder heights ranging from 66 to 71 cm and weights from 25 to 34 kg, although weights can be above and below these average weights. Greyhounds have very short fur, which is easy to maintain. There are approximately 30 recognized color forms, of which variations of white, brindle, fawn (pale tan to dark deer-red), black, red, and blue (gray) can appear uniquely or in combination. Greyhounds are dolichocephalic, with a skull which is relatively long in comparison to its breadth, and an elongated muzzle.

Temperament
Greyhounds live most happily as pets in quiet environments. They do well in families with children, as long as the children are taught to treat the dog properly with politeness and appropriate respect. Greyhounds have a sensitive nature, and gentle commands work best as training methods.

Occasionally, a Greyhound may bark; however, they are generally not barkers, which is beneficial in suburban environments, and they are usually as friendly to strangers as they are with their own families. A 2008 University of Pennsylvania study found that Greyhounds are one of the least aggressive dog breeds towards strangers, owners, and other dogs.

A survey of those adopting rescue Greyhounds found that Greyhound adoptions have higher short term adoption success than shelters. The survey also found reported hyperactivity levels to be below that of shelter dogs.

In Victoria, Australia it is illegal for racing Greyhounds to be uncontrolled and without a muzzle in public. This only applies to active racing Greyhounds, any retired Greyhounds or those used for other purposes as exempt.

Greyhounds tend to be out-going, happy and sociable with people and seem to relish human contact, even following owners from room to room at home (known colloquially as being a "Velcro dog"). Small animals including cats may be the subject of prey-driven behaviour by Greyhounds.

Coursing
The original primary use of Greyhounds, both in the British Isles and on the Continent of Europe, was in the coursing of deer for meat and sport; later, specifically in Britain, they specialized in competition hare coursing. Some Greyhounds are still used for coursing, although artificial lure sports like lure coursing and racing are far more common and popular. Many leading 300- to 550-yard sprinters have bloodlines traceable back through Irish sires, within a few generations of racers that won events such as the Irish Coursing Derby or the Irish Cup.

Racing
Until the early 20th century, Greyhounds were principally bred and trained for hunting and coursing. During the 1920s, modern greyhound racing was introduced into the United States, England (1926), Northern Ireland (1927), Scotland (1927), and the Republic of Ireland (1927). Australia also has a significant racing culture.

In the United States, aside from professional racing, many Greyhounds enjoy success on the amateur race track. Organizations like the Large Gazehound Racing Association (LGRA) and the National Oval Track Racing Association (NOTRA) provide opportunities for Greyhounds to compete.

Companion
Historically, the Greyhound has, since its first appearance as a hunting type and breed, enjoyed a specific degree of fame and definition in Western literature, heraldry and art as the most elegant or noble companion and hunter of the canine world. In modern times, the professional racing industry, with its large numbers of track-bred greyhounds, as well as international adoption programs aimed at re-homing dogs has redefined the breed as a sporting dog that will supply friendly companionship in its retirement. This has been prevalent in recent years due to track closures in the United States. Outside the racing industry and coursing community, the Kennel Clubs' registered breed still enjoys a modest following as a show dog and pet.

Health and physiology
A 2024 UK study found a life expectancy of 11.5 years for the breed compared to an average of 12.7 for purebreeds and 12 for crossbreeds. A 2005 Swedish study of insurance data found 60% of Greyhounds died by the age of 10, higher than the overall rate of 35% of dogs dying by the age of 10.

The key to the speed of a Greyhound can be found in its light but muscular build, large heart, highest percentage of fast twitch muscle of any breed, double suspension gallop, and extreme flexibility of its spine. "Double suspension rotary gallop" describes the fastest running gait of the Greyhound in which all four feet are free from the ground in two phases, contracted and extended, during each full stride.

Due to the Greyhound's unique physiology and anatomy, a veterinarian who understands the issues relevant to the breed is generally needed when the dogs need treatment, particularly when anesthesia is required. Greyhounds cannot metabolize barbiturate-based anesthesia in the same way that other breeds can because their livers have lower amounts of oxidative enzymes. Greyhounds demonstrate unusual blood chemistry, which can be misread by veterinarians not familiar with the breed and can result in an incorrect diagnosis.

Greyhounds are very sensitive to insecticides. Many vets do not recommend the use of flea collars or flea spray on Greyhounds if the product is pyrethrin-based. Products like Advantage, Frontline, Lufenuron, and Amitraz are safe for use on Greyhounds, however, and are very effective in controlling fleas and ticks.

Greyhounds have higher levels of red blood cells than other breeds. Since red blood cells carry oxygen to the muscles, this higher level allows the hound to move larger quantities of oxygen faster from the lungs to the muscles. Conversely, Greyhounds have lower levels of platelets than other breeds.

Delayed haemorrhage following trauma or routine surgery is more common in Greyhounds, with one study reporting significant haemorrhage in 26% of Greyhounds following routine gonadectomy, compared to 0-2% in other dog breeds. This is often termed greyhound fibrinolytic syndrome or breed-associated hyperfibrinolysis, where in there is a disorder of the fibrinolysis system without derangement of the primary or secondary coagulation systems, and is also not related to platelet count. In this syndrome there is initial adequate hemostasis following trauma or routine surgical procedures, however 36–48 hours later the site undergoes inappropriate hyperfibrinolysis. This results in delayed bleeding which can result in significant morbidity and mortality. Standard pre-operative blood work does not identify those at risk It is distinct from common bleeding disorders in other breeds such as von Willebrand's disease, which is uncommon in Greyhounds. Although high-quality research data are lacking, it is thought that this condition can be prevented and treated by administering antifibrinolytic medication such as tranexamic acid via the oral or parenteral route. Intensive care and blood product administration may also be required in severe cases.

Greyhounds do not have undercoats and thus are less likely to trigger dog allergies in humans (they are sometimes incorrectly referred to as "hypoallergenic"). The lack of an undercoat, coupled with a general lack of body fat, also makes Greyhounds more susceptible to extreme temperatures (both hot and cold); because of this, they must be housed inside. Some Greyhounds are susceptible to corns on their paw pads; a variety of methods are used to treat them.

Thyroxine levels in the Greyhound are below the normal reference range for dogs; thyroxine response to thyroid-stimulating hormone is also lowered. This can impact testing for thyroid disease but it is not a concern for health.

Origins
"The true origin of the greyhound is unsure, but drawings of findings from the Çatalhöyük site in Turkey (6000 BC), the finding of a greyhound-like dog in a funeral vase in the town of Fusa in Iran (4200 BC) or in rock art in Tassili (dated at 5000 – 2000 BC) indicate that the greyhound is indeed one of the oldest breeds of dog.".

The ancient skeletal remains of a dog identified as being of the greyhound/saluki form were excavated at Tell Brak in modern Syria, and dated as being approximately 4,000 years old. Dogs that look similar to Salukis and Greyhounds were increasingly depicted on Egyptian tombs from the Middle Kingdom (2134 BC–1785 BC) onward.

Historical literature by Arrian on the vertragus (from the Latin vertragus, a word of Celtic origin), the first recorded sighthound in Europe and possible antecedent of the Greyhound, suggested that its origin lies with the Celts from Eastern Europe or Eurasia. Systematic archaeozoology of Britain conducted in 1974 ruled out the existence of a true greyhound-type in Britain prior to the Roman occupation, which was further confirmed in 2000. Written evidence from the early period of Roman occupation, the Vindolanda tablets (No. 594), demonstrate that the occupying troops from Continental Europe either had with them in the North of England, or certainly knew of, the vertragus and its hunting use.

During the Middle Ages, greyhounds could only be owned by rulers and nobles, having long been associated with heraldic symbols of the ruling class in England, France, and the Czech lands.

The earliest archaeological discovery found conclusively to be a greyhound specifically was at the Chotěbuz fort in the Czech Republic. This comprised sighthound type "gracile" bones dating from the 8th to 9th century AD. These bones matched those of a 70 cm high "greyhound", and were also genetically compared with the modern Greyhound and other sighthounds, and found to be almost completely identical with the modern Greyhound breed, with the exception of only four deletions and one substitution in the DNA sequences, which were interpreted as differences probably arising from 11 centuries of breeding of this type of dog.

All modern pedigree Greyhounds derive from the Greyhound stock recorded and registered first in private studbooks in the 18th century, then in public studbooks in the 19th century, which ultimately were registered with coursing, racing, and kennel club authorities of the United Kingdom. Historically, these sighthounds were used primarily for hunting in the open where their pursuit speed and keen eyesight were essential.

Etymology
The name "Greyhound" is generally believed to come from the Old English 'grighund'. Hund is the antecedent of the modern "hound", but the meaning of grig is undetermined, other than in reference to dogs in Old English and Old Norse. The word "hund" is still used for dogs in general in Scandinavian languages today. Its origin does not appear to have any common root with the modern word "grey" for color, and indeed the Greyhound is seen with a wide variety of coat colors. The lighter colors, patch-like markings and white appeared in the breed that was once ordinarily grey in color.

The Greyhound is the only dog mentioned by name in the Bible (זַרְזִיר מׇתְנַיִם, zarir mosna'im) in. Many versions, including the Jewish Publication Society and King James Version, name the Greyhound as one of the "three that are stately of stride". However, some newer biblical translations, including the New International Version, have changed this to 'strutting rooster', which appears to be an alternative translation. However, the Douay–Rheims Bible translation from the late 4th-century Latin Vulgate into English translates this term as "a cock".

According to Pokorny, the English term 'Greyhound' does not mean "grey dog/hound", but simply "fair dog". Subsequent words have been derived from the Proto-Indo-European root *g'her- "shine, twinkle": English 'grey', Old High German gris "grey, old", Old Icelandic griss "piglet, pig", Old Icelandic gryja "to dawn", gryjandi "morning twilight", Old Irish grian "sun", Old Church Slavonic zorja "morning twilight, brightness". The common sense of these words is "to shine; bright".

In 1928, the first winner of Best in Show at Crufts was breeder/owner Mr. H. Whitley's Greyhound Primley Sceptre. Greyhounds have won the award three times in total, the most recent being in 1956.

Historically, English Greyhounds were grouped: two for coursing, as a "Brace", three for hunting, as a "Leash", otherwise known as a "couple and a half".

title: Greyhound