Gram-positive anthrax bacteria (purple rods) in cerebrospinal fluid sample. If present, a Gram-negative bacterial species would appear pink. (The other cells are white blood cells).

Gram staining (or Gram's method) is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls.^[1] Image File history File links Gram stained cerebrospinal fluid showing gram-positive anthrax baccilli (purple rods). ... Image File history File links Gram stained cerebrospinal fluid showing gram-positive anthrax baccilli (purple rods). ... Cerebrospinal fluid (CSF), Liquor cerebrospinalis, is a clear bodily fluid that occupies the subarachnoid space in the brain (the space between the skull and the cerebral cortex—more specifically, between the arachnoid and pia layers of the meninges). ... A scanning electron microscope image of normal circulating human blood. ... In philosophy generally, empiricism is a theory of knowledge emphasizing the role of experience in the formation of ideas, while discounting the notion of innate ideas. ... Phyla/Divisions Actinobacteria Aquificae Bacteroidetes/Chlorobi Chlamydiae/Verrucomicrobia Chloroflexi Chrysiogenetes Cyanobacteria Deferribacteres Deinococcus-Thermus Dictyoglomi Fibrobacteres/Acidobacteria Firmicutes Fusobacteria Gemmatimonadetes Nitrospirae Omnibacteria Planctomycetes Proteobacteria Spirochaetes Thermodesulfobacteria Thermomicrobia Thermotogae Bacteria (singular, bacterium) are a major group of living organisms. ... Gram-positive bacteria are those that are stained dark blue or violet by gram staining, in contrast to gram-negative bacteria, which are not affected by the stain. ... Bacteria that are Gram-negative are not stained dark blue or violet by Gram staining, in contrast to Gram-positive bacteria. ... A cell wall is a fairly rigid layer surrounding a cell, located external to the cell membrane, that provides the cell with structural support, protection, and a filtering mechanism. ...

The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique in 1884 to discriminate between pneumococci and Klebsiella pneumoniae bacteria.^[2] Professor Hans Christian Gram Hans Christian Joachim Gram (September 13, 1853 - November 14, 1938) was a Danish bacteriologist. ... Year 1884 (MDCCCLXXXIV) was a leap year starting on Tuesday (link will display the full calendar) of the Gregorian calendar (or a leap year starting on Sunday of the 12-day-slower Julian calendar). ... Binomial name Streptococcus pneumoniae Streptococcus pneumoniae is a species of Streptococcus that is a major human pathogen. ... Binomial name Klebsiella pneumoniae (Schroeter 1886) Trevisan 1887 Klebsiella pneumoniae is a Gram-negative, nonmotile, encapsulated, lactose-fermenting, facultatively anaerobic, rod-shaped bacterium found in the normal flora of the mouth, skin, and intestines. ...

Uses

Research

Gram staining is one of the most useful staining procedures in the traditional bacteriological laboratory.^[3] The technique is used as a tool for the differentiation of Gram-positive and Gram-negative bacteria, as a first step to determine the identity of a particular bacterial sample.^[4] Microbiology (in Greek micron = small and biologia = studying life) is the study of microorganisms, including unicellular (single-celled) eukaryotes and prokaryotes, fungi, and viruses. ... For other uses of lab, see Lab. ...

The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, however. It is of extremely limited use in environmental microbiology, and has been largely superseded by molecular techniques even in the medical microbiology lab. Given that some organisms are gram-variable (i.e. they may stain either negative or positive), and that some organisms are not susceptible to either stain used by the Gram technique, its true utility to researchers should be considered limited and specific. 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 biopsy when infection is suspected. It yields results much more quickly than culture, and is 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.^[3][5] Bodily fluids are fluids, which are generally excreted or secreted from the human body. ... Brain biopsy A biopsy (in Greek: bios = life and opsy = look/appearance) is a medical test involving the removal of cells or tissues for examination. ... An infection is the detrimental colonization of a host organism by a foreign species. ... A microbiological culture is a way to determine the cause of infectious disease by letting the agent multiply (reproduce) in predetermined media. ... Cerebrospinal fluid (CSF), Liquor cerebrospinalis, is a clear bodily fluid that occupies the subarachnoid space in the brain (the space between the skull and the cerebral cortex—more specifically, between the arachnoid and pia layers of the meninges). ... Meningitis is the inflammation of the protective membranes covering the central nervous system, known collectively as the meninges. ... Synovial fluid is a thin, stringy fluid found in the cavities of synovial joints. ... Septic arthritis is the proliferation of bacteria in joints and resultant inflammation. ...

As a general rule of thumb (which has exceptions), Gram-negative bacteria are more pathogenic due to their outer membrane structure, which is often hidden by a capsule or slime layer. This effectively hides the bacterial cell antigens and acts as "camouflage" since the human immune system recognises foreign body by its antigens; if the antigens are hidden, it is more difficult for the body to detect the invader. The presence of a capsule will often increase the virulence of a pathogen. Additionally, Gram-negative bacteria have lipopolysaccharide in their outer membrane, an endotoxin which increases the severity of inflammation. This inflammation may be so severe that septic shock may occur. Gram-positive infections are generally less severe because the human body does not contain peptidoglycan; in fact, humans produce an enzyme (lysozyme) that attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also frequently much more susceptible to beta-lactam antibiotics, such as penicillin. A pathogen or infectious agent is a biological agent that causes disease or illness to its host. ... A Slime layer in bacteria is an easily removed, diffuse, unorganised layer of extracellular material that surrounds a bacteria cell. ... This article does not cite any references or sources. ... A scanning electron microscope image of a single neutrophil (yellow), engulfing anthrax bacteria (orange). ... This article does not cite any references or sources. ... The word capsule (from the Latin capsula, a small box), has many similar meanings in English: In botany, a capsule is a type of dry fruit as in the poppy, iris, foxglove, etc. ... A pathogen or infectious agent is a biological agent that causes disease or illness to its host. ... Lipopolysaccharide (captions are in French) Lipopolysaccharide (LPS) is a large molecule consisting of a lipid and a polysaccharide (carbohydrate) joined by a covalent bond. ... Endotoxins are potentially toxic, natural compounds found inside pathogens such as bacteria. ... An abscess on the skin, showing the redness and swelling characteristic of inflammation. ... Septic shock is a serious medical condition causing such effects as multiple organ failure and death in response to infection and sepsis. ... Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids that forms a homogeneous layer outside the plasma membrane of eubacteria. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... Lysozyme single crystal. ... β-lactam antibiotics are a broad class of antibiotics which include penicillin derivatives, cephalosporins, monobactams, carbapenems and β-lactamase inhibitors; basically any antibiotic agent which contains a β-lactam nucleus in its molecular structure. ... Penicillin nucleus Penicillin (sometimes abbreviated PCN) refers to a group of β-lactam antibiotics used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms. ...

Exceptions to the rule include branching and filamentous Gram-positive bacteria such as Mycobacterium tuberculosis and other agents of tuberculosis, or Nocardia species, the agents of nocardiosis and some types of actinomycetoma. These organisms present unique problems in diagnosis and treatment, and special stains such as the Ziehl-Neelsen stain and the Kinyoun stain are used in their laboratory workup. Binomial name Mycobacterium tuberculosis Zopf 1883 Mycobacterium tuberculosis is the bacterium that causes most cases of tuberculosis[1]. It was first described on March 24, 1882 by Robert Koch, who subsequently received the Nobel Prize in physiology or medicine for this discovery in 1905. ... Tuberculosis (abbreviated as TB for tubercle bacillus) is a common and deadly infectious disease caused by mycobacteria, mainly Mycobacterium tuberculosis. ... Nocardia is a genus of Gram-positive, catalase-positive, rod-shaped bacteria; some species are pathogenic (nocardiosis). ... Actinomycetoma is a disease affecting the skin and connective tissue. ... The Ziehl-Neelsen stain, also known as the acid-fast stain, was first described by two german doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1894), a pathologist. ...

Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (90% of cell wall), which stain purple and Gram-negative bacteria have a thinner layer (10% of cell wall), which stain pink. Gram-negative bacteria also have an additional outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space. There are four basic steps of the Gram stain, which include applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture, followed by the addition of a mordant (Gram's iodine), rapid decolorization with alcohol or acetone, and counterstaining with safranin or basic fuchsin. Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids that forms a homogeneous layer outside the plasma membrane of eubacteria. ... A polyunsaturated triglyceride. ... Structure of Methyl Violet 2B Methyl violet is the name given to a group of similar chemicals used as pH indicators and dyes. ... Look up Mordant in Wiktionary, the free dictionary. ... General Name, Symbol, Number iodine, I, 53 Chemical series halogens Group, Period, Block 17, 5, p Appearance violet-dark gray, lustrous Standard atomic weight 126. ... Grain alcohol redirects here. ... The chemical compound acetone (also known as propanone, dimethyl ketone, 2-propanone, propan-2-one and β-ketopropane) is the simplest representative of the ketones. ... Safranin (also Safranin O or basic red 2) is a biological stain used in histology and cytology. ... Fuchsine Fuchsine, fuchsin, rosanilin, or rosaniline hydrochloride is a magenta dye with chemical formula C19H17N3·HCl. ...

Crystal violet (CV) dissociates in aqueous solutions into CV⁺ and chloride (Cl^–) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV⁺ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodine (I^– or I₃^–) interacts with CV⁺ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of 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 membrane and the 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.^[6][7]

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, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. 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^[8] In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain. Species A. naeslundii Actinomyces is a genus of Gram-positive bacteria. ... Species See text. ... Species See text. ... Binomial name Propionibacterium acnes Propionibacterium acnes is a bacterium that is linked to the skin condition acne. ... Species Bacillus anthracis Bacillus cereus Bacillus coagulans Bacillus globigii Bacillus licheniformis Bacillus natto Bacillus subtilis Bacillus sphaericus Bacillus thuringiensis etc. ... Species Clostridium acetobutylicum Clostridium aerotolerans Clostridium botulinum Clostridium colicanis Clostridium difficile Clostridium formicaceticum Clostridium novyi Clostridium perfringens Clostridium sordelli Clostridium tetani Clostridium piliforme Clostridium tyrobutyricum etc. ...

Gram staining protocol

Wikibooks Transwiki has more about this subject:

Gram staining

1. Make a slide of tissue or body fluid that is to be stained. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide.
2. Add the primary stain crystal violet and incubate 1 minute. This step colors all cells violet.
3. Add Gram's iodine, for 1 min. It is not a stain; it is a mordant. It doesn't give color directly to the bacteria but it fixes the crystal violet to the bacterial cell wall. All cells remain violet.
4. Wash with ethanol and acetone, the Decolorizer. If the bacteria is Gram-positive it will retain the primary stain. If it is Gram-negative it will lose the primary stain and appear colorless.
5. Add the secondary stain, safranin, and incubate 1 min, then wash with water for a maximum of 5 seconds. If the bacteria is Gram-positive then the cell will retain the primary stain, will not take the secondary stain, and will appear black-violet. If the bacteria is Gram-negative then the cell will lose the primary stain, take secondary stain, and will appear red-pink.

Gram Stain is 2 g of 90% crystal violet dissolved in 20 ml of 95% ethyl alcohol. Image File history File links Wikibooks-logo-en. ... Structure of Methyl Violet 2B Methyl violet is the name given to a group of similar chemicals used as pH indicators and dyes. ... Look up Mordant in Wiktionary, the free dictionary. ... Safranin (also Safranin O or basic red 2) is a biological stain used in histology and cytology. ... Ethyl alcohol, also known as ethanol or grain alcohol, is a flammable, colorless chemical compound, one of the alcohols that is most often found in alcoholic beverages. ...

Gram's iodine is 1 g of iodine, 2 g of potassium iodide, dissolved in 300 ml of distilled water. General Name, Symbol, Number iodine, I, 53 Chemical series halogens Group, Period, Block 17, 5, p Appearance violet-dark gray, lustrous Standard atomic weight 126. ... Potassium iodide is a white crystalline salt with chemical formula KI, used in photography and radiation treatment. ... Bottle for Distilled water in the Real Farmacia in Madrid. ...

Decolorizer is 50% ethyl alcohol, 50% acetone.[1]

In addition it is now common to use basic fuchsin instead of safranin. Fuchsine Fuchsine, fuchsin, rosanilin, or rosaniline hydrochloride is a magenta dye with chemical formula C19H17N3·HCl. ...

See also

• Bacterial cell structure
• Staining (biology)

Bacteria, despite their apparent simplicity contain a well developed cell structure which is responsible for many of their unique biological properties. ... Staining is a biochemical technique of adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. ...

References

Professor Hans Christian Gram Hans Christian Joachim Gram (September 13, 1853 - November 14, 1938) was a Danish bacteriologist. ... PDF is an abbreviation with several meanings: Portable Document Format Post-doctoral fellowship Probability density function There also is an electronic design automation company named PDF Solutions. ... --> Year 2007 (MMVII) is now the current year, a common year starting on Monday of the Gregorian calendar and the AD/CE era. ... February 17 is the 48th day of the year in the Gregorian calendar. ... PDF is an abbreviation with several meanings: Portable Document Format Post-doctoral fellowship Probability density function There also is an electronic design automation company named PDF Solutions. ... --> Year 2007 (MMVII) is now the current year, a common year starting on Monday of the Gregorian calendar and the AD/CE era. ... February 17 is the 48th day of the year in the Gregorian calendar. ... PDF is an abbreviation with several meanings: Portable Document Format Post-doctoral fellowship Probability density function There also is an electronic design automation company named PDF Solutions. ... --> Year 2007 (MMVII) is now the current year, a common year starting on Monday of the Gregorian calendar and the AD/CE era. ... February 17 is the 48th day of the year in the Gregorian calendar. ...

External links

• More examples of Gram staining
• Gram staining video