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 based on the chemical and physical properties of their cell walls. Cerebrospinal fluid (CSF) 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). ... White blood cells (also called leukocytes or immune cells) are a component of blood. ... Empiricism (greek εμπειρισμός, from empirical, latin experientia - the experience), is the philosophical doctrine that all human knowledge comes at first from senses and experience. ... 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. ...

The method is named after the inventor, the Danish scientist Hans Christian Gram (1853-1938), who developed the technique in 1884 to discriminate between pneumococci and Klebsiella pneumoniae bacteria. Hans Christian Joachim Gram (September 13, 1853 - November 14, 1928) was a Danish bacteriologist. ... Binomial name Streptococcus pneumoniae Streptococcus pneumoniae is a species of Streptococcus that is a major human pathogen. ... Klebsiella pneumoniae is a gram-negative rod-shaped bacteria, and clinically the most important member of the Klebsiella genus of Enterobacteriaceae. ...

Uses

Gram stains are performed on body fluid or biopsy when infection is suspected. It yields results much quicker 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. It necessitates the 24 hour staffing of microbiological laboratories in hospitals. Bodily fluids are fluids, which are generally excreted or secreted from the human body. ... 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) 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). ... Inferior view of a brain with meningitis caused by Haemophilus influenzae. ... Synovial fluid is a thick, stringy fluid found in the cavities of synovial joints. ... Septic arthritis is the proliferation of bacteria in joints and resultant inflammation. ...

Mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan which is capable of retaining the violet dye/iodine complex. Gram-negative bacteria have a thin cell wall made of a layer of peptidoglycan. In addition to an inner membrane, they also have an outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space. Peptidoglycan, also known as murein, is a substance that forms a homogeneous layer lying outside the plasma membrane in prokaryotes. ... Figure 1: Structure of a Lipid. ...

The decolorizing mixture causes dehydration of the multilayered peptidoglycan in the Gram-positive cell wall, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. But in Gram-negative bacteria, the decolorizing mixture acts as a lipid solvent and dissolves the outer membrane of the Gram-negative cell wall. The thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the Gram-negative cell is decolorized. The decolorization step is the crucial one, and requires some degree of skill, as Gram-positivity is not an all-or-none phenomenon.

As a rule of thumb (which has exceptions), Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer which hides the antigens of the cell and so acts as "camouflage" - the human body recognises a foreign body by its antigens; if they are hidden, it becomes harder for the body to detect the invader. Often the presence of a capsule will increase the virulence of a pathogen. Additionally, Gram-negative bacteria have lipopolysaccharide in their outer membrane. Lipopolysaccharide is 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, and in fact the human body produces an enzyme called lysozyme which attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also much more susceptible to beta-lactam antibiotics, such as penicillin. A Slime layer in bacteria is an easily removed, diffuse, unorganised layer of extracellular material that surrounds a bacteria cell. ... 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 literally birth of pain from the Greek παθογένεια) is a biological agent that can cause disease to its host. ... A lipopolysaccharide (LPS) is a large molecule that contains both lipid and a carbohydrate. ... Endotoxin is part of the outer membrane of the cell wall of Gram-negative bacteria. ... Inflammation is the first response of the immune system to infection or irritation and may be referred to as the innate cascade. ... Septic shock is a serious medical condition causing such effects as multiple organ failure and death in response to infection and sepsis. ... Ribbon diagram of the catalytically perfect enzyme TIM. Factor D enzyme crystal prevents the immune system from inappropriately running out of control. ... Categories: Stub | EC 3. ... β-lactam antibiotics are a broad class of antibiotics including penicillin derivatives, cephalosporins, monobactams, carbapenems and β-lactamase inhibitors; basically any antibiotic agent which contains a β-lactam nucleus in its molecular structure. ... Penicillin is a β-lactam antibiotic used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms. ...

Procedure

A description of Gram staining

1. First, an inoculum is taken from a culture using an inoculation loop and put on a slide. If the culture is solid, it is diluted by adding a drop of water on the slide and mixing with the loop. It is important here to take a very small inoculum so that the end result is a sparse single layer of bacteria. It is a common mistake for beginners to put way too much inoculum at this step.
2. The specimen is heat-fixed by passing the slide through a bunsen flame a few times, without allowing the slide to become hot to the touch.
3. A basic dye, crystal violet or gentian violet, is used to stain the slide. This dye is taken up by both Gram-positive and Gram-negative bacteria. Allow to stain for 1 minute. The slide should look purple to the unaided eye, and if examined microscopically at this point both Gram-positive and Gram-negative bacteria are purple. Lugol can also be used instead of crystal violet.
4. Rinse off with water for a maximum of 5 seconds.
5. Add iodine solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye.
6. Rinse with water.
7. Apply 95% ethanol or a mixture of acetone and alcohol several times until no more colour appears to come from the sample. This washes away all the unbound basic dye, (usually crystal violet) and leaves Gram-positive organisms stained purple and Gram-negative organisms unstained (colourless).
8. Rinse with water.
9. Apply a suitable counterstain. Opinions vary as to the best choice but suitable stains include safranin or fuchsin. This stain is taken up by both Gram-positive and Gram-negative organisms, but does not alter the colour of Gram-positive organism much, as they are already purple. It does, however, make the Gram-negative organisms pinkish-red.
10. Blot gently and allow to dry. Do not smear.

An inoculation loop (sometimes called smear loop) is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. ... Microscope slides and cover slips. ... A bunsen burner with needle valve. ... For the battery, see alkaline battery The word alkali can mean:- In chemistry, an alkali is a specific type of base, formed as a carbonate, hydroxide or other ionic salt of an alkali metal or alkali earth metal element. ... Structure of Methyl Violet 2B Methyl violet is the name given to a group of similar chemicals used as pH indicators and dyes. ... Gentian violet is an antifungal agent. ... General Name, Symbol, Number iodine, I, 53 Chemical series halogens Group, Period, Block 17, 5, p Appearance violet-dark gray, lustrous Atomic mass 126. ... // Diluting Concentrated Acids Dilution of concentrated acid should always be done in a fume cupboard. ... A mordant is a substance used to set dyes. ... Ethanol, also known as ethyl alcohol or grain alcohol, is a flammable, colorless chemical compound, one of the alcohols that is most often found in alcoholic beverages. ... For an alternate use of acetone, see Acetone (music). ... In general usage, alcohol (from Arabic al-khwl الكحول, or al-ghawl الغول) refers almost always to ethanol, also known as grain alcohol, and often to any beverage that contains ethanol (see alcoholic beverage). ... For the battery, see alkaline battery The word alkali can mean:- In chemistry, an alkali is a specific type of base, formed as a carbonate, hydroxide or other ionic salt of an alkali metal or alkali earth metal element. ... 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. ... Safranin (also Safranin O or basic red 2) is a biological stain used in histology and cytology. ... Fuchsine or rosaniline hydrochloride is a magenta dye with chemical formula C19H17N3·HCl and CAS number 569-61-9. ...

Interpretation

Inspect the slide under a microscope 1852 microscope Compound microscope made by John Cuff in 1750 A microscope (Greek: micron = small and scopos = aim) is an instrument for viewing objects that are too small to be seen by the naked or unaided eye. ...

• Gram-positive organisms will appear blue-black or purple.
• Gram-negative organisms will appear red or pink.

Organisms that cannot reliably be differentiated by this staining technique are said to be Gram-variable 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. ...

See also

• Staining (biology)

For staining in the sense of wood staining, see Wood staining. ...

References

• Gram HC. Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpreparäten. Fortschr Med 1884;2:185-89.
• Bergey, John G. Holt, Noel R. Krieg, Peter H.A. Sneath (1994). Bergey's Manual of Determinative Bacteriology, 9th ed.. Lippincott Williams & Wilkins. ISBN 0-683-00603-7.
• Michael T. Madigan, John Martinko, Jack Parker (2004). Brock Biology of Microorganisms, 10th Edition. Lippincott Williams & Wilkins. ISBN 0-130-66271-2.