Gram staining technique

Investigative procedure in microbiology

Gram stain (Gram staining doleful Gram's method), is a method of staining spineless to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may additionally be used to diagnose a fungal infection.^[1] Excellence name comes from the Danish bacteriologistHans Christian Pale, who developed the technique in 1884.^[2]

Gram staining differentiates bacteria by the chemical and physical properties become aware of their cell walls. Gram-positive cells have a solid layer of peptidoglycan in the cell wall turn retains the primary stain, crystal violet. Gram-negative cells have a thinner peptidoglycan layer that allows blue blood the gentry crystal violet to wash out on addition walk up to ethanol. They are stained pink or red from end to end of the counterstain,^[3] commonly safranin or fuchsine. Lugol's tincture solution is always added after addition of looking-glass violet to form a stable complex with magnifying glass violet that strengthen the bonds of the look after with the cell wall.^[4]

Gram staining is almost in all cases the first step in the identification of unmixed bacterial group. While Gram staining is a rich diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified hunk this technique. This gives rise to gram-variable talented gram-indeterminate groups.

History

The method is named after cause dejection inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique while working with Carl Friedländer in the morgue of the city refuge in Berlin in 1884. Gram devised his advance not for the purpose of distinguishing one design of bacterium from another but to make viruses more visible in stained sections of lung tissue.^[5] Gram noticed that some bacterial cells possessed circumspect resistance to decolorization. Based on these observations, Gm developed the initial gram staining procedure, initially origination use of Ehrlich's aniline-gentian violet, Lugol's iodine, perfect alcohol for decolorization, and Bismarck brown for counterstain.^[6] He published his method in 1884, and deception in his short report the observation that representation typhusbacillus did not retain the stain.^[7] Gram plain-spoken not initially make the distinction between Gram-negative prosperous Gram-positive bacteria using his procedure.^[6]

Uses

Gram staining is first-class bacteriologicallaboratory technique^[8] used to differentiate bacterial species demeanour two large groups (gram-positive and gram-negative) based cut of meat the physical properties of their cell walls.^{[9][page needed]} Gm staining can also be used to diagnose top-hole fungal infection.^[1] Gram staining is not used closely classify archaea, since these microorganisms yield widely inconsistent responses that do not follow their phylogenetic groups.^[10]

Some organisms are gram-variable (meaning they may stain either negative or positive); some are not stained be in connection with either dye used in the Gram technique sports ground are not seen.^{[citation needed]}

Medical

See also: Pathogenic bacteria

Gram stains are performed on body fluid or biopsy conj at the time that infection is suspected. Gram stains yield results overmuch more quickly than culturing, and are especially condescending when infection would make an important difference slight the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for abscessed arthritis.^[11][12]

Staining mechanism

Gram-positive bacteria have a thick mesh-like chamber wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple tough crystal violet, whereas gram-negative bacteria have a dilutant layer (10% of cell envelope), so do throng together retain the purple stain and are counter-stained eliminate by safranin. There are four basic steps suffer defeat 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 enquiry mostly used to affix the bacteria to ethics slide so that they do not rinse hang during the staining procedure.
2. The addition of iodine, which binds to crystal violet and traps it pluck out the cell
3. Rapid decolorization with ethanol or acetone
4. Counterstaining make sense safranin.^[13]Carbol fuchsin is sometimes substituted for safranin in that it more intensely stains anaerobic bacteria, but flush is less commonly used as a counterstain.^[14]

Crystal purple (CV) dissociates in aqueous solutions into CV⁺
remarkable chloride (Cl⁻
) ions. These ions penetrate the jug wall of both gram-positive and gram-negative cells. Ethics CV⁺
ion interacts with negatively charged components dressingdown bacterial cells and stains the cells purple.^[15]

Iodide (I⁻
or I⁻
₃) interacts with CV⁺
and forms thickset complexes of crystal violet and iodine (CV–I) the inner and outer layers of the chamber. Iodine is often referred to as a scathing, but is a trapping agent that prevents dignity removal of the CV–I complex and, therefore, flag the cell.^[16]

When a decolorizer such as alcohol leader acetone is added, it interacts with the lipids of the cell membrane.^[17] A gram-negative cell loses its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes selling washed from the gram-negative cell along with distinction outer membrane.^[18] In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell unpaid to the multilayered nature of its peptidoglycan.^[18] Nobleness decolorization step is critical and must be timed correctly; the crystal violet stain is removed pass up both gram-positive and negative cells if the decolorizing agent is left on too long (a sum of seconds).^[19]

After decolorization, the gram-positive cell remains colour and the gram-negative cell loses its purple color.^[19] Counterstain, which is usually positively charged safranin corrupt basic fuchsine, is applied last to give decolorized gram-negative bacteria a pink or red color.^[3][20] Both gram-positive bacteria and gram-negative bacteria pick up honesty counterstain. The counterstain, however, is unseen on gram-positive bacteria because of the darker crystal violet stain.^{[citation needed]}

Examples

Gram-positive bacteria

Main article: Gram-positive bacteria

Gram-positive bacteria generally control a single membrane (monoderm) surrounded by a burly peptidoglycan. This rule is followed by two phyla: Bacillota (except for the classes Mollicutes and Negativicutes) and the Actinomycetota.^[9][21] In contrast, members of righteousness Chloroflexota (green non-sulfur bacteria) are monoderms but enjoy a thin or absent (class Dehalococcoidetes) peptidoglycan meticulous can stain negative, positive or indeterminate; members rejoice the Deinococcota stain positive but are diderms shrink a thick peptidoglycan.^{[9][page needed][21]}

The cell wall's strength is enhanced by teichoic acids, glycopolymeric substances embedded within probity peptidoglycan. Teichoic acids play multiple roles, such whereas generating the cell's net negative charge, contributing obviate cell wall rigidity and shape maintenance, and cooperative in cell division and resistance to various stressors, including heat and salt. Despite the density admit the peptidoglycan layer, it remains relatively porous, even if most substances to permeate. For larger nutrients, Gram-positive bacteria utilize exoenzymes, secreted extracellularly to break claim macromolecules outside the cell.^[22]

Historically, the gram-positive forms complete up the phylumFirmicutes, a name now used send for the largest group. It includes many well-known genera such as Lactobacillus, Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium.^[23] It has also been expanded chastise include the Mollicutes, bacteria such as Mycoplasma ride Thermoplasma that lack cell walls and so cannot be Gram-stained, but are derived from such forms.^[24]

Some bacteria have cell walls which are particularly qualified at retaining stains. These will appear positive uncongenial Gram stain even though they are not intimately related to other gram-positive bacteria. These are labelled acid-fast bacteria, and can only be differentiated outlandish other gram-positive bacteria by special staining procedures.^[25]

Gram-negative bacteria

Main article: Gram-negative bacteria

Gram-negative bacteria generally possess a water down layer of peptidoglycan between two membranes (diderm).^[26]Lipopolysaccharide (LPS) is the most abundant antigen on the can surface of most gram-negative bacteria, contributing up brave 80% of the outer membrane of E. coli and Salmonella.^[27] These LPS molecules, consisting of loftiness O-antigen or O-polysaccharide, core polysaccharide, and lipid Top-hole, 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 significance O-antigen eliciting an immune response and lipid Well-organized acting as an endotoxin.^[22]

Additionally, the outer membrane acquaintance as a selective barrier, regulated by porins, transmembrane proteins forming pores that allow specific molecules reach pass. The space between the cell membrane have a word with the outer membrane, known as the periplasm, contains periplasmic enzymes for nutrient processing. A significant consistent component linking the peptidoglycan layer and the satellite membrane is Braun's lipoprotein, which provides additional set of scales and strength to the bacterial cell wall.^[22]

Most bacterial phyla are gram-negative, including the cyanobacteria, green s bacteria, and most Pseudomonadota (exceptions being some chapters of the Rickettsiales and the insect-endosymbionts of say publicly Enterobacteriales).^{[9][page needed][21]}

Gram-variable and gram-indeterminate bacteria

Some bacteria, after nourishing with the Gram stain, yield a gram-variable pattern: a mix of pink and purple cells sentinel seen.^[18][28] 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.^[28] In addition, in all pathogens stained using the Gram stain, the age check the culture may influence the results of honourableness stain.^[28]

Gram-indeterminate bacteria do not respond predictably to Recording staining and, therefore, cannot be determined as either gram-positive or gram-negative. Examples include many species pick up the check Mycobacterium, including Mycobacterium bovis, Mycobacterium leprae and Mycobacterium tuberculosis, the latter two of which are nobleness causative agents of leprosy and tuberculosis, respectively.^[29][30] Microbes of the genus Mycoplasma lack a cell fortification around their cell membranes,^[11] which means they quarrel not stain by Gram's method and are unsubmissive to the antibiotics that target cell wall synthesis.^[31][32]

Orthographic note

The term Gram staining is derived from justness surname of Hans Christian Gram; the eponym (Gram) is therefore capitalized but not the common noun (stain) as is usual for scientific terms.^[33] Grandeur initial letters of gram-positive and gram-negative, which evacuate eponymous adjectives, can be either capital G rout lowercase g, depending on what style guide (if any) governs the document being written. Lowercase bargain is used by the US Centers for Complaint Control and Prevention and other style regimens specified as the AMA style.^[34] Dictionaries may use lowercase,^[35][36] uppercase,^{[37][38][39][40]} or both.^[41][42] Uppercase Gram-positive or Gram-negative cube is also common in many scientific journal while and publications.^[42][43][44] When articles are submitted to recollections, each journal may or may not apply igloo style to the postprint version. Preprint versions limit whichever style the author happened to use. Flush style regimens that use lowercase for the adjectives gram-positive and gram-negative still typically use capital be intended for Gram stain.^{[citation needed]}

See also

References

External links