In formal descriptions of the germ-fighting powers of antibacterial and biocidal products, the terms "Gram positive" and "Gram negative" are used as a way to categorize bacteria. While there are estimated to be over 10,000 species of bacteria, they can be categorized into a few helpful categories.
One of those categories has to do with the structure of the cell membrane. All the known bacteria fit into one of two categories of cell membrane structure: Gram-positive or Gram-negative. But what does that mean? Let's first look at where "Gram" comes from. In this case, Gram - with a capital G - refers to the Danish bacteriologist Hans Christian Gram. In 1884, Gram devised a test to identify whether or not a bacteria had a peptidoglycan (a mesh-like layer of sugars and amino acids) wall. In his test, a dye was introduced to the bacteria. If the bacteria had a thick peptidoglycan cell wall, it absorbed the dye and turned purple - it tested positive for peptidoglycan. If it did not turn purple, it tested negative for peptidoglycan, meaning, its peptidoglycan layer was thin. As this method was adopted, the resulting categories were called "Gram positive" and "Gram negative." This method of "Gram staining" is still a widely-used, standard procedure in microbiology. Now we can look at some of the most important differences between Gram-positive and Gram-negative bacteria in the fight against HAIs. The reason EPA public health claims, and as a result, products, clarify that testing includes both Gram-positive and Gram-negative bacteria is that they have different levels of resistance to cleansing products, different reactions to dry surfaces, and other important distinctions. Gram-positive bacteria, those species with peptidoglycan outer layers, are easier to kill - their thick peptidoglycan layer absorbs antibiotics and cleaning products easily. In contrast, their many-membraned cousins resist this intrusion with their multi-layered structure. Therefore, infection prevention techniques must ensure that they can breach the thick peptidoglycan layer of the Gram-positive bacteria but also get through the many layers of the Gram-negative bacteria.However thin their peptidoglycan layer, Gram-negative bacteria are protected from certain physical assaults because they do not absorb foreign materials that surround it (including Gram's purple dye). Imagine a spacecraft with a series of airlocks. Any intruder would have to make their way through these airlocks before entering the ship. Such is the case with gram-negative bacteria. Their additional membrane allows them to control what reaches the inner airlock, enabling them to sequester or even remove threats in that space between the membranes (periplasmic space) before it reaches the cell itself. As a result, Gram-negative bacteria are not destroyed by certain detergents which easily kill Gram-positive bacteria. While thick, the Gram-positive bacteria's membrane absorbs foreign materials (Gram's dye), even those that prove toxic to its insides. This makes them easier to destroy with certain detergents. As a result, only certain cleansers are approved for use to eliminate bacteria - because it must kill both Gram-positive and Gram-negative bacteria. Gram-negative bacteria cannot survive as long as Gram-positive bacteria on dry surfaces (while both survive a surprisingly long time). This makes certain species more dangerous between routine cleaning, since they can survive and even multiply on dry surfaces. However, the long survival time of many pathogens means hospitals must use novel technologies to eradicate bacteria between routine cleanings. Finally, Gram-negative bacteria are more intrinsically resistant to antibiotics - they don't absorb the toxin into their insides. Their ability to resist traditional antibiotics make them more dangerous in hospital settings, where patients are weaker and bacteria are stronger. New and very expensive antibiotics have been developed to combat these resistant species, but there remain some superbugs (MDROs) that nothing can kill. Not only do the Gram-negative bacteria's natural defenses keep out these antibiotics, some even have an acquired resistance to antibiotics that make it to their inner cell bodies. Gram-positive and Gram-negative bacteria exist everywhere, but pose unique threats to hospitalized patients with weak immune systems. Gram-positive bacteria cause tremendous problems and are the focus of many eradication efforts, but meanwhile, Gram-negative bacteria have been developing dangerous resistance and are therefore classified by the CDC as a more serious threat. For this reason, the need for new technologies that kill bacteria, both Gram-positive and Gram-negative, are essential to make hospitals safer for everyone.
Editor's Note: This post was originally published in August 2015 and has been updated for freshness, accuracy and comprehensiveness. Gram stain or Gram staining, also called Gram's method, is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.[1] 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,[2] 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 preliminary identification of a bacterial organism. 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. 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.[3] He published his method in 1884, and included in his short report the observation that the typhus bacillus did not retain the stain.[4] Gram stain of Candida albicans from a vaginal swab. The small oval chlamydospores are 2–4 µm in diameter. Gram staining is a bacteriological laboratory technique[5] used to differentiate bacterial species into two large groups (gram-positive and gram-negative) based on the physical properties of their cell walls.[6][page needed] Gram staining is not used to classify archaea, formerly archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.[7] 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. 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 informative than differential staining.[citation needed] MedicalGram 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.[8][9] Purple-stained gram-positive (left) and pink-stained gram-negative (right) 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:
Crystal violet (CV) dissociates in aqueous solutions into CV+ Iodide (I− When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane.[14] 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.[15] 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.[15] 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).[16] After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color.[16] Counterstain, which is usually positively charged safranin or basic fuchsine, is applied last to give decolorized gram-negative bacteria a pink or red color.[2][17] 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 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.[6][18] 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.[6][page needed][18] 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.[19] 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.[20] 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.[21] Gram-negative bacteriaGram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderm).[22] 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.[23] 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).[6][page needed][18] Gram-variable and Gram-indeterminate bacteriaSome bacteria, after staining with the Gram stain, yield a gram-variable pattern: a mix of pink and purple cells are seen.[15][24] 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.[24] In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.[24] 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.[25][26] Bacteria of the genus Mycoplasma lack a cell wall around their cell membranes, [8] which means they do not stain by Gram's method and are resistant to the antibiotics that target cell wall synthesis.[27][28] 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.[29] 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.[30] Dictionaries may use lowercase,[31][32] uppercase,[33][34][35][36] or both.[37][38] Uppercase Gram-positive or Gram-negative usage is also common in many scientific journal articles and publications.[38][39][40] 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.
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