microbiologists use the gram stain to aid in the identification of bacteria. what is the major difference between gram-positive and gram-negative bacteria?
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get microbiologists use the gram stain to aid in the identification of bacteria. what is the major difference between gram-positive and gram-negative bacteria? from EN Bilgi.
Use of the gram stain in microbiology
The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be "gram-positive," whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be "gram-negative." This stain …
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Abstract
The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be "gram-positive," whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be "gram-negative." This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.
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Gram stain
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.
Contents
History[edit]
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]
Uses[edit]
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.
Medical[edit]
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.[8][9]
Staining mechanism[edit]
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.[12]
Iodide (−
or −
3) 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.[13]
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.
Examples[edit]
Gram-positive bacteria[edit]
Gram-positive bacteria generally have a single membrane (monoderm) surrounded by a thick peptidoglycan. This rule is followed by two phyla: Firmicutes (except for the classes Mollicutes and Negativicutes) and the Actinobacteria.[6][18] In contrast, members of the Chloroflexi (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 Deinococcus–Thermus 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 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 bacteria[edit]
Gram-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 Proteobacteria (exceptions being some members of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).[6]page needed[18]
Gram-variable and Gram-indeterminate bacteria[edit]
Some 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, Mycobactrium 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]
Orthographic note[edit]
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.
See also[edit]
References[edit]
External links[edit]
Gram Staining
The Gram staining is one of the most crucial staining techniques in microbiology. It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882, mainly to identify organisms causing pneumonia.[1] Often the first test performed, gram staining involves the use of crystal violet or methylene blue as the primary color.[2] The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms.
Introduction
The Gram staining is one of the most crucial staining techniques in microbiology. It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882, mainly to identify organisms causing pneumonia.[1] Often the first test performed, gram staining involves the use of crystal violet or methylene blue as the primary color.[2] The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms.
The first step in gram staining is the use of crystal violet dye for the slide's initial staining. The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye. Subsequently, a decolorizer, often solvent of ethanol and acetone, is used to remove the dye. The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment.[3] Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content.[4]
Initially, all bacteria take up crystal violet dye; however, with the use of solvent, the lipid layer from gram-negative organisms is dissolved. With the dissolution of the lipid layer, gram negatives lose the primary stain. In contrast, solvent dehydrates the gram-positive cell walls with the closure of pores preventing diffusion of violet-iodine complex, and thus, bacteria remain stained.[5] The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria.[6]
The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative bacteria pink color for easier identification. It is also known as counterstain. Some laboratories use safranin as a counterstain; however, basic fuchsin stains gram-negative organisms more intensely than safranin. Similarly, Hemophilus spp., Legionella app, and some anaerobic bacteria stain poorly with safranin.
Specimen Collection
Various clinical specimens can be used to perform Gram staining. Some of the commonly used specimens are sputum, blood, cerebrospinal fluid, ascitic fluid, synovial fluid, pleural fluid, and urine, etc. Swabs from nostrils, throat, rectum, wound, and cervix, etc. can also be used. The collection of specimens should always be in sterile containers.
Procedures
Types of equipment needed for Gram staining include:
Reagents needed for Gram staining include:
Procedure
1. Preparation of a slide smear:
2. Gram staining:
3. Microscopic examination of slide:
Various modifications of gram staining are used, such as Atkin gram stain, and Burke gram stain, etc.
Indications
Gram staining is indicated whenever a bacterial infection is suspected for easy and early diagnosis.[8]
Potential Diagnosis
Gram staining aids in the diagnosis of a disease or a pathologic condition.
Examples of gram-positive organisms are:
Normal and Critical Findings
A normal finding in a sterile body fluid should be the absence of any pathologic organism in the smear. The organisms are identified based on color and shape. Gram-positive organisms are either purple or blue in color, while gram-negative organisms are either pink or red in color. Bacilli are rod-shaped, while cocci are spherical.
Findings on gram stain that suggest underlying bacterial infections:
(Please note: Moraxella spp., and Acinetobacter spp., are often diplococcal in morphology. Acinetobacter can sometimes appear as Gram-positive cocci, and they can be pleomorphic.
Gram variable organisms: these organisms do not group into either gram-positive or gram-negative organisms.
Interfering Factors
If the specimen collection is not sterile, multiple organisms can contaminate the specimen. Similarly, improper specimen collection and prior use of antibiotics can interfere with the growth of organisms. During the interpretation of the Gram stain, as described by the World Health Organization in 2003, the following steps should be followed:
1. General nature of the smear requires analysis under low power magnification (10X)
2. Low power magnification should be utilized to note the following:
3. Oil immersion examination of multiple fields is necessary to note the following:
Complications
The interpretation of slides can be difficult if the microscopic smear is thick and clumped. Decolorization time should have very close monitoring to avoid under-decolorization or over-decolorization. Thicker smears require longer decolorizing time. Similarly, cultures should undergo evaluation while they are still fresh. Old cultures tend to lose the peptidoglycan cell walls, which predisposes gram-positive cells to be gram-negative or gram variable. Gram stain is not useful for organisms without a cell wall like Mycoplasma species, and for smaller bacteria like Chlamydia and Rickettsia species.
Gram stain may not falsely reveal organisms in the following scenario:
Sometimes results of Gram-stain may not match the final results of cultures and could potentially lead to inappropriate use of antibiotics.[10]
Clinical Significance
Gram stain is often the initial diagnostic test for the evaluation of infections. The use of Gram stain facilitates the rapid use of appropriate antibiotics. However, genetic sequences and molecular techniques are more specific than classic gram stain.
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