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Gram stain, is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.
In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall.
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Confusion can arise because some authors use the acronym RT-PCR to denote real-time PCR. In this article, RT-PCR will denote Reverse Transcription PCR. Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as EtBr, which is also an abbreviation for bromoethane. To avoid confusion, some laboratories have used the abbreviation EthBr for this salt. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle. The high incidence of antimicrobial resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Despite its reputation as a mutagen, tests have shown it to have low mutagenicity without metabolic activation.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology, in cytology, and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.
Flow cytometry (FC) is a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles.
The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the periplasmic space in Gram-negative bacteria. Using cryo-electron microscopy it has been found that a much smaller periplasmic space is also present in Gram-positive bacteria, between cell wall and the plasma membrane. The periplasm may constitute up to 40% of the total cell volume of gram-negative bacteria, but is a much smaller percentage in gram-positive bacteria.
DAPI, or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA. It is used extensively in fluorescence microscopy. As DAPI can pass through an intact cell membrane, it can be used to stain both live and fixed cells, though it passes through the membrane less efficiently in live cells and therefore provides a marker for membrane viability.
Propidium iodide is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a fluorescent excitation maximum of 493 nm (blue-green), and an emission maximum of 636 nm (red). After binding DNA, the quantum yield of PI is enhanced 20-30 fold, and the excitation/emission maximum of PI is shifted to 535 nm (green) / 617 nm (orange-red). Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis, or in microscopy to visualize the nucleus and other DNA-containing organelles. Propidium Iodide is not membrane-permeable, making it useful to differentiate necrotic, apoptotic and healthy cells based on membrane integrity. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining. PI is widely used in fluorescence staining and visualization of the plant cell wall.
Carbol fuchsin, carbol-fuchsin, carbolfuchsin, or Castellani's paint is a mixture of phenol and basic fuchsin that is used in bacterial staining procedures. It is commonly used in the staining of mycobacteria because it has an affinity for the mycolic acids found in their cell membranes.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.
Filamentation is the anomalous growth of certain bacteria, such as Escherichia coli, in which cells continue to elongate but do not divide. The cells that result from elongation without division have multiple chromosomal copies.
A bacterium, despite its simplicity, contains a well-developed cell structure which is responsible for some of its unique biological structures and pathogenicity. Many structural features are unique to bacteria and are not found among archaea or eukaryotes. Because of the simplicity of bacteria relative to larger organisms and the ease with which they can be manipulated experimentally, the cell structure of bacteria has been well studied, revealing many biochemical principles that have been subsequently applied to other organisms.
Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by intercalation, or RNA via electrostatic attractions. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and fluorescein have a maximum excitation at 502nm and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as lysosomes and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of phagocytosis of apoptotic cells. Acridine orange is used in epifluorescence microscopy and flow cytometry. The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows the dye to differentiate between various types of cells. The shift in maximum excitation and emission wavelengths provides a foundation to predict the wavelength at which the cells will stain.
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DNA-dye-complex best absorbs 497 nanometer blue light and emits green light. The stain preferentially binds to double-stranded DNA, but will stain single-stranded (ss) DNA with lower performance. SYBR Green can also stain RNA with a lower performance than ssDNA.
Bacteria are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. Bacteria inhabit soil, water, acidic hot springs, radioactive waste, and the deep biosphere of Earth's crust. Bacteria play a vital role in many stages of the nutrient cycle by recycling nutrients and the fixation of nitrogen from the atmosphere. The nutrient cycle includes the decomposition of dead bodies; bacteria are responsible for the putrefaction stage in this process. In the biological communities surrounding hydrothermal vents and cold seeps, extremophile bacteria provide the nutrients needed to sustain life by converting dissolved compounds, such as hydrogen sulphide and methane, to energy. Bacteria also live in mutualistic, commensal and parasitic relationships with plants and animals. Most bacteria have not been characterised and there are many species that cannot be grown in the laboratory. The study of bacteria is known as bacteriology, a branch of microbiology.
Propidium monoazide (PMA) is a photoreactive DNA-binding dye that preferentially binds to dsDNA. It is used to detect viable microorganisms by qPCR. Visible light induces a photoreaction of the chemical that will lead to a covalent bond with PMA and the dsDNA. The mechanism of DNA modification by PMA can be seen in this protocol. This process renders the DNA insoluble and results in its loss during subsequent genomic DNA extraction. Theoretically, dead microorganisms lose their capability to maintain their membranes intact, which leaves the "naked" DNA in the cytosol ready to react with PMA. DNA of living organisms are not exposed to the PMA, as they have an intact cell membrane. After treatment with the chemical, only the DNA from living bacteria is usable in qPCR, allowing to obtain only the amplified DNA of living organisms. This is helpful in determining which pathogens are active in specific samples. The main use of PMA is in Viability PCR but the same principle can be applied in flow cytometry or fluorescence microscopy.
SYBR Safe is a cyanine dye used as a nucleic acid stain in molecular biology. SYBR Safe is one of a number of SYBR dyes made by the Life Technologies Corporation. SYBR Safe binds to DNA. The resulting DNA-dye-complex absorbs blue light and emits green light.
Bacterioplankton counting is the estimation of the abundance of bacterioplankton in a specific body of water, which is useful information to marine microbiologists. Various counting methodologies have been developed over the years to determine the number present in the water being observed. Methods used for counting bacterioplankton include epifluorescence microscopy, flow cytometry, measures of productivity through frequency of dividing cells (FDC), thymidine incorporation, and leucine incorporation.
SYBR Gold is an asymmetrical cyanine dye. It can be used as a stain for double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold is the most sensitive fluorescent stain of the SYBR family of dyes for the detection of nucleic acids. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific
References
- ↑ Lee H, Woo ER, Lee DG (July 2015). "Glochidioboside Kills Pathogenic Bacteria by Membrane Perturbation". Current Microbiology. 71 (1): 1–7. doi:10.1007/s00284-015-0807-9. PMID 25820208. S2CID 14011127.
- ↑ Gaforio JJ, Serrano MJ, Ortega E, Algarra I, Alvarez de Cienfuegos G (June 2002). "Use of SYTOX green dye in the flow cytometric analysis of bacterial phagocytosis". Cytometry. 48 (2): 93–96. doi:10.1002/cyto.10107. PMID 12116370.
- ↑ Lebaron P, Catala P, Parthuisot N (July 1998). "Effectiveness of SYTOX Green stain for bacterial viability assessment". Applied and Environmental Microbiology. 64 (7): 2697–2700. Bibcode:1998ApEnM..64.2697L. doi:10.1128/AEM.64.7.2697-2700.1998. PMC 106447 . PMID 9647851.
- ↑ Bakshi S, Choi H, Rangarajan N, Barns KJ, Bratton BP, Weisshaar JC (August 2014). Parales RE (ed.). "Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack". Applied and Environmental Microbiology. 80 (16): 4977–4986. Bibcode:2014ApEnM..80.4977B. doi:10.1128/AEM.00989-14. PMC 4135745 . PMID 24907320.
- ↑ Domahidy, Farkas; Kovacs, Beatrix; Levente, cseri; Katona, Gergely; Rozsa, Balazs J.; Mucsi, Zoltan; Kovacs, Ervin (3 May 2024). "Comprehensive Study of Thiazole-Orange-Based DNA Dyes". ChemPhotoChem. x (x): e202400080. doi:10.1002/cptc.202400080.
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