Schaeffer–Fulton Stain
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Schaeffer–Fulton Stain
The Schaeffer–Fulton stain is a technique designed to isolate endospores by staining any present endospores green, and any other bacterial bodies red. The primary stain is malachite green, and the counterstain is safranin, which dyes any other bacterial bodies red. Endospores cannot be stained by normal staining procedures because their walls are practically impermeable to all chemicals. The Schaeffer- Fulton endospore stain uses heat to drive the primary stain(malachite green) into the endospore. After cooling, the slide is decolorized with water and counterstained with safranin. Procedure Using an aseptic technique, bacteria are placed on a slide and heat fixed. The slide is then suspended over a water bath with some sort of porus paper over it, so that the slide is steamed. Malachite green is applied to the slide, which can penetrate the tough walls of the endospores, staining them green. After five minutes, the slide is removed from the steam, and the paper towel is removed. ...
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Bacillus Subtilis Spore
''Bacillus'' (Latin "stick") is a genus of Gram-positive, rod-shaped bacteria, a member of the phylum ''Bacillota'', with 266 named species. The term is also used to describe the shape (rod) of other so-shaped bacteria; and the plural ''Bacilli'' is the name of the class of bacteria to which this genus belongs. ''Bacillus'' species can be either obligate aerobes which are dependent on oxygen, or facultative anaerobes which can survive in the absence of oxygen. Cultured ''Bacillus'' species test positive for the enzyme catalase if oxygen has been used or is present. ''Bacillus'' can reduce themselves to oval endospores and can remain in this dormant state for years. The endospore of one species from Morocco is reported to have survived being heated to 420 °C. Endospore formation is usually triggered by a lack of nutrients: the bacterium divides within its cell wall, and one side then engulfs the other. They are not true spores (i.e., not an offspring). Endospore formation ...
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Endospore
An endospore is a dormant, tough, and non-reproductive structure produced by some bacteria in the phylum Bacillota. The name "endospore" is suggestive of a spore or seed-like form (''endo'' means 'within'), but it is not a true spore (i.e., not an offspring). It is a stripped-down, dormant form to which the bacterium can reduce itself. Endospore formation is usually triggered by a lack of nutrients, and usually occurs in gram-positive bacteria. In endospore formation, the bacterium divides within its cell wall, and one side then engulfs the other. Endospores enable bacteria to lie dormant for extended periods, even centuries. There are many reports of spores remaining viable over 10,000 years, and revival of spores millions of years old has been claimed. There is one report of viable spores of ''Bacillus marismortui'' in salt crystals approximately 250 million years old. When the environment becomes more favorable, the endospore can reactivate itself into a vegetative state. Mos ...
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Malachite Green
Malachite green is an organic compound that is used as a dyestuff and controversially as an antimicrobial in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper. Despite its name the dye is not prepared from the mineral malachite; the name just comes from the similarity of color. Structures and properties Malachite green is classified in the dyestuff industry as a triarylmethane dye and also using in pigment industry. Formally, malachite green refers to the chloride salt 6H5C(C6H4N(CH3)2)2l, although the term malachite green is used loosely and often just refers to the colored cation. The oxalate salt is also marketed. The anions have no effect on the color. The intense green color of the cation results from a strong absorption band at 621 nm ( extinction coefficient of ). Malachite green is prepared by the condensation of benzaldehyde and dimethylaniline to give leuco malachite green (LMG): :C6H5CHO + C6H5N(CH3)2 ...
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Safranin
Safranin (Safranin O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endospore staining. It can also be used for the detection of cartilage, mucin and mast cell granules. Safranin typically has the chemical structure shown at right (sometimes described as dimethyl safranin). There is also trimethyl safranin, which has an added methyl group in the ''ortho-'' position (see Arene substitution pattern) of the lower ring. Both compounds behave essentially identically in biological staining applications, and most manufacturers of safranin do not distinguish between the two. Commercial safranin preparations often contain a blend of both types. Safranin is also used as redox indicator in analytical chemistry. Safranines Safranines are the azonium compounds of symmetrical 2,8-dimethyl-3,7-diaminophenazine. They are o ...
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Heat Fixation
In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability. Tissue fixation is a critical step in the preparation of histological sections, its broad objective being to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections. This allows the investigation of the tissues' structure, which is determined by the shapes and sizes of such macromolecules (in and around cells) as proteins and nucleic acids. Purposes In performing their protective role, fixatives denature proteins by coagulation, by forming additive compounds, or by a combination of coagulation and additive processes. A compound that adds chemically to macromolecules stabilizes structure most effectively if it is able to combine with par ...
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Steaming
Steaming is a method of cooking using steam. This is often done with a food steamer, a kitchen appliance made specifically to cook food with steam, but food can also be steamed in a wok. In the American southwest, steam pits used for cooking have been found dating back about 5,000 years. Steaming is considered a healthy cooking technique that can be used for many kinds of foods. Because steaming can be achieved by heating less water or liquid, and because of the excellent thermodynamic heat transfer properties of steam, steaming can be as fast, or faster, than cooking in boiling water, as well as being more energy efficient. History Some of the world's earliest examples of steam cooking were found in China's Yellow River Valley, early steam cookers made of stoneware have been found dating back as far as 5,000 BCE. And also in Gunma Prefecture, Japan, created during the Stone Age. Some of the second earliest examples of steam cooking have been found in Italy and Sardinia, cre ...
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Bibulous Paper
Blotting paper, called bibulous paper, is a highly absorbent type of paper or other material. It is used to absorb an excess of liquid substances (such as ink or oil) from the surface of writing paper or objects. Blotting paper referred to as bibulous paper is mainly used in microscopy to remove excess liquids from the slide before viewing. Blotting paper has also been sold as a cosmetic to aid in the removal of skin oils and makeup. Manufacture Blotting paper is made from different materials of varying thickness, softness, etc. depending on the application. It is often made of cotton and manufactured on special paper machines. Blotting paper is reputed to be first referred to in the English language in the 15th century but there is a tradition in Norfolk, England that it was invented by accident at Lyng Mill on the River Wensum. It is reported that a Berkshire (England) paper mill worker failed to add sizing to a batch of paper that was being produced. The batch was discarded ...
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Optical Microscope
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast. The object is placed on a stage and may be directly viewed through one or two eyepieces on the microscope. In high-power microscopes, both eyepieces typically show the same image, but with a stereo microscope, slightly different images are used to create a 3-D effect. A camera is typically used to capture the image (micrograph). The sample can be lit in a variety of ways. Transparent objects can be lit from below and solid objects can be lit with light coming through ( bright field) or around (dark field) the objective lens. Polarised ...
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Middlebury College
Middlebury College is a private liberal arts college in Middlebury, Vermont. Founded in 1800 by Congregationalists, Middlebury was the first operating college or university in Vermont. The college currently enrolls 2,858 undergraduates from all 50 states and 74 countries and offers 44 majors in the arts, humanities, literature, foreign languages, social sciences, and natural sciences, as well as joint engineering programs with Columbia University, Dartmouth College, and Rensselaer Polytechnic Institute. In addition to its undergraduate liberal arts program, the school also has graduate schools, the Middlebury College Language Schools, the Bread Loaf School of English, and the Middlebury Institute of International Studies at Monterey, as well as its C.V. Starr-Middlebury Schools Abroad international programs. It is the among the ''Little Ivies'', an unofficial group of academically selective liberal arts colleges, mostly in the northeastern United States. Middlebury is known f ...
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Moeller Stain
Moeller staining involves the use of a steamed dye reagent in order to increase the stainability of endospores. Carbol fuchsin is the primary stain used in this method. Endospores are stained red, while the counterstain methylene blue stains the vegetative bacteria blue. Endospores are surrounded by a highly resistant spore coat, which is highly resistant to excessive heat, freezing, desiccation, as well as chemical agents. More importantly, for identification, spores are resistant to commonly employed staining techniques; therefore alternative staining methods are required. Method Carbol fuchsin is applied to a heat-fixed slide. The slide is then heated over a bunsen burner, or suspended over a hot water bath, covered with a paper towel, and steamed for 3 minutes. The slide is rinsed with acidified ethanol, and counter-stained with Methylene blue. An improved method involves the addition of the surfactant Tergitol 7 to the carbol fuchsin stain, and the omission of the steamin ...
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Staining
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissues), in cytology (microscopic study of cells), 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 (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells), or organelles within individual cells. In biochemistry, it involves adding a class-specific ( DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent tagging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis. Light microscopes are used for viewin ...
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Microbiology Techniques
Microbiology () is the scientific study of microorganisms, those being unicellular (single cell), multicellular (cell colony), or acellular (lacking cells). Microbiology encompasses numerous sub-disciplines including virology, bacteriology, protistology, mycology, immunology, and parasitology. Eukaryotic microorganisms possess membrane-bound organelles and include fungi and protists, whereas prokaryotic organisms—all of which are microorganisms—are conventionally classified as lacking membrane-bound organelles and include Bacteria and Archaea. Microbiologists traditionally relied on culture, staining, and microscopy. However, less than 1% of the microorganisms present in common environments can be cultured in isolation using current means. Microbiologists often rely on molecular biology tools such as DNA sequence based identification, for example the 16S rRNA gene sequence used for bacteria identification. Viruses have been variably classified as organisms, as they have bee ...
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