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Gram stain or Gram staining, also called Gram's method, is a method of staining used to distinguish and classify bacterial species into two large groups. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique.
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 cell wall.
Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane.
An agar plate is a Petri dish that contains agar as a solid growth medium plus nutrients, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses disease at a microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.
Haemophilus ducreyi is a fastidious gram-negative coccobacillus bacteria, which causes the sexually transmitted disease chancroid, a major cause of genital ulceration in developing countries characterized by painful sores on the genitalia. Chancroid starts as an erythematous papular lesion that breaks down into a painful bleeding ulcer with a necrotic base and ragged edge.
The HACEK organisms are a group of fastidious Gram-negative bacteria that are an unusual cause of infective endocarditis, which is an inflammation of the heart due to bacterial infection. HACEK is an abbreviation of the initials of the genera of this group of bacteria: Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella. The HACEK organisms are a normal part of the human microbiota, living in the oral-pharyngeal region.
Septic arthritis, also known as joint infection or infectious arthritis, is the invasion of a joint by an infectious agent resulting in joint inflammation. Symptoms typically include redness, heat and pain in a single joint associated with a decreased ability to move the joint. Onset is usually rapid. Other symptoms may include fever, weakness and headache. Occasionally, more than one joint may be involved.
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.
The cell envelope comprises the inner cell membrane and the cell wall of a bacterium. In gram-negative bacteria an outer membrane is also included. This envelope is not present in the Mollicutes where the cell wall is absent.
Ziehl-Neelsen staining is a type of Acid-fast stain, first introduced by Paul Ehrlich. Ziehl–Neelsen staining is a bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. It is named for two German doctors who modified the stain: the bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898).
Crystal violet or gentian violet is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria. Crystal violet has antibacterial, antifungal, and anthelmintic properties and was formerly important as a topical antiseptic. The medical use of the dye has been largely superseded by more modern drugs, although it is still listed by the World Health Organization.
A growth medium or culture medium is a solid, liquid or semi-solid designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens. Different types of media are used for growing different types of cells.
A counterstain is a stain with colour contrasting to the principal stain, making the stained structure easily visible using a microscope.
Differential Staining is a staining process which uses more than one chemical stain. Using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organism.
Acridine orange is an organic compound. It is used as a nucleic acid-selective fluorescent cationic dye useful for cell cycle determination. Being cell-permeable, it interacts with DNA and RNA by intercalation or electrostatic attractions respectively. When bound to DNA, it is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm (green). When it associates with RNA, the excitation maximum shifts to 460 nm (blue) and the emission maximum shifts to 650 nm (red). Acridine orange will also enter acidic compartments such as lysosomes where it becomes protonated and sequestered. Within these low pH vesicles the dye emits red fluorescence when excited by blue light. Thus, acridine orange can be used to visualize primary lysosomes and phagolysosomes that may include products of phagocytosis of apoptotic cells. The dye is often used in epifluorescence microscopy and flow cytometry.
The Löwenstein–Jensen medium, more commonly known as LJ medium, is a growth medium specially used for culture of Mycobacterium species, notably Mycobacterium tuberculosis.
Endospore Staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample, which can be useful for classifying bacteria. Within bacteria, endospores are protective structures used to survive extreme conditions, but this protective nature makes them difficult to stain using normal techniques such as simple staining and Gram staining. Special techniques for endospore staining include the Schaeffer–Fulton stain and the Moeller stain.
A fastidious organism is any organism that has complex or particular nutritional requirements. In other words, a fastidious organism will only grow when specific nutrients are included in its diet. The more restrictive term fastidious microorganism is used in microbiology to describe microorganisms that will grow only if special nutrients are present in their culture medium. Thus fastidiousness is often practically defined as being difficult to culture, by any method yet tried. An example of a fastidious bacterium is Neisseria gonorrhoeae, which requires blood or hemoglobin and several amino acids and vitamins to grow. Other examples include Campylobacter spp. and Helicobacter spp., which are capnophilic – require elevated CO2 – among other requirements. Fastidious organisms are not inherently "weak"—they can flourish and thrive in their particular ecological niche with its particular nutrients, temperature, and absence of competitors, and they can be quite difficult to kill off. But they are difficult to culture simply because it is difficult to accurately simulate their natural milieu in a culture medium. For example, Treponema pallidum is not easy to culture, yet it is resilient in its preferred environment, being difficult to eradicate from all tissues of a person with syphilis.
In microbiology, the term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment, for example in water or soil flora, or from living beings with skin flora, oral flora or gut flora, in order to identify the microbe(s) of interest. Historically, the laboratory techniques of isolation first developed in the field of bacteriology and parasitology, before those in virology during the 20th century. Methods of microbial isolation have drastically changed over the past 50 years, from a labor perspective with increasing mechanization, and in regard to the technology involved, and hence speed and accuracy.
References
- P. Bruneval et al.. "Detection of fastidious bacteria in cardiac valves in cases of blood culture negative endocarditis." Journal of Clinical Pathology. 54:238-240 (2001).
- D.F. Gimenez. "Staining Rickettsiae in yolksack cultures". Stain Technol39:135–40 (1964).
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