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Isolation (microbiology)

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 (during the 19th century), 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.

History

The laboratory techniques of isolating microbes first developed during the 19th century in the field of bacteriology and parasitology using light microscopy. Proper isolation techniques of virology did not exist prior to the 20th century. The methods of microbial isolation have drastically changed over the past 50 years, from a labor perspective with increasing mechanization, and in regard to the technologies involved, and with it speed and accuracy.

General techniques

In order to isolate a microbe 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, one has to separate it from the mix. This can be achieved in two ways; Traditionally microbes have been cultured in order to identify the microbe(s) of interest based on its growth characteristics. Depending on the expected density and viability of microbes present in a liquid sample, physical methods to increase the gradient as for example serial dilution or centrifugation may be chosen. In order to isolate organisms in materials with high microbial content, such as sewage, soil or stool, serial dilutions will increase the chance of separating a mixture. In a liquid medium with few or no expected organisms, from an area that is normally sterile (such as CSF, blood inside the circulatory system) centrifugation, decanting the supernatant and using only the sediment will increase the chance to grow and isolate bacteria or the usually cell-associated viruses. If one expects or looks for a particularly fastidious organism, the microbiological culture and isolation techniques will have to be geared towards that microbe. For example, a bacterium that dies when exposed to air, can only be isolated if the sample is carried and processed under airless or anaerobic conditions. A bacterium that dies when exposed to room temperature (thermophilic) requires a pre-warmed transport container, and a microbe that dries and dies when carried on a cotton swab will need a viral transport medium before it can be cultured successfully. More recently, microbes have been isolated without culturing them. Samples are inoculated into microtiter plates or cartridges extracting their particular genetic material (DNA or RNA) which can be used to identifying them.

Bacterial and fungal culture

Inoculation

Laboratory technicians inoculate the sample onto certain solid agar plates with the streak plate method or into liquid culture medium, depending what the objective of the isolation is:

Incubation

After the sample is inoculated into or onto the choice media, they are under the appropriate atmospheric settings, such as aerobic, anaerobic or microaerophilic conditions or with added carbon dioxide (5%), at different temperature settings, for example 37 °C in an incubator or in a refrigerator for cold enrichment, under appropriate light, for example strictly without light wrapped in paper or in a dark bottle for scotochromogen mycobacteria, and for different lengths of time, because different bacteria grow at a different speed, varying from hours ( Escherichia coli) to weeks (e.g. mycobacteria). At regular, serial intervals laboratory technicians and microbiologists inspect the media for signs of visible growth and record it. The inspection again has to occur under conditions favoring the isolate's survival, i.e. in an 'anaerobic chamber' for anaerobe bacteria for example, and under conditions that do not threaten the person looking at the plates from being infected by a particularly infectious microbe, i.e. under a biological safety cabinet for Yersinia pestis (plague) or Bacillus anthracis (anthrax) for example.

Identification

When bacteria have visibly grown, they are often still mixed. The identification of a microbe depends upon the isolation of an individual colony, as biochemical testing of a microbe to determine its different physiological features depends on a pure culture. To make a subculture, one again works in aseptic technique in microbiology, lifting a single colony off the agar surface with a loop and streaks the material into the 4 quadrants of an agar plate or all over if the colony was singular and did not look mixed. Gram staining the raw sample before incubation or staining freshly grown colony material helps to determine if a colony consists of uniformly appearing bacteria or is mixed, and the color, and shape of bacteria allow a first classification based on morphology. In clinical microbiology numerous other staining techniques for particular organisms are used (acid fast bacterial stain for mycobacteria). Immunological staining techniques, such as direct immunofluorescence have been developed for medically important pathogens that are slow growing ( Auramine-rhodamine stain for mycobacteria) or difficult to grow (such as Legionella pneumophila species) and where the test result would alter standard management and empirical therapy. Biochemical testing of bacteria involves a set of agars in vials to separate motile from non-motile bacteria. In 1970 a miniaturized version was developed, called the analytical profile index. Successful identification via e.g. genome sequencing and genomics depends on pure cultures.

Bacteria, culture-independent

The most rapid method to isolate and identify bacteria is by sequencing their 16S rRNA gene, which has been PCR-amplified beforehand. Since most bacteria cannot be grown with conventional methods (particularly environmental or soil bacteria) metagenomics or metatranscriptomics are used, shotgun sequencing or PCR directed sequencing of the genome. Sequencing with mass spectrometry as in Matrix-assisted laser desorption/ionization (MALDI-TOF MS) is used in the analysis of clinical specimens to look for pathogens. Whole genome sequencing is an option for a singular organism that cannot be sufficiently characterized for identification. Small DNA microarrays can also be used for identification

References

"green air" © 2007 - Ingo Malchow, Webdesign Neustrelitz
This article based upon the http://en.wikipedia.org/wiki/Isolation_(microbiology), the free encyclopaedia Wikipedia and is licensed under the GNU Free Documentation License.
Further informations available on the list of authors and history: http://en.wikipedia.org/w/index.php?title=Isolation_(microbiology)&action=history
presented by: Ingo Malchow, Mirower Bogen 22, 17235 Neustrelitz, Germany