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.
HistoryThe 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 techniquesIn 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
InoculationLaboratory 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:
- If one wants to isolate only a particular group of bacteria, such as Group A Streptococcus from a throat swab, one can use a selective medium that will suppress the growth of concomitant bacteria expected in the mix (by antibiotics present in the agar), so that only Streptococci are "selected", i.e. visibly stand out. To isolate fungi, Sabouraud agar can be used. Alternatively, lethal conditions for streptococci and gram negative bacteria like high salt concentrations in Mannitol salt agar favor survival of any staphylococci present in a sample of gut bacteria, and phenol red in the agar acts as a ph indicator showing if the bacteria are able to ferment mannitol by excreting acid into the medium. In other agar substances are added to exploit an organism's ability to produce a visible pigment (e.g. granada medium for Group B Streptococcus)which changes the bacterial colony's color, or to dissolve blood agar by hemolysis so that they can be more easily spotted. Some bacteria like Legionella species require particular nutrients or toxin binding as in charcoal to grow and therefore media such as Buffered charcoal yeast extract agar must be used.
- If one wants to isolate as many or all strains possible, different nutrient media as well as enriched media, such as blood agar and chocolate agar and anaerobic culture media such as thioglycolate broth need to be inoculated. To enumerate the growth, bacteria can be suspended in molten agar before it becomes solid, and then poured into petri dishes, the so-called 'pour plate method' which is used in environmental microbiology and food microbiology (e.g. dairy testing) to establish the so-called 'aerobic plate count'.