Isolation (microbiology)

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:

• 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'.

Incubation

After the sample is inoculated into or onto the choice media, they are incubated 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.