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 allows for visualization of the bacteria's cell wall composition based on the color the bacteria stains after a series of staining and decolorization steps. This staining process allows for the identification of
gram-negative and
gram positive bacteria. Gram-negative bacteria will stain a pink color due to the thin layer of peptidoglycan. If a bacteria stains purple, due to the thick layer of peptidoglycan, the bacteria is a gram-positive bacteria. 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. ==Culture-independent identification of bacteria==