LESSON 10: CULTURE METHODS Flashcards
A population of bacteria grown in the laboratory i
CULTURE
contains only one single type
pure culture
contains two or more
different bacteria
mix culture
bacterial cultures must be periodically transferred, or
to new media to keep the bacterial population growing
subcultured
means using practices and procedures to
prevent contamination from pathogens.
It involves applying the strictest rules to
minimize the risk of infection.
aseptic technique
Indications for various culture methods:
Isolate bacteria in pure culture and identify the same by performing various tests.
2. Demonstrate biochemical, antigenic, and other phenotypic and genomic
properties of the isolated colonies.
3. Demonstrate susceptibility of the isolated bacteria to antibiotics, bacteriophages, bacteriocins, etc.
4. Prepare antigens for various uses.
5. Maintain stock culture.
6. Estimate viable counts.
Culture method
The most effective way to isolate a single type of
bacteria from a source that contains many by diluting the individual cells by
spreading them over the surface of an agar plate using a platinum or inoculating
loop of 2–4 mm diameter.
Streak plate methods
On the plate single cells reproduce and create millions
of clones,
which all pile up on top of the original cell
Streak plate culture
The piles of bacterial cells
observed after an incubation period are
colonies
Also called as carpet culture, this method is used for
(a)
Antibiotic susceptibility testing by disk diffusion method,
(b) Bacteriophage typing,
(c) For preparation of bacterial antigens and vaccines.
Lawn culture
Procedure: Lawn cultures are prepared by flooding the surface of the plate
with a liquid culture or suspension of the bacterium, pipetting off the excess
inoculum and incubating the plate. Alternatively, the surface of the plate
maybe inoculated by applying a swab soaked in the bacterial culture or
suspension. After incubation, lawn culture provides a uniform growth of the
bacterium.
b) Antibiotic sensitivity testing
Provides a pure growth of bacteria for carrying
out slide agglutination and other diagnostic tests.
It is carried out in tubes usually
containing slanted nutrient agar slopes.
This method is used for
a) mainly for demonstration of gelatin
liquefaction,
(b) demonstration of oxygen requirement of the bacterium under
study,
(c) for the maintenance of stock cultures,
(d) to study motility of bacteria in
semisolid agar
stab culture
The preparation of the stab cultures, a suitable medium such as
nutrient gelatin or glucose agar is punctured with a long, straight, charged
wire into the center of the medium and withdrawing it in the same line to
avoid splitting the medium. The medium is allowed to set, with the tube in the
upright position, providing a flat surface at the top of the medium.
The pour-plate culture is used to determine approximate
number of viable organisms in liquids, such as water or urine.
Pour plate culture
Procedure: This method is carried out in tubes, each containing 15 mL of
molten agar. The molten agar in tubes is left to cool in a water bath at 45°C. The inoculum to be tested is diluted in serial dilution. Then 1 mL each of
diluted inoculum is added to each tube of molten agar and mixe
Pour plate methods
- Bacterial sample mixed with warm agar (45-50’C)
- sample poured onto sterile plate
- sample Swirled to mix, allowed solidify
- plate incubated until bacterial colonies grow
a deep culture of agar or gelatin through which the inoculum is
evenly distributed by shaking before the medium is solidified and
which is used
chiefly for the demonstration of anaerobic colonies
shake culture
This method is used for
(a) blood culture and for sterility,
(b)
dilution in the medium, or
(c) large yields culture.
However, liquid cultures does
not provide a pure culture from mixed inocula—the major disadvantage, nor
identify a bacteria
Liquid culture
For cultivation of aerobes the incubation is done in an incubator under normal
atmospheric condition.
Incubation of cultures at 37°C is standard practice in the
culture of bacteria pathogenic to man
Aerobic Culture
Culture in an Atmosphere with Added Carbon
Dioxide Some organisms, such as Brucella abortus and capnophilic streptococci,require extra CO2 in the air in which they are grown and others, such as the
pneumococcus and gonococcus, grow better in air supplemented with 5 to 10
percent CO2
Require incubation without oxygen and differ in their
requirement and sensitivity to oxygen.
Anaerobic bacteria
will not grow from small
inocula unless oxygen is absent and the Eh of the medium is low
Obligate anaerobes
METHODS OF ISOLATING PURE CULTURE
a method routinely employed in clinical bacteriology and enables
the isolation of distinct colonies which may be picked out, if necessary for further
purification and study
Surface plating
are widely used for the isolation of
pathogens from specimens such as feces, with varied flora.
Enrichment, selective and indicator
Selective media such as tellurite media for the diphtheria
bacillus, have been devised so that, the majority of organisms likely to be associated
with those for which the media are used will not grow,
and the isolation of pure
cultures is thus facilitated.
selective media
a such as selenite broth for Salmonella
sp, favor the multiplication of particular species as a step towards their isolation in
pure culture.
enrichement media
: Indicator media, such as Willis and Hobbs medium for
Clostridium sp,
contain ingredients that change in appearance with particular
organisms and so assist their isolation
Indicator media
can be used to separate spores from vegetative bacilli but does not guarantee that
spores will germinate under subsequent cultural conditions.
Heating at 65°C for 30 minutes or at higher temperatures for shorter periods
Pure
cultures may be obtained by pretreatment of specimens with appropriate bactericidal
substances which destroy the unwanted bacteria.
This method is the standard
practice for the isolation of tubercle bacilli from sputum and other clinical specimens, by treatment with alkali, acid or other substances to which most commensals are
susceptible but tubercle bacilli are resistant.
Pretreatment of specimens with appropriate bactericidal s
The temperature
and atmosphere chosen for a culture automatically preclude the growth of many
bacteria.
Separation of bacteria with different temperature optima
Incubation at 37°C, used for most medically important bacteria, is too warm
for some air contaminants, which subsequently appear as colonies when plates are
kept at room temperature.
Separation of bacteria with different temperature optima
Some pathogens are selectively favoured by growth at
temperatures above 37ºC.
Only thermophilic bacteria grow at 60ºC.
Obligate aerobes and
anaerobes may be separated by cultivation under aerobic or anaerobic condition.
Cultivation under aerobic or anaerobic condition
Strict anaerobes will not grow in air and most facultative anaerobes grow less
vigorously under anaerobic than under aerobic condition.
. Cultivation under aerobic or anaerobic condition
This consists of a tube of semisolid agar, with a narrow tube open at both
ends placed in the center of the medium in such a way that it projects above the
level of the agar. Inoculation of the mixture is made into the central tube. On
incubation, subculture is taken from the surface of the medium in the outer tube
because the motile bacteria alone traverse the agar and appear at the top of the
medium outside the central tube.
- Separation of motile from non-motile bacteria can be effected using Cragie’s tube
also serves the same purpose, inoculation being performed in one
limb and the subculture taken from the other. This method can also be used to obtain
phase variants in Salmonella species.
U-tube
Pathogenic bacteria may be isolated from mixtures by inoculation into
appropriate animals due to the fact that laboratory animals are highly susceptible to
certain organisms for example, the mouse to the pneumococcus
Animal inoculation:
If a mixture of organisms containing the pneumococcus, e.g.
in sputum, is inoculated subcutaneously into a mouse, the animal dies of
pneumococcal septicemia in 12 to 48 hours and the organism can be obtained in
pure culture from the heart blood.
Pneumococcus
can be distinguished from other aerobic
sporulating bacilli by inoculation into mice or guinea pIG
ANTHRAx BACILLI
produce a fatal septicemia and may be cultured pure from the heart blood
Anthrax bacilli
Can be isolated from
contaminating organisms by inoculation of an infected specimen into a guinea pig.
Is found in a pure state in the resulting lesion.
Tubercle bacillus
Of differing sizes may be separated by the use of selective filters.
Are widely used for separating g viruses from bacteria
filters
