Culture methods

Culture methods employed depend on the purpose for which they are intended.
Culture methods are mainly employed to;
1. isolate bacteria in pure culture;
2. demonstrate their properties;
3. Obtain sufficient growth for preparation of antigens and for other tests;
4. type isolates by methods such as bacteriophage and bacteriocin susceptibility,
5. determine sensitivity to antibiotics,
6. estimate viable counts; and
7. maintain stock cultures.
The methods of culture used ordinarily in the laboratory are streak, lawn, stroke,
stab, plate and liquid cultures. Special methods are employed for culturing
anaerobic bacteria.
1. The streak culture
The streak culture (surface plating) is routinely employed for the isolation of
bacteria in pure culture from clinical specimens. A platinum loop is charged with
the specimen to be cultured.Owing to the high cost of platinum, loops for routine
work are made of Nichrome resistance wire(24 SWG size). One loopful of the
specimen is transferred onto the surface of a well dried plate,on which it is spread
over a small area at the periphery. The inoculum is then distributed thinly over
the plate by streaking it with the series of parallel lines, in different segments Of
the plate. The loop should be flamed and cooled between the different sets of
streaks. On incubation growth may be confluent at the site of original inoculation
but progressively thinner, and well separated colonies are obtained over the final
series of streaks.
2. Lawn or carpet culture
Lawn or carpet culture provides a uniform surface growth of the bacterium and is
useful for bacteriophage typing and antibiotic sensitivity testing (disc method). It
may also be employed when a large amount of growth is required on solid media
as, for instance, in the preparation of bacterial antigens and vaccines. 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 may be inoculated applying a swab
soaked in the bacterial culture or suspension.
3. Stroke culture
The stroke culture is made in tubes containing agar slope(slant) and is employed
for providing a pure growth of the bacterium for slide agglutination and other
diagnostic tests.
4. Stab cultures
Stab cultures are prepared by puncturing a suitable medium such as nutrient
gelatin or glucose agar with a long, straight, charged Wire. The medium is allowed
to set, With the tube in the upright position, providing a flat surface at the top of
the medium. Stab cultures arc employed mainly for demonstration of gelatin
liquefaction and oxygen requirement of the bacterium under study. They are also
used in the maintenance of stock cultures.
5.Pour plate method
In this method the original sample is diluted several times to reduce the microbial
population sufficiently to obtain separate colonies when plated. The small
volumes of several diluted samples are mixed with liquid agar that has been
cooled to about 45 degrees Celsius and the mixtures are poured immediately into
sterile culture dishes. After agar has hardened, each cell is fixed in place and
forms an individual colony. Plates containing between 30 and 300 colonies are
counted. The total number of colonies equals the number of viable
microorganisms in the diluted sample. Colonies growing on the agar plates can be
used to inoculate fresh medium and prepare pure cultures.
6.Spread plate method

7. Sweep plate method

In the sweep plate method, the edges of the Petri dish containing the culture
media are rubbed over the fabric, with the medium facing it. The dust particles
stirred up from the cloth settled on the culture medium and colonies develop on
dictation. They can be counted and estimated.
8. Liquid cultures
Liquid cultures in tubes, bottles or flasks may be inoculated by touching with a
charged loop or by adding the inoculum with pipettes or syringes.Large inoculum
can be employed in liquid cultures and hence this method is adopted for blood
culture and for sterility tests, were the concentration of bacteria in the inoculum
is expected to be small. Liquid cultures are preferable for inocula containing
antibiotics and other inhibitory substances, as these are rendered ineffective by
dilution in the medium. Liquid cultures are also preferred when large yields are
desired, the yield being enhanced by agitation, aeration, addition of nutrients and
removal of toxic metabolites. The major disadvantage of liquid culture is that it
does not provide a pure culture from mixer inoculum.
ANAEROBIC CULTURE METHODS
Anaerobic bacteria differ in their requirements of and sensitivity to oxygen. Some,
such as Histolyticum are aerotolerant and may produce growth on the surface of
aerobic plates, while others such as C. tetani. are strict anaerobes and form
surface growth free if the O2 tention is less than two mmHg.Following methods
are used for anaerobic culture
1.McIntosh and Fildes anaerobic jar
This is the most reliable and widely used anaerobic method. It consists of a stout
glass or metal jar with a metal lid that can be clamped airtight with a screw. The
lid has two tubes with taps, one acting as the gas inlet and other as the outlet. ,
which can be connected to an electric supply. leading from the terminals and
suspended by stout wires on the underside of the lid is a small grooved porcelain
spool around which is wrapped a layer of palladinised asbestos. Inoculated
culture plates are placed inside the jar, with the medium in the bottom half of the
plates, and the lid clamped tight. The outlet tube is connected to vacuum pump
and the air inside is evacuated.The outlet tap is then closed and the inlet tube
connected to a hydrogen supply. After the jar is filled with hydrogen, the electric
terminals are connected to a current supply so that the palladinised asbestos is
heated. This acts as a catalyst for the combination of hydrogen with the residual
oxygen present in the jar. This method ensures complete anaerobiosis but carries
the risk of explosion, which may occur. This risk can be eliminated by modification
of the catalyst. Alumina pellets coated with palladium in a gauze sachet
suspended from the lid of the jar act as a catalyst at room temperature, as long as
the sachet is kept dry.
2. Gaspak
This is now the method of choice for anaerobiosis.The Gaspak is commercially
available as a disposable envelope. containing chemicals which generate
hydrogen and carbon dioxide on addition of water. After the inoculated plates are
kept in the jar, the Gaspak envelope, With water added, is placed inside and the
lid screwed light.Hydrogen and carbon dioxide are liberated and the presence of a
cold catalyst in the envelope permits the combination of hydrogen and oxygen to
produce an anaerobic environment. The Gaspak is simple and effective,
eliminating the need for drawing a vacuum and adding hydrogen.
3.Pre-reduced anaerobic system (PRAS):
For fastidious anaerobes, particularly for quantitative cultures,pre-reduced media
are available,
4.Anaerobic chamber ('glove box’)
The anaerobic chamber is an airtight, glass-fronted cabinet filled with inert gas,
with an entry lock for the introduction and removal of materials, and gloves for
the hands.
An indicator should be used for verifying the anaerobic condition in the jars.
Reduced methylene blue is generally used for this purpose. It remains
colourless anaerobically but turns blue on exposure to oxygen.
5.Other methods of anaerobiasis:
a) Reducing agents: Reduction of oxygen in the medium is achieved by the use of
various reducing agents, including 1% glucose, 0.1% thioglycollate, 0.1% ascorbic
acid and 0.05% cysteine.
b)Broth is an easily prepared anaerobic medium which pieces of red hot metallic
iron are introduced.It is then layered over with sterile Vaseline. Brought the
containing fresh animal tissue, such as rabbit kidney, spleen, testis or heart
supports the growth of many anaerobes.
c)Thioglycolate broth with hemin and vitamin K is an enriched liquid medium for
culturing anaerobic and microaerophilic bacteria.
d) The addition of a small quantity of agar enhances the anaerobic capacity of the
medium by slowing the diffusion of oxygen in it.
e)Robertson's cooked meat medium is probably the most widely used fluid
medium for the culture of anaerobes. It consists of fat-free minced cooked
meat in broth. It permits the growth of even strict anaerobes and indicates their
saccharolytic or proteolytic activities, by the meat being turned red or black,
respectively.