Culture Media & Culture Methods

CULTURE MEDIA &
CULTURE
METHODS
 What is
culture?
 Bacteria have to be grown (cultured)
for them to be identified.
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CULTURE METHODS
The indications for culture are:
– To isolate bacteria in pure cultures.
– To demonstrate their properties.
– To obtain sufficient growth for the preparation of
antigens.
– To determine sensitivity to antibiotics.
– To estimate viable counts.
– Maintain stock cultures.
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Colony – macroscopically visible collection
of millions of bacteria originating from a
single bacterial cell.
Cooked cut potato by Robert Koch

– earliest solid medium
Gelatin – not satisfactory
- liquefy at 24oC
Types of culture media
I. Based on their consistency
a) solid medium
b) liquid medium
c) semi solid medium
II. Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
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Special media
– Enriched media
– Enrichment media
– Selective media
– Indicator media
– Differential media
– Sugar media
– Transport media
– Media for biochemical reactions
III. Based on Oxygen requirement

- Aerobic media
- Anaerobic media
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Solid media – contains 2% agar
Colony morphology, pigmentation, hemolysis can
be appreciated.
Eg: Nutrient agar, Blood agar
Liquid media – no agar.

For inoculum preparation, Blood culture, for the
isolation of pathogens from a mixture.
Eg: Nutrient broth
Semi solid medium – 0.5% agar.

Eg: Motility medium
Simple media / basal media
- Eg: NB, NA
- NB consists of peptone, meat
extract, NaCl,
- NB + 2% agar = Nutrient agar

Complex media
Media other than basal media.
They have added ingredients.
Provide special nutrients
Synthetic or defined media

Media prepared from pure chemical
substances and its exact composition is
known
Eg: peptone water – 1% peptone +
0.5%
N a C l i n w ater kwar
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Dr. As hi sh Ja w ar Parul Sevashram
Enriched media
Substances like blood, serum, egg
are added to the basal medium.
Used to grow bacteria that are exacting
in their nutritional needs.
Eg: Blood agar, Chocolate agar

Enrichment media
Liquid media used to
isolate pathogens from a
mixed culture.
Media is incorporated
with inhibitory substances
to suppress the unwanted
organism.
Eg:
– Selenite F Broth – for the
isolation of Salmonella, Shigella
– Alkaline Peptone Water – for
Dr. AshVisihbJraiwoarckahroleraPearul Sevashram 14
Selective media
The inhibitory substance is added to a
solid media.
Eg:
Mac Conkey’s medium for gram
negative bacteria
TCBS – for V.cholerae
LJ medium – M.tuberculosis
Wilson and Blair medium – S.typhi
Potassium tellurite medium –
Diphtheria bacilli
Indicator media
These media contain an indicator
which changes its colour when a
bacterium grows in them.
Eg:
– Blood agar
– Mac Conkey’s medium
– Christensen’s urease medium

Differential media
A media which has substances
incorporated in it enabling it to distinguish
between bacteria.
Eg: Mac Conkey’s medium
– Peptone
– Lactose
– Agar
– Neutral red
– Taurocholate
Distinguish between lactose fermenters
&
Sugar media
Media containing any
fermentable substance.
Eg: glucose, arabinose, lactose,
starch etc.
Media consists of 1% of the sugar
in peptone water.
Contain a small tube (Durham’s tube)
for the detection of gas by the bacteria.

Transport media
Media used for transporting
the samples.
Delicate organisms may
not survive the time taken
for transporting the
specimen without a
transport media.
Eg:
– Stuart’s medium – non nutrient
soft agar gel containing a
reducing agent
Dr.
– Buffered glycerol saline –Hospital
Parul Sevashram 25
Anaerobic media
These media are used to grow anaerobic
organisms.
Eg: Robertson’s cooked meat medium,
Thioglycolate medium.

Culture methods include:
Streak culture
Lawn culture
Stroke culture
Stab culture
Pour plate method
Liquid culture
Anaerobic culture
methods

STREAK CULTURE
Used for the isolation of bacteria in pure culture
from clinical specimens.
Platinum wire or Nichrome wire is used.
One loopful of the specimen is transferred
onto the surface of a well dried plate.
Spread over a small area at the periphery.
The inoculum is then distributed thinly over the
plate by streaking it with a loop in a series
of parallel lines in different segments of the
plate.
On incubation, separated colonies are obtained
over the last series of streaks.
LAWN CULTURE
Provides a uniform surface growth of the
bacterium.
Uses
– For bacteriophage typing.
– Antibiotic sensitivity testing.
– In the preparation of bacterial antigens and
vaccines.
Lawn cultures are prepared by flooding the
surface of the plate with a liquid suspension of
the bacterium.

Dr. Ashish JawarkaAr ntibioPatircul
STROKE CULTURE
Stroke culture is made in
tubes containing agar slope /
slant.
Uses
– Provide a pure growth
of bacterium for slide
agglutination and other
diagnostic tests.
STAB CULTURE
Prepared by puncturing a suitable
medium
– gelatin or glucose agar with a long,
straight, charged wire.
Uses
– Demonstration of gelatin
liquefaction.
– Oxygen requirements of the bacterium
under study.
– Maintenance of stoke cultures.
Gelatin Oxidation –
liquefaction Fermentation medium

POUR PLATE CULTURE
Agar medium is melted (15 ml) and cooled to
45oC.
1 ml of the inoculum is added to the molten
agar.
Mix well and pour to a sterile petri dish.
Allow it to set.
Incubate at 37oC, colonies will be distributed
throughout the depth of the medium.
Uses
– Gives an estimate of the viable bacterial count
in a suspension.
– For the quantitative urine cultures.
LIQUID CULTURES
Liquid cultures are inoculated by touching
with a charged loop or by adding the inoculum
with pipettes or syringes.
Uses
– Blood culture
– Sterility tests
– Continuous culture methods
Disadvantage
– It does not provide a pure culture from mixed
inocula.

Blood culture
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Parul Sevashram Hospital 29
ANAEROBIC CULTURE METHODS
Anaerobic bacteria differ in their requirement
and sensitivity to oxygen.
Cl.tetani is a strict anaerobe – grows at an
oxygen tension < 2 mm Hg.
Methods:
– Production of vacuum
– Displacement of oxygen with other
gases
– Chemical method
– Biological method
– Reduction of medium
Production of vacuum:
Incubate the cultures in a
vacuum desiccator.
Displacement of oxygen with

other gases
Displacement of oxygen with
hydrogen, nitrogen, helium or CO2.
Eg: Candle jar

Chemical method
 Alkaline pyrogallol absorbs
oxygen.
McIntosh – Fildes’ anaerobic jar
Consists of a metal jar or glass jar with a metal
lid which can be clamped air tight.
The lid has 2 tubes – gas inlet and gas outlet
The lid has two terminals – connected to
electrical supply.
Under the lid – small grooved porcelain spool,
wrapped with a layer of palladinised
asbestos.

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CULTURE MEDIA &

Cooked cut potato by Robert Koch

III. Based on Oxygen requirement

Liquid media – no agar.

Semi solid medium – 0.5% agar.

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Synthetic or defined media

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Displacement of oxygen with

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