Culture Media and

Bacterial Culture
methods

DR.C. P. PRINCE
HOD & Associate Professor
Department of Microbiology
Mother Theresa Post Graduate & Research Institute of Health
Sciences
(Government of Puducherry Institution)
 Cultivation/Culturing of Bacteria

A microbial culture, is a method of

multiplying microorganisms by
letting them to reproduce in
predetermined culture media under
controlled laboratory conditions.
( Growing Bacteria in
Laboratory)
Microbial cultures are used to
determine the type of organism, its
abundance in the sample being
tested, or both.
 Purpose of culturing
 Isolation of bacteria ( pure culture)
 Diagnosis of infectious diseases
 Properties of bacteria i.e. culturing bacteria is the
initial step in studying its morphology and its
identification.
 Maintenance of stock cultures.
 Estimate viable counts. Water , air, milk testing
 To test for antibiotic sensitivity.
 To create antigens for laboratory use.
 Vaccine preparation
 Sterility testing
 Preparation of pharmaceutical products like
antibiotics, enzymes, toxins etc
 Certain genetic studies and manipulations of the cells
also need that bacteria to be cultured in vitro.
 Culturing on solid media is another convenient way of
separating bacteria in mixture.
Culture Media

An artificial culture media must provide similar

environmental and nutritional conditions that
exist in the natural habitat of a bacterium.
A culture medium contains water, a source of
carbon & energy, source of nitrogen, trace
elements and some growth factors.
The pH of the medium must be set accordingly.
Uses:
*Enrich the number of bacteria.
*Select for certain bacteria and suppress
others.
*Differentiate among different kinds of
bacteria.
Agar

 Solidifying agent used for preparation of solid

culture medium
 Agar, a polysaccharide extracted from marine
algae, is used to solidify a specific nutrient
solution.
 Unlike other gelling agent, it is not easily
degraded by many bacteria.
 It is not easily destroyed at higher
temperatures, and therefore it can be sterilized
by heating, the process which also liquefies it.
 Once solidified, agar medium will remain solid
 Pure culture

In the laboratory bacteria are isolated

and grown in pure culture in order to
study the functions of a particular
specie.
 A pure culture is a population of cells or
growing in the absence of other species
or types. A pure culture may originate
from a single cell or single organism, in
which case the cells are genetic clones
of one another
Medium containing only one type of
bacterial growth
Classification of Culture Media

Bacterial culture media can be

classified in at least three ways.
1. Consistency
2. Nutritional component
3. Functional use
Classification based on
consistency
1. Liquid media.
2. Solid media.
3. Semi solid media.
Classification based on Nutritional Components

1. Simple media.

2. Complex media.
3. Synthetic or chemically
defined media.
Classification based on Functional Use or
Application

1. Enriched media.

2. Selective media.
3. Differential media.
4. Transport media.
5. Indicator media.
6. Anaerobic media.
BASIC MEDIA

These are simple media used

to support the growth of
microorganisms that do not
have special nutritional
requirements.
They include nutrient broth,
peptone water, and nutrient
agar.
 ENRICHED MEDIA
 Prepared by the addition of substances such
as blood, serum or egg to a basic medium.
Used for cultivation of fastidious organisms
that cannot grow on simple media and need
highly nutritive substances for growth.
Used for culturing sterile body fluids such as
blood or CSF, where the finding of any
organisms = infection due to that organism.
And also for primary identification of
microorganisms e.g. haemolysis on blood
e.g. blood agar, chocolate agar, Loeffler’s
serum slope
Blood agar

sterilede-fibrinated sheep or human 5-

10%. blood + melted nutrient agar at
55°C
Red opaque solid medium.
N. agar is sterilized in autoclave at
121°C for 30 min. Blood is added under
complete aseptic condition at 45- .55 oC
Supports the growth of most delicate
organisms e.g- .Streptococcus pyogenes
Identifying bacteria according to their
haemolytic-action on the red cells
Chocolate agar

Prepared as blood agar followed

by raising the temperature to l00
°C for 2 min to rupture red cells
and release nutrients as X and V
factors
Brown opaque solid medium.
Sterilized as blood agar.
Used for the isolation of Neisseria
meningitides,
Haemophilus .influenza and
Streptococcus pneumonae
Loeffler's serum slope

Opaque whitish solid

medium.
The medium is solidified in
hot air inspissator at.. 75°C
for 2 hr for 2 successive days
Uses : Culture of
Corynebacterium diphtheria
SELECTIVE MEDIA

Solid media that contain

substances (e.g. Bile salts or
other chemicals, dyes,
antibiotics) which inhibit the
growth of one organism to allow
the growth of another .
Used when culturing a specimen
from a site having a normal
microbial flora to prevent
unwanted contaminants
overgrowing a pathogen.
Lowenstein Jensen
medium
Selective medium for
Mycobacterium tuberculosis
Contains beaten eggs
+malachite green
Green opaque solid medium.
Sterilized in hot air
inspissator at 75 °C for 2hr
for 2 successive days
MacLeod's tellurite blood
agar(TBA)
Blood agar + 0.02-0.04% K
tellurite.
Red opaque solid medium. .
Selective medium Used for
isolation of Corynebacterium
diphtheriae .
Modified Thayer-Martin
agar
Chocolate agar + vancomycin
+ colistin +nystatin
Brown opaque-solid medium.
Selective medium for Used
for Isolation of Neisseria from
non-sterile specimens.
Thiosulphate citrate bile sucrose agar
(TCBS )

Alkaline agar + sucrose +

thiosulphate + citrate
and .bromothymol blue
indicator
Greenish transparent solid
medium.
Sterilized in autoclave at 121°C
for 30 min.
Selective medium Used for
isolation of Vibrio cholerae.
Deoxycholate citrate agar
(DCA)
Na deoxycholate and citrate
+ Agar + lactose + neutral
red indicator.
Reddish semi transparent
solid medium.
Selective medium Used for
isolation of Shigella and
Salmonella.
XLD Media

Agar + lactose + phenol red

indicator + ferric citrate +
desoxycholate + xylose +
lysine +sucrose + yeast
extract
Reddish semi transparent
solid medium.
Selective medium Used for
isolation of Shigella and
Salmonella
ENRICHMENT MEDIA
Fluid media that contain substances
which favour the growth of wanted
organisms on the expense of others.
Usually used as a preliminary step for
isolation of pathogens before
subculturing on solid selective media.
Examples are:
Selenite broth for isolation of Salmonella
and Shigella species from faeces
Tetrathionate broth for isolation of
Salmonella from faeces
Alkaline peptone water for isolation of
Vibrio cholerea
INDICATOR (DIFFERENTIAL MEDIA)

These are media to which

dyes or other substances
(Indicators()are added to
differentiate microorganisms.
Indicators change colour
when acid is produced
following fermentation of a
specific carbohydrate e.g.
MacConkey's agar medium.
MacConkey's agar medium

 Peptone, agar, lactose, bile

salt and neutral red indicator.
Reddish transparent solid
medium
Detects lactose fermenting
and non lactose fermenting
bacteria
IDENTIFICATION MEDIA

These include media to which

substrates or chemicals are added to
help identify bacteria isolated on
primary cultures. i.e. organisms
identified must be first isolated in pure
culture.
Organisms are mainly identified by a
change in the colour of the medium
and or the production of gas.
They include peptone water sugars,
litmus milk, and gelatin media.
TRANSPORT MEDIA

Semisolid media that contain ingredients

to prevent the overgrowth of commensal
& ensure the survival of aerobic and
anaerobic pathogens when specimens
cannot be cultured immediately.
Examples:
1- Cary-Blair medium for preserving
enteric pathogens.
2- Stuarts and Amies transport medium
for ensuring the viability of gonococci
3- Thioglycollate broth and deep agar
for- 3 anaerobic organisms
CULTURE MEDIA FOR ANAEROBES

Media for anaerobes is the same as

media for aerobes except that:
1. They are richer in organic
constituents .
2. Contain reducing agents (cysteine
& haemin).
3. Contain a redox indicator .
The inoculated media are incubated
in anaerobic environment using
anaerobic gas pack .
Robertson's cooked meat medium

Anaerobic enrichment media

cooked minced meat to which
broth is added
Anaerobiosis is achieved
through (reducing substances
in the meat).e.g. haemin and
glutathione
Sterilized in autoclave at
121°C-for 30 min
Anaerobic GasPak System

A method for the exclusion of

oxygen from a sealed jar used
for incubation of anaerobic
cultures in a non-reducing
medium .
Robertson's cooked meat
medium
Anaerobic Culture Methods

Production of a vacuum
Displacement of Oxygen with
other gases
Absorption of Oxygen by
chemical or biological methods
By using reducing agents
McIntosh & Filde’s Jar
Anaerobic GasPak System
Displacement of Oxygen
Anaerobic Glove Chamber
Anaerobic Glove Chamber
Nutrient broth
L-J medium
TCBS
XLD Agar
MacConkey's agar medium
Robertson's cooked meat
medium
Culture Methods

Streak culture
Lawn culture
Stroke culture
Stab culture
Pour plate method
Streak culture

 Used for the isolation of bacteria in pure

culture from clinical specimens.
 Platinum wire is used.
 One loop full 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.
The streak-plate method to
obtain pure cultures
Streak culture
Lawn Culture

Provides a uniform surface growth of

the bacterium.
Lawn cultures are prepared by
flooding the surface of the plate with
a liquid suspension of the bacterium
Uses
– For bacteriophage typing.
– Antibiotic sensitivity testing.
– In the preparation of bacterial
antigens and vaccines.
Lawn Culture
Stroke Culture

• Stroke culture is made in tubes

containing agar slope / slant.
Uses:
Provides a pure growth of
bacterium for slide agglutination
and other diagnostic tests.
Stroke Culture
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 stock cultures.
Pour Plate Culture

1 ml of the innoculum is added to

the molten agar.
 Mix well and pour to a sterile Petri
dish.
 Allow it to set.
Uses:
– Gives an estimate of the viable
bacterial count in a suspension.
– For the quantitative urine cultures
Thanks