Culture Media and Methods

CULTURE MEDIA AND
CULTURE METHODS
CULTURE MEDIUM
• Culture medium – An environment which support the growth
of microorganisms.
• Consist of nutrients which support the growth of
microorganisms.
• Microbes can use the nutrients of culture media as their food.
• First medium prepared was Meat –Infusion Broth by Robert
Koach.
• A culture media is a liquid or gel/solid designed to support
the growth of microorganisms or cell.
• Culture media are required to grow the organisms from
infected material to identify the causative agent.
Need for Culture media:
• Bacteria: mixed population in nature
• By appropriate procedures they have to be grown
separately (isolated) on culture media and obtained as
pure culture for study
• Medium → Nutrients → support growth
Culture medium
Liquid medium Solid medium

Liquid medium:
• Diffused growth
• No characteristics for identification
• Difficult to isolate
• Earliest liquid medium: urine or meat broth used by Louis
Pasteur
Solid medium:
• Distinct colony morphology
• Characteristics → easy to identify
• Colony – macroscopically visible collection of millions of
bacteria originating from a single bacterial cell
• Earliest solid medium:
Cooked cut potato by Robert Koch
• Gelatin - not satisfactory
- liquefy at 24oC
Agar
• Universally used for preparing solid medium
• It contains mainly long –chain polysaccharide,
a small amount of protein-like material and
inorganic salts.
• Obtained from seaweed: Gelidium
• No nutritive value
• Not affected by the growth of the bacteria.
• Melts at 98°C & sets /solidifies at 42°C
• 2% agar is employed in solid medium
Types of culture media
I. Based on their consistency/Physical state
a) Solid medium
b) Liquid medium
c) Semi solid medium
II. Based on the constituents/ nutritional factors
a) Simple medium
b) Complex medium
c) Synthetic or defined medium
d) Special media
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
Solid medium
Liquid Semi-solid medium

medium
Simple media / basal media:
• Most common in routine diagnostic laboratories
Eg: Nutrient Broth, Nutrient Agar
• This is simple with no added ingredients.
• NB consists of peptone, meat extract, NaCl,
water
• NB + 0.5% Glucose = Glucose Broth
• NB + 2% agar = Nutrient agar
• Agar conc. Reduced (0.2 - 0.5%) = Semi-solid
medium
Complex media
• Media other than basal media.
• They have added complex ingredients such as yeast
extract or casein hydrolysate, which consist of a mixture
of many chemical species in unknown proportions
• Provide special nutrients for the growth of the
bacterium.
Synthetic or defined media
• Media prepared from pure chemical substances.
• The exact composition of the medium is known.
• Used for special studies, eg. metabolic requirements
• Eg: peptone water- (1% peptone + 0.5% NaCl in water)
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
• Blood agar - Streptococcus
• Chocolate agar – It is heated blood agar used for isolation of
Neisseria and Haemophilus influenza
• Loeffler’s serum slope Corynebacterium Diptheriae
Enrichment media
• Liquid media used to isolate pathogens from a mixed
culture.
• Stimulate growth of desired bacterium
Inhibit growth of unwanted
bacterium
• Media is incorporated with inhibitory substances to
suppress the unwanted organism → increase in numbers of
desired bacteria.
• Eg:
Selenite F Broth – for the isolation of Salmonella, Shigella
Tetrathionate Broth – inhibit coliforms
Alkaline Peptone Water – for Vibrio cholerae
Selenite F Broth Tetrathionate Alkaline Peptone
Broth water
Selective media
• The inhibitory substance is added to a solid media

• Increase in number of colonies of desired bacterium Eg:
• Deoxycholate citrate medium for dysentery bacilli
Salmonella and Shigella.
• Mac Conkey’s medium for gram negative bacteria-
Coliform bacteria.
• Thiosulfate citrate bile salts sucrose (TCBS)agar – for
Vibrio cholerae
• Lowenstein Jensen -LJ medium – Mycobacterium
tuberculosis
Mac Conkey’s medium Thiosulfate citrate bile
salts sucrose (TCBS)agar
Deoxycholate citrate agar LJ media
Indicator media
• Media contain an indicator which changes its colour
when a bacterium grows in them.
• Eg:
Wilson-Blair medium – Salmonella typhi forms
black colonies (reduces sulphite to sulphide).
McLeod’s medium (Potassium tellurite to mettalic
telurium)–Diphtheria bacilli– Clostridium diptheriae.
Wilson-Blair Medium McLeod’s medium

Urease producing
bacteria
Urease
Urea → CO2 + NH3
NH3 → Medium turns pink

Urease medium
Blood agar:
shows three types of Hemolysis
α Hemolysis
β
Hemolysis
γ Hemolysis
Differential media
• Substances incorporated in it enabling it to distinguish
between bacteria.
• Eg: Mac Conkey’s medium
– Peptone
– Lactose
– Agar
– Neutral red
– Sodium Taurocholate
• Distinguish between lactose fermenters & non lactose
fermenters.
MacConkey agar:
• Lactose fermenters – Pink colonies
• Non lactose fermenters – colourless colonies
Sugar media
• Media containing any fermentable substance
• Eg: glucose, arabinose, lactose, starch etc.
• Media consists:
1% of the sugar in peptone water + Indicator
• 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 & charcoal
• used for Gonnococci
– Buffered glycerol saline – enteric bacilli
Anaerobic media
• These media are used to grow anaerobic organisms.
• Eg: Robertson’s cooked meat medium, Thioglycolate
medium.
CULTURE METHODS
• Culture methods employed depend on the purpose for
which they are intended.
• Purposes:
– To isolate bacteria in pure cultures.
– To demonstrate their properties.
– To obtain sufficient growth for the preparation of
antigens and for other tests.
– For bacteriophage & bacteriocin susceptibility.
– To determine sensitivity to antibiotics.
– To estimate viable counts.
– Maintain stock cultures.
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.
Antibiotic sensitivity testing
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 stock cultures.
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 bottles
ANAEROBIC CULTURE METHODS
• Anaerobic bacteria differ in their requirement and
sensitivity to oxygen.
• Clostridium 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 desiccators.
Displacement of oxygen with other gases

• Displacement of oxygen with hydrogen,
nitrogen, helium or CO2.
• Eg: Candle jar
Chemical method
• Alkaline pyrogallol absorbs oxygen.
• Chromium and Sulphuric acid
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.
Working:
• Inoculated plates are placed inside the jar and the lid
clamped air tight.
• The outlet tube is connected to a vacuum pump and the air
inside is evacuated.
• The outlet tap is then closed and the inlet tube is
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.
• Act as a catalyst for the combination of hydrogen with
residual oxygen.
Gaspak
• Commercially available disposable envelope.
• Contains chemicals which generate H2 and CO2
on addition of water.
• Cold catalyst – permits combination
of Hydrogen & Oxygen
• Indicator is used – reduced
methylene blue.
– Colourless – anaerobically
– Blue colour – on exposure to oxygen
Biological method
• Absorption of oxygen by incubation with aerobic

bacteria, germinating seeds or chopped
vegetables.
Reduction of oxygen
• By using reducing agents – 1% glucose,
0.1% Thioglycolate
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CULTURE MEDIA AND

Liquid medium Solid medium

III. Based on Oxygen requirement

Liquid Semi-solid medium

• The inhibitory substance is added to a solid media

Wilson-Blair Medium McLeod’s medium

Urea → CO2 + NH3

NH3 → Medium turns pink

• Media used for transporting the samples.

Displacement of oxygen with other gases

McIntosh – Fildes’ anaerobic jar

• Absorption of oxygen by incubation with aerobic

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