Dr.T.V.
Rao MD
Major Contribution to Culture
Media
Dr.T.V.Rao MD
Agar - Agar Frau Hesse
Dr.T.V.Rao MD
Agar Agar
Solid medium is made by
adding Agar
Agar is obtained from Sea
weeds New Zealand agar is
more
Agar contain long chain
poly saccharides.Inoranic
salts and protein like
substance
Melts at 980c and sets at
420c
Dr.T.V.Rao MD
Dr.T.V.Rao MD
Agar - Agar
Complex polysaccharide
Used as solidifying agent for culture
media in Petri plates, slants, and deeps
Generally not metabolized by microbes
Liquefies at 98C
Solidifies ~42C
Dr.T.V.Rao MD
Media and Culture
Media: Nutrients (agar, pH indicators, proteins
and carbohydrates) used to grow organisms
outside of their natural habitats
Culture: The propagation of microorganisms using
various media
Dr.T.V.Rao MD
Culture media
Used to grow bacteria
Can be used to:
Enrich the numbers of bacteria
Select for certain bacteria and suppress
others
Differentiate among different kinds of
bacteria
Dr.T.V.Rao MD
Culture and Medium
Culture is the term given to microorganisms that
are cultivated in the lab for the purpose of
identifying and studying them.
Medium is the term given to the combination of
ingredients that will support the growth and
cultivation of microorganisms by providing all the
essential nutrients required for the growth (that
is, multiplication) in order to cultivate these
microorganisms in large numbers to study them.
Need for Culture Media
It is usually essential to obtain a culture by
growing the organism in an artificial medium.
If more than one species or type of organism
are present each requires to be carefully
separated or isolated in pure culture.
Several organism need the determination of
Antibiotic sensitivity pattern for optimal
antibiotic selection
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Basic requirements of
culture media
Nutrients
- Energy source
- Carbon source
- Nitrogen source
Mineral salts Sulphate, phosphates, chlorides
& carbonates of K, Mg & Ca.
A suitable pH 7.2 7.4
Accessory growth factors
- Tryptophan for Salmonella typhi
- X & V factors for H. influenzae
Dr.T.V.Rao MD
Classification of Culture media
Based on the consistency:
Peptone water, Nutrient broth
Semisolid -- Nutrient agar stabs
Solid
-- Blood agar, Serum agar
Liquid
-- Based on Oxygen requirement:
-- Aerobic medium
-- Anaerobic media
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Pouring the Culture
Plates
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Pouring a plate
Dr.T.V.Rao MD
http://www.biotopics.co.uk/microbes/pourp2.gif
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Petri dish with Media
Plate: provide large surface
for isolation and
observation of colonies
Using a sterile loop or a
sterile swab streak your
sample on the petri plate
Important let your
sterilized loop cool before
you pick up your sample
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Aerobic Media
Simple media
Complex media
May be Synthetic or Defined Medium
- Enriched media
- Differential media
- Enrichment media
- Selective media
Semisynthetic Medium
- Sugar media
- Transport media
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Aerobic media
Simple media- consists of only basic
necessities
Liquid media
- Peptone water(1% peptone +0.5%Nacl +
100 ml water)
- Nutrient broth ( peptone water + 1% meat
extract
Solid media
- Nutrient agar (nutrient broth + 2% Agar)
Use: To grow non-fastidious
microorganisms
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17
Liquid Medium
Difficult to identify
all types of
organisms
Suitable for
isolation of
bacteria from
Blood culturing
and water analysis
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Peptone Water
Peptone contain partially digested
proteins
Proteases
Polypeptides
Aminoacids
Inorganic salts
Phosphates
Potassium and Magnesium
Riboflavin
Meat exract called as Lab lemco
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Nutrient Agar
Contain 2% agar added
to Nutrient agar
commonly used
Concentration can be
increased to 6% to
prevent swarming
Can be reduced to 05%
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Pigment producing
Staphylococci
on Nutrient Agar
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Defined Media
Defined media are media composed of
pure ingredients in carefully measured
concentrations dissolved in double
distilled water i.e., the exact chemical
composition of the medium is known.
Typically, they contain a simple sugar as
the carbon and energy source, an
inorganic nitrogen source, various
mineral salts and if necessary growth
factors (purified amino acids, vitamins,
purines and pyrimidine's
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Selective / Differential Media
Selective/differential media are media based
on either of the two categories above
supplemented with growth-promoting or
growth-inhibiting additives. The additives
may be species- or organism-selective (e.g.,
a specific substrate, or an inhibitor such as
Cyclohexamide (artidione) which inhibits all
eukaryotic growth and is typically used to
prevent fungal growth in mixed cultures).
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Salmonella Shigella agar
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TCBS medium
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Complex media
Enriched media: Blood agar
Nutrient agar + 5 to 10% sheep blood
Melt the sterile nutrient agar by steaming, cool, to
450 c
Add the blood aseptically with constant shaking
Mix the blood with molten nutrient agar thoroughly
but gently avoiding froth formation
Immediately pour in to the Petri dishes or tubes
and allow to set
Use: To cultivate all the fastidious organisms
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Enriched Medium
To culture medium
Blood serum or egg
are added to
medium eg Blood
agar, Chocolate
agar
Egg based medium
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Different types of hemolysis
on Blood Agar
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Other Enrichments Chocolate Agar
Several organic
materials are added
to the basic
constituents of the
Medium such as
Blood, yeast, yeast
extract etc
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Chocolate agar
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Differential Medium
MacConkey agar
Mac
Bringing out different
characters of bacteria
their atypical characters
Mac Conkeys medium
Contain peptone, Lactose
Agar, Neutral red and
taurocholate and show
growth of Lactose
fermenters as pink
colored colonies
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Lactose fermenting Mucoid
colonies on MacConkey Agar
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MacConkey agar
MacConkey agar is
useful medium for
cultivation of coliforms
It contains a bile salt to
inhibit non intestinal
bacteria
Lactose in combination
with Neutral red
distinguish the lactose
fermenting from the non
lactose fermenting
Salmonella and
Dysentery group
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Lactose fermenting and Non
lactose fermenting
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Enrichment Medium
If the sample contain more
than one type of bacteria,
undesired bacteria grwoth
can be reduced or
eliminated.
The desired organism is
facilitated to grow
Eg Tetrathionate broth
Selenite F broth
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Selective media
Serve the same purpose as
Enrichment media but are solid in
consistency
- Wilson & Blairs medium - Lowenstein Jensens medium
Use: To cultivate Salmonella,
Shigella &
Mycobacteria
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Selective Medium
Deoxycholate citrate agar
Inhibitory
substances to
solid medium
as in
Deoxycholate
citrate agar
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Deoxycholate citrate Agar
Suitable for isolation of
dysentery bacilli, food
poisoning Salmonella and
S.paratyphi B, and less so, but
superior to MacConkey agar for
S. typhi.
It is a heat sensitive medium It
should not be autoclaved or
remelted
When prepared from
commercial medium it should
be dissolved and sterilized at
1000c for a short period
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Indicator Medium Wilson-Blair
medium
Indicate by change
of color Sulphite to
sulphide in WilsonBlair medium
S.typhi reduces
sulphite to sulphide
in the presence of
Glucose
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Carbohydrate media
Peptone water 100 ml, Desired sugar 1 gm%
and Andrade's indicator 0.005% soln(1ml)
Dissolve the desired carbohydrate in peptone
water and steam for 30 min or sterilize by
filtration.
Distribute into sterile test tube containing
inverted Durhams tubes to detect gas
production and steam for 30 min
Use: To test the fermenting ability of an
organism
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Carbohydrate media
Peptone water 100 ml,
Desired sugar 1 gm% and
Andrade's indicator
0.005% soln(1ml)
Dissolve the desired
carbohydrate in peptone
water and steam for 30
min or sterilize by
filtration.
Use: To test the
fermenting ability of an
organism
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Carbohydrate media
Peptone water 100 ml,
Desired sugar 1 gm% and
Andrade's indicator
0.005% soln(1ml)
Dissolve the desired
carbohydrate in peptone
water and steam for 30
min or sterilize by
filtration.
Use: To test the
fermenting ability of an
organism
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42
Sugar Medium
Sugars are fermenting substances
Monosaccharide peptone, arabinose, xylose and hexose's, dextrose
and mannose
Disaccharides Sucrose and Lactose
Polysaccharides Starch and Inulin
Alcohols Glycerol. Sorbitol
Sugar medium contain 1% sugar
Durhams tube indicates production of gas
Hiss Serum sugars apartDr.T.V.Rao
from sugar ,MD
serum is added.
Sugar Medium
Sugar medium
contain 1% sugar
Durhams tube
indicates production
of gas
Hiss Serum sugars
apart from sugar ,
serum is added.
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Urease Test
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Loffler's serum slope
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Lowenstein Jensen Medium
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Lowenstein-Jensens medium
Mineral salt soln
- 600ml
Malachite green soln - 20ml
(2gm% in D.water)
Beaten egg
- 1000ml
(20-22 eggs)
Mix the above
Distribute in Mc Cartney bottles
Sterilize by Inspissation
Use: To cultivate Mycobacteria
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Transport Medium
Stuarts medium
contain reducing
agents to prevent
oxidation.
Charcoal to
neutralize certain
bacterial inhibitors
to Gonococci,
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Hiss Serum Sugars
Sugar Medium with Serum enrichment
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Anaerobic Medium
Robertsons cooked
meat medium
Thioglyclolate liquid
medium
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Anaerobic Culture Methods
Anaerobic jar
Anaerobic
jar
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Figure 6.5
Sabouraud's Dextrose Agar
Dextrose
Neopeptone
Agar
- 4 gm%
- 1 gm%
- 1.5 gm%
Distilled water - 100 ml
Dissolve the ingredients by heating in a water bath, cool
and adjust pH to 5.4
Autoclave and dispense 20 ml amount in test tubes
Use: For the cultivation
of Fungi
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MD
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Sabouraud's Dextrose agar
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Sterilization of culture media
Media
are
sterilized
in
the
autoclave
at
1210 c for 15 under 15lbs of Pressure
Heat-labile substances like serum & sugar
solutions must be sterilized by free-steam
or filtration
Egg containing media -- LowensteinJensens medium, Loffler's serum slope by
inspissation
Discarded culture plates are to be
sterilized by autoclaving prior to washing
Dr.T.V.Rao MD
Storage of culture media
Prepared media in individual
screw capped bottles can be
stored for weeks at room temp
Poured plates deteriorate
quickly and often contaminated,
hence cold storage is necessary
For smaller labs domestic
refrigerators & for larger labs
insulated cold room(4-5oc)
Deep freeze refrigerators for
preservation of sera, antibiotics
& amino acids (-10 to - 400c)
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Colonies of Bacteria in Pure
Culture
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Complex Medium
Complex media are rich in nutrients, they
contain water soluble extracts of plant or
animal tissue (e.g., enzymatic ally digested
animal proteins such as peptone and
tryptone). Usually a sugar, often glucose is
added to serve as the main carbon and energy
source. The combination of extracts and sugar
creates a medium which is rich in minerals
and organic nutrients, but since the exact
composition is unknown, the medium is called
complex
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58
Blood culture
Liquid Medium
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Muller Hinton Agar for
Antibiotic Testing
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Blood Agar for Antibiotic
Testing
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Programme Created by
Dr.T.V.Rao MD for Medical and
Paramedical students in
Microbiology
Email
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