Culture Media (Types, Preparation &
Sterilization)
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Microorganisms (MOs) are ubiquitous.
Culture: growth in media containing living
MOs.
◦ Pure culture Vs Mixed culture.
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Culture media: solution which contains
nutrients needed for bacterial growth.
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Water.
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Source of C.
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Source of N.
◦ To dissolve materials to be transported across cytoplasmic membrane.
◦ Required for the construction of all organic molecules.
◦ Usually glucose.
◦ Examples:
 Proteins: broken down into amino acids.
 Beef extract: broken down into nucleic acids.
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Buffer system.
◦ Most bacteria grow at pH 7.0.
◦ This is achieved by using a buffer system;
 Carbonate buffer.
 Phosphate buffer.
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Source of minerals.
◦ Required in small amounts (Iron, Sulfur, phosphorus… etc)
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Media can be classified according to any of
the following criteria:
◦ Composition.
◦ Content / percent of agar.
◦ Purpose.
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Agar:
◦ The usual solidifying agent for media.
◦ A hydrocolloid derived from red algae.
◦ Properties:
 Melting point: 100 degrees.
 Remains liquid until cooled to 40 degrees.
 Cannot be metabolized by most bacteria.
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Culture media can be classified into two
categories according to their composition;
◦ Chemically-defined (Synthetic).
 The exact chemical composition is known.
 Prepared from biochemicals off the shelf.
 Example: Minimal Salt Medium.
◦ Complex (Undefined).
 The exact composition is not known.
 Usually contain complex materials of biological origin, such
as;
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Blood.
Milk.
Yeast extract.
Beef extract.
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According to percent of agar:
◦ Broth media (liquid).
 No agar is added.
 Example: Trypticase soy broth media.
◦ Semisolid media.
 <1% agar.
 Example: Motility media.
◦ Solid media.
 1.5-1.8% agar.
 Example: Trypticase soy agar.
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Solid media are divided into three forms:
◦ Slanted agar position.
◦ Deep agar position.
◦ Agar plate media.
o Provide large surface area.
o Used for isolation and subculture purposes.
o Always incubated in inverted position to avoid
condensation that forms on the surface of petridishes.
water
Slant agar:
-Used for maintenance and
preservation of pure cultures for
subculture purposes.
Deep agar:
-Used for storage.
-Also used for studying the
gaseous requirements for MOs.
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According to purpose:
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All purpose medium (simple).
Enriched medium.
Differential medium.
Selective medium.
Selective-Differential medium.
Enrichment-Differential medium.
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These media support growth of most
bacteria.
They don’t have inhibitors or pH indicators.
Examples:
◦ Trypticase Soy broth.
◦ Nutrient broth.
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These media contain:
◦ The base growth medium.
◦ Special supplements.
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Examples:
◦ Blood Agar (also differential).
◦ Chocolate Agar.
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More than one type of MOs grow on it.
Separation is based on color and other
characteristic differences.
Example:
◦ Blood Agar.
 It separates bacteria according to the type of
hemolysis. (Next Slide)
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Some bacteria have hemolytic substances
which breakdown red blood cells (Hemolysin).
Types of hemolysis:
◦ Alpha (partial) hemolysis.
◦ Beta (complete) hemolysis.
◦ Gamma or no hemolysis.
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Alpha hemolysis:
 Greenish zone surrounding colonies.
 These bacteria have hydrogen peroxide which oxidizes
Hb into MetHb.
 Examples:
 Streptococcus pneumoniae.
 Streptococcus viridans.
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Beta hemolysis:
 Yellow zone surrounding colonies.
 These bacteria contain Streptolysin (an exotoxin).
 Streptolysin O:
 Oxygen sensitive, results in β-hemolysis under the surface.
 Streptolysin S:
 Oxygen stable, results in β-hemolysis on the surface.
 Example:
 Streptococcus pyogenes / GAS (Group A Streptococci).
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They allow the growth of one or more
species.
They also suppress the growth of other
species, by one of the following:
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Incorporating dyes.
Antibiotics.
Adjusting pH.
Bile salts.
Example:
◦ Salmonella-Shigella agar (S-S)
 This medium contains Bile salts and brilliant green dye
as selective agents.
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Containing inhibitors (Selective) and pH
indicators (Differential).
Examples:
◦ MacConkey agar.
◦ Mannitol Salt agar.
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Selective;
◦ It contains Bile salts and Crystal violet.
◦ Prevent the growth of Gram +ve bacteria.
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Differential;
◦ Lactose fermenter Vs Non-lactose fermenter.
◦ It contains lactose & Neutral red (a dye).
◦ If bacteria ferment lactose, the medium will acidify
and the dye turns red.
◦ If bacteria don’t ferment lactose, no color change
occurs.
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Examples of Lactose fermenters:
◦ Escherichia coli, Enterobacter and Klebsiella
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Examples of Non-Lactose fermenters:
◦ Salmonella , Proteus species , Yersinia ,
Pseudomonas aeruginosa and Shigella
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Is a selective differential medium, because it
contains:
◦ High concentration of salt;
 Only Staphylococcal species will tolerate.
◦ Mannitol;
 S. aureus, unlike other staphylococcal species will
ferment mannitol and change the pH.
◦ A pH indicator dye;
 A yellow halo will surround S. aureus colonies due to
changes in pH.