Transcript 7-8Culture_Media

Culture Media
COMPONENTS
PREPARATION
INOCULATION
Culture Media
 a source of energy and certain environmental
conditions in order to grow and produce bacteria.
 Depending on the type and combination of nutrients,
different categories of media can be made.
Types of Media
 Basal or complex media.
 Enriched media.
 Selective media.
 Deferential media.
Common Ingredients
Water: essential for bacterial growth.
Peptone : from hydrolyzed animal or plant protein
Meat extract: provide amino acids, vitamins, minerals.
Yeast extract: used as bacterial growth stimulant.
Mineral salts: essential for bacterial enzyme activity, Sulfur,
phosphorus, iron, potassium.
Carbohydrates: simple or complex sugars as source of
energy and carbon.
Agar
It’s solidifying agent of the medium. A gelatinous Inert
polysaccharide material, derived from sea-weed or
certain marine algae. It is Resistant to microbial
action. Non toxic to bacteria. Dissolves at 100 c°,
Solidifies when cooled below 45 c°. Use 1-2%
concentration.
Forms of Media
1) Liquid form: called broth. Without agar, used to grow bacteria in a large quantity.
Growth of bacteria
No growth
turbidity
clear
2)solid:
 plate: used mostly to culture organisms
 slant: a tube containing solid media that was left to solidify at an angle. used to keep
the bacteria for long period of time (3 months)
 Deep agar : agar solidified at bottom of tube. used to keep the bacteria for long time.
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Semi-solid agar deep: same as agar deep except it contains less agar (0.5% agar) to
allow motility of organisms.
 Slant Agar
 Plate Agar
 Deep Agar
Media preparation
Equipment:
 Media powder.
 Water (100 ml).
 Balance.
 Flask ( larger than the
size of media volume).
 Weighing plate.
 Weighing spatula
 Cylinder.
 Bacti-cinerator.
 Autovalve.
 Sterile empty Petri dishes.
 Autoclave tape.
 Aluminum foil.
Procedure
 Measure out the required volume of the media,
e.g. 40 gm of media for 1000 ml of water, let’s say u want to
prepare 100 ml of media
40
1000
!!
100
= 4 gm of media for 100 ml of water.
 Put the media powder in the flask.
 Add the water onto the media.
 Mix well.
 Cover the flask with aluminum foil.
 Stick the autoclave tape on the flask and use it for
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labeling.
Sterilize the media in autoclave 15-20min at 12oC°.
Leave to cool at room temperature.
Pour the media in petri dish.
Leave to solidified at RT.
Inoculation
 It is streaking bacteria on agar plate, allow the bacteria to
grow to produce isolated colonies, and pure culture.
Pure culture: culture containing a single species of organism.
Mixed culture: culture containing more than one species of
organism.
Contamination culture: a bacterial culture that has acquired
unwanted organisms.
 In order to obtain well-isolated colonies, the quadrant
streak technique should be used.
Quadrant streak technique
Equipment:
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Bacti-cinerator.
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Loop.
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Subculture media.
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Agar media plane.

Marker.
PROCEDURE
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Sterile the loop by bacti-cineratoe until it is red then allow to
cool.
Take a loopful of bacteria from the subculture media
Immediately streak the inoculating loop VERY gently over a
quarter of the plate around 4-5 lines (quadrant 1).
Sterile the loop then allow to cool.
Go back to the edge of the area 1, extend the streaks into the
second quarter of the plate (quadrant 2).
Sterile the loop then allow to cool.
Go back to the edge of the area 2, extend the streaks into the
third quarter of the plate (quadrant 3).
 Sterile the loop then allow to cool.
 Go back to the edge of the area 3, extend the streaks into the
forth quarter of the plate in zig zag lines (quadrant 4).
 let the bacteria to grow at 37 C° for 24 hr in the incubator.
Types of media
BASAL OR COMPLEX MEDIA.
ENRICHED MEDIA.
SELECTIVE MEDIA.
DEFERENTIAL MEDIA.
Basal or complex media: Nutrient Agar(NA)
It contains nutrients that
allows non fastidious or
Non pathogenic organisms
To grow.
Notice the shape, margin,
elevation, color, size,
Smell of organism.
Notice pigment production by organism
Enriched Media: Blood Agar (BA)/chocolate agar(Choc)
It contains simple nutrients
and additional requirements
such as blood, serum to
allow fastidious or
pathogenic organisms to
grow.
Selective Media: Mac Conkey agar(MAC)
It contains inhibiting
agents that inhibit some
organisms and allows
others to grow.
Inhibiting agents :
Bile salts, crystal violet.
It inhibits gram positive
organisms, allows growth of
gram negative organisms.
Differential Media
Cysteine Lactose Electrolyte Deficient Agar (CLED)
It contains a sugar and Indicator
if organism ferments sugar an acid is
produced, this will change the color of
indicator.
Indicator: is Bromothymol blue.
Sugar is lactose.
If organism ferments lactose it will give
yellow color, if it does not ferment
lactose no changes occurs, colorless.
Selective and Differential Media: MAC
Sugar: is lactose.
Indicator: is neutral red.
If the organism ferment lactose,
It will give pink color =LF.
If organism does not ferment
Lactose, no change in color ,
Colorless= NLF
Selective and Differential
(EMB(Eosin Methylene Blue
As Mac Conkey, inhibits G(+ve)
organisms, allows growth of
G(-ve) organisms.
Indicator: is Methylene Blue
And Eosin.
Lactose fermentor organsim
Appears dark purple while
non lactose fermentor appears
colorless.
E.coli produces green
Metallic sheen( LF)
Selective and deferential: Mannitol Salt Agar( MSA)
 Selective: as it contains 7.5%
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salt
Only organisms that can
tolerate high salt conc. can
grow
Differential :as it contains
mannitol sugar and phenol red
indicator .
The organism that ferments
mannitol produces acid thus
color changes to yellow
If organism does not ferment
mannitol no change in color
Differential Media
Blood Agar
- Types of hemolysis:
α Hemolysis
β Hemolysis
γ Hemolysis
Types of Hemolysis
α hemolysis
Incomplete hemolysis
greenish color around
colonies
Types of hemolysis
β hemolysis
Complete hemolysis
Clear area around
colonies.
Swarming of Proteus on BA
Swarming appear as
spreading rose on BA
plate.