Culture media …

 It is artificially prepared media provide

nutrient materials essential for bacterial
growth and multiplication .

 It my be either solid or liquid.

 Bacteria are grown in the lab for
. several purposes
1. To study their properties .
2. To prepare antigens, toxins, vaccine.
3. To isolate them from pathological materials …
sputum, pus, urine, blood .
4. Isolated bacteria further identified and their
antibiotic sensitivity is determined that help in
line of treatment .
 Culture media …

Culture
media

Selective
Ordinary Enriched and Enrichment Sugar media
differential
 Simple or ordinary media :

1. Fluid media :
 Peptone water.
Structure :1% peptone and 0.5% NaCl.
Sterilized by : autoclave .
Use : used a base for sugar media , indole
production test and nutrient broth .
• Nutrient broth .
Structure : it is a meat extract +1%peptone
. +0.5% NaCl
. Sterilized by : autoclave
. Use : transfer media
It is clear yellowish fluid and become turbid with
. bacterial growth
 . Solid media .2
 Nutrient agar :
Structure :
it is composed of nutrient broth +agar agar (solidifying agent ) .
Sterilized by : autoclave .
Use :
 as a base of different types of media .
 For growth of different types of bacteria. e.g. staph
endopigment ,pseudomonas,proteus .
Preparation :
the agar melt at 100°c and remain liquid till it solidifies at 45°c .
Types :
agar slope ,deep agar and agar plates.
Enriched media ………
Some bacteria are fastidious and their growth
require presence of highly nutritive substance
ex: milk, blood ,serum or egg .
 Blood agar :
Structure : nutrient agar +10% sheep RBCs .
Sterilization :
Blood is already sterile .
N. agar autoclave .
Use :
Some bacteria release haemolysin toxin cause haemolysis of RBCs and
their colonies surrounded by clear zone of haemolysis (β) .
Other bacteria cause greenish discoloration of blood
ex:viridans streptococci and pneumococci .
Preparation :
the sterile blood is added to sterile agar at a temp. of
55°c ,then it poured in plates
Chocolate agar

Structure : It is a heated blood agar .

It prepared like blood agar then the temp. of the
medium raised to 100°c before pouring in the
plates .
During heating red cells are ruptured ,liberate
hematin and glutathione brown color.
Sterilization : as blood agar.
Use : for Neisseria and haemophilus group .
 … loffler's serum
 composition :
3 parts of sterile ox sheep ,horse serum + 1part of
nutrient broth .
 use : for diphtheria bacilli .
 Sterilized by : Inspissation .
…Dorset egg medium
 It composed f 3 parts of fresh beaten egg +1
part of tap water.
 Used for growth of TB.
 Sterilized by Inspissation.
 The media is yellow opaque present as slopes in
screw capped bottles to prevent loss of humidity
during long incubation period (4 – 8 ) weeks.
Milk agar …
 It composed of nutrient agar +10% pasteurized milk .
 Used for demonstration of staph. endopigment .
 Sterilized by:
Nutrient agar by autoclave.
Milk by pasteurization.
 Intended learning
…objectives
 What is the meaning of culture media.
 Reasons for culturing .
 Types of culture media :
1. Selective media .
2. Indicator media .
3. Enrichment media .
 selective media
 These media contain substance
(chemical, dyes or antibiotic ) that inhibit
all types of bacteria except a few types
of bacteria .
 They facilitate the isolation of particular
species from a mixed inoculum.
 Loẅenstien Jensen media

 Use :cultivation of tubercle bacilli .

 Composition :
Beaten egg ,mineral salts and malachite
green “ that inhibit growth of bacteria
other than T.B. “
 Sterilization :
Inspissation at 80°c for 1hr on 3 successive
days .
 Blood tellurite
 Use : for corynebacterium diphtheriae (black to
gray colonies).
 Composition :
Blood agar with 0.03%potassium tellurite as selective
substance.
 Sterilization :as blood agar.
 T.C.B.S.
 Use : for vibrio cholera.
 Composition :
1. Selective substance : thiosulphate
,citrate and bile salts.
2. Test sugar : sucrose .
3. Indicator : thymol and bromothymol blue that
give yellow color in acidic medium.
 Sterilization :by steaming at 100°c for 30
minutes on 3 successive days.
 ……… Other media
: Thayer martin medium
.Used for Neisseria gonorrhoeae

: Desoxycholate citrate
Used for Shigella ,salmonella food
poisoning group and salmonella
. paratyphi B
 ……Differential and indicator media
MacConkey's medium .
 Use: it can differentiate between two major
groups of enteric bacteria.
The lactose fermenters :
which give pink colonies ex.E.coli and klebsiella.
The non lactose fermenters:
which give pale yellow colonies
ex. Salmonella and Shigella.
: It composed of
•Peptone as a nutrient .
•Agar as a solidifying agent .
•Lactose as a test sugar .
•Bile salts as a selective substance that inhibit non
intestinal bacteria.
•Neutral red as indicator that changed pink in
acidic medium due to lactose fermentation.

Sterilization : by steaming .
 Triple sugar iron (T.S.I)
Composition :
 0.1% glucose, 1% sucrose, 1%lactose .
 Phenol red (indicator).
 Ferrous sulphate for detection of H2S
production.
 Non inoculated medium is red in color with
pH=7.4
 The medium is poured in test tubes in the form
of slants with a deep butt.
Sterilization : by steaming .
Bacterial reaction :
 Red alkaline slant and yellow acidic butt
……… Shigella and salmonella.
 Yellow acidic slant and yellow acidic butt
……… E.coli and kelbsiella.
 Red alkaline slant and red alkaline butt
……… pseudomonas aeruginosa.
 Redalkaline slant and black butt. H2s
producing salmonella and some sp. of
proteus .
 .Eosin-methylene blue (EMB)
 Composition :
it contain lactose and a mixture of eosin and
methylene blue.
 Used for: lactose fermenters .
 E.coli show small dark colonies with green
metallic sheen.
 Klebsiella :large pink mucoid colonies.
 Sterilization : by autoclave .
 .Bisthmus sulphite agar
 Composition :
Bisthmus sulphite and brilliant green that is
the selective substance .
 Used for :
Salmonella that produce black to brown
colonies
 sterilization : by steaming .
 Mannitol salt agar
It composed of :
 7.5 % NaCl :it is selective substance for staphylococci that can
grow in NaCl conc. That highly enough to be inhibitory to many
other bacteria .
 Phenol red as indicator.
 Mannitol as test sugar.
It use:
Pathogenic staph. colonies surrounded by yellow
halo indicating mannitol fermentation .
Sterilization : Steaming .
 Indicator media
 Simmon's citrate medium .
 Christensen's urea medium .
 T.S.I
 . Simmon's citrate
 Composition :
Agar, citrate and bromothymol blue (indicator) .
 Use:
To test the ability of the organism to utilize citrate as
a sole source of carbon. e.g.
E.coli not utilize citrate (-ve) of green color.
Klebsiella utilize citrate (+ve) of blue color.
Sterilization : autoclave
 Christensen's urea medium
Composition and sterilization :
 5%urea solution by filtration .
 Urea base by autoclave .
 95% nutrient agar by autoclave.
Use :
For Urease positive organisms e.g.proteus.
Urease enzyme splits urea with release of
ammonia that causes alkalinity and rise of pH
of the surrounded medium that will turn pink .
 … Enrichment media
 They are fluid media contain substance
that stimulate growth of some organisms
and not other .
 Two media commonly used in isolation of
salmonella and Shigella from stool sample
are selenite F broth and tetrathionate
broth .
 ………Sugar media
 Use :
To test the biochemical reaction of the bacteria and so its
identification.
 Consists of :
 Peptone water.
 1% sugar to be tested.
 1% Andrade's indicator is colorless at alkaline pH and
pink/red color at acid pH.
 Durham's tube :
Small inverted tube open at its lower end is put in the bottom
of the tube.
Gas production is denoted by collection of bubble of gas in the
closed upper end of the tube.
 Sterilization : steaming .
 …… Anaerobic cultivation
 Is essential for some bacteria that cannot
grow in presence of oxygen ex.
Clostridia ,actinomyces, lactobacilli and
bacteroids .
 It include anaerobic gaspak system and
McIntosh metal jar.
 Also include anaerobic culture media.
 ..Robertson's cooked meat medium
Composition :
 Cooked meat to which the broth is
added ,the meat contains reducing
substance e.g. haematin and glutathione
that maintain anaerobic condition .
 Used for: clostridium and bacteroides
sp.
 Sterilized : by autoclave
 ……Thioglycolate broth
Composition :
Na thioglycolate and broth .
 Reducing substance :
Na thioglycolate .
Sterilization :autoclave
 Deep agar
 Poured in vertical position ,so it allow
growth of m.o at the bottom of the media
where there is low O2 tension.
 Sterilized by: autoclave .