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 .
peptone+0.5 %NaCl % 1 composition

colourless colour

.Autoclave at 121 for 20-30 min Sterilization

Base for other media-1 Use

Alkaline peptone water(pH=9)-2

For isolation of Vibrio cholerae
.indole production test-3
• 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
peptone+0.5 %NaCl+meat % 1 composition
extract
Yellowish and become turbid when colour
.bacteria grow on it

.Autoclave at 121 for 20-30 min Sterilization

Transfer media &increase No. of-1 Use
m.o when sample small
 . 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.
By plating out to obtain separate colonies from
.associated bacteria
.The media is incubated for 24-48 hrs at suitable temp
c 35-37

The growth of the organism allows the study of the

:following characters
.size of the colonies (small, large) -1
.contour of the colonies (flat, convex) -2
.surface of the colonies (smooth, rough) -3
.consistency of the colonies (mucoid, granular) -4
optical characters of the colonies (opaque, -5
.transparent)
.odour (protus has offensive odour) -6
effect of growth on the media e.g. hemolysis on -7
blood agar
Nutrient broth+Agar Agar composition
.as solidifying agent

Yellowish colour
.semitransparent
Autoclave at 121 Sterilization
.for 20-30 min
Uses
: Nutrient agar-1
a-Staph. Endopigments
Staph albus
whitewhite
Staph citrus lemon
yellow
Staph aureusgolden
yellow
-b 

B-greenish exopigment 

of Pseudomonas
”c-Swarming of proteus”Successive waves of growth
because it is highly motile
D)Anthracoid:show opaque,rough,granular with irregular 
fimbriate edge(medusa head appearance)
Nutrient agar slope -2
Enriched media ………

Some bacteria are fastidious and

their growth require presence of
highly nutritive substance ex: milk,
blood ,serum or egg .
Blood agar)1 Dorset egg)4

chocolate agar )2

Milk agar)3 loffler’s serum )5

 :Blood agar-1

Nutrient agar +10% blood composition

Opaque red colour
.Agar by Autoclave Sterilization
.Blood is already sterile
.highly nutritive medium -1 Use
.to study the haemolytic activity -2
β-haemolysis…complete zone of
haemolysis due to production of
haemolysin which cause haemolysis
,,,to the RBCs.ex Staph aureus
Strept pyogenes.,,,Bacillus
.anthracoids
Alpha-haemolysis…(green
discoloration) ex:viridans
streptococci,,,,due to production of
H2O2 which convert hamoglobin to
met-hb(green in colour)

ɣ-haemolysis or no haemolysis
ex:strpt fecalis
Blood agar :haemolysis (3 types)
A-beta haemolysis “complete haemolysis” with clear zone
around colonies due to production of haemolysin ex. Staph
aureus,Strept pyogenes, Anthracoids
Blood agar showing B-haemolysis
chocolate agar-2
Nutrient agar +10% blood composition
Opaque brown colour
.Agar by Autoclave Sterilization
.Blood is already sterile
.highly nutritive medium -1 Use
for isolation of -2
Pneumococci….greish whie colonies
Chocolate agar: small greyish white colonies
parts of sterile serum + 1 part 3 composition
.of glucose broth

.Opaque white slope colour

Inspissation at 80 c for 2 h on 3 Sterilization
.suuccessive days

.FOR C.diphtheriae-1 Use

 :Milk agar-4
Nutrient agar +10% sterile milk composition

Opaque white colour

.Agar by Autoclave Sterilization
.Milk by pasteurization

for demonstration of -1 Use

.endopigments of staphylococci
Nutrient agar (simple media) or milk agar (Enriched
:media)
.Staph aureus  golden yellow endopigments
Staph citrus  lemon yellow endopigments
Staph albus  white endopigments
 :Dorset egg-5
part of tap water + 3 parts of 1 composition
.fresh beaten egg

.Opaque yellow colour

.inspissation Sterilization

.for the growth of T.B bacilli Use

 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 .
Beaten egg+ mineral salts + Malachite green composition
..as selective substance

Opaque green colour

inspissation Sterilization

Selective for T.B Uses

.Non inoculated
 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.
Blood agar + 0.03 % potassium tellurite as composition
selective substance for Corynebacterim
diphtheriae

.Red colour
.Autoclave for nutrient Sterilization
.Blood,potassium telluritete is sterile

Selective for c. diphtheriae that reduce tellurite Uses

to tellurime leading to black colonies due to
.reduction of tellurite to tellurium
thiosulphate+citrate+bile salt+sucrose* composition
Bile salt is the selective substance*
Bromothymol blue & thymol as indicator to*
.give yellow colour at acidic PH
green..semitransparent colour
Tyndalization Sterilization

Selective for Vibrio cholerae Uses

Indicator media
(differential)
These media contain substances that
change colour visibly as aresult of certain
.bacterial growth
Nutrient agar+Lactose as test sugar+Neutral red as . Comp
indicator+peptone water
.Bile salt as Selective substance.to inhibit non intestinal bacteria

.orange transparent colour

Tyndalization .Steriliz

.Selective for Enterobacteriaceae Uses

Lactose fermenter (E.coli,Klebsiella,citrobacter,enterobacter)
…..acidic pH….pink colonies
Non Lactose fermenter (Salmonella,,shigella;;proteus).. pale
yellow colonies
 Enterobacteriaceae
MacConkey agar
Lactose fermenters (Rose pink colonies)
E.coli, Klebsiella, Citrobacter, Enterobacter
Lactose non fermenters (pale yellow) Salmonella, shigella,
proteus, yersinia
Nutrient agar+ Lactose as test sugar+ mixture of . Comp
.Eosine and methylene blue

‫ لون الكركديه‬Dark pink colour

Tyndalization .Steriliz

E.coli….small dark dry colonies with green metallic Uses

.sheen
.Klebsiella….Large mucoid moist colonies
 E.M.B medium

E.M.B
E.coli: small dry E.M.B
rough Klebsiella: large
Colonies with green mucoid pink
metallic sheen colonies
Nutrient agar+ Brilliant geen and Bismuth . Comp
.sulphite as selective substance

.Slightly green,opaque colour

autoclave .Steriliz

Salmonella……Black to brown colonies due to Uses

reduction of sulphite to sulphide
Nutrient agar+Mannitol as test sugar+phenol composition
red as indicator+7.5 % Na Cl as selective
.substance

.transparent pink colour

Tyndalization Sterilization
Selective for staphylococci that can grow on Uses
high salt conc. Differentiate pathogenic
staph(staph aureus) from non pathogenic
Staph aureus ferment mannitol so acidic ph
will change the colour to yellow colonies
surrounded by a yellow zoned
 Triple sugar iron
Nutrient agar+beef extract, yeast extract, Comp
phenol red indicator, ferrous sulphate for .
detection of H2S production+ 0.1 % glucose,
1%lactose, 1% sucrose
Red slobe colour
Tyndalization Steriliz
.
Uses
Bacteria that ferment glucose only will release small amount of
acid and give yellow butt (acidic) and red slant (alkaline) ex:
salmonella and shigella

Bacteria that ferment lactose and or sucrose will release large

amount of acid and give yellow slant (acidic) and yellow
butt(acidic) ex: E. coli, Klebsiella. Gas may appear in the
.bottom

Bacteria with no carbohydrate fermenting activity wll give red

slant and red butt ex: pseudomonas
Bacteria that ferment glucose only with H2S production give red
.slant(alkaline) and black butt
.ex: proteus , salmonella para typhi B
TSI
 Simmon’s citrate medium
,Nutrient agar+ citrate, methylene blue indicator . Comp

green colour

autoclaving .Steriliz

Non inoculated….green slope Uses

Citrate +ve….blue (klebsiella)
.Citrate –ve…..green (E.coli)
 Christensen’s urea medium
Nutrient agar +urea base, urea solution, phenol . Comp
red indicator
Pale yellow colour

Autoclaving for urea base, Filteration for urea .Steriliz

.solution
urease +ve….pink (klebsiella, proteus) Uses
Utease enzyme split urea to ammonia so
increase ph of surrounding media & change of
colour of the indicator to pink
 Entichement media
These are selective+enriched media
E.g. selenite broth & tetrathionate broth
They inhibit all coliform bacilli except typhoid
& paratyphoid groups so allow their
.isolation from stool samples
 Sugar media & biochemical reaction
Peptone water+1% of the test sugar as comp
glucose, lactose,..+1% of Andrades indicator(it
is colourless in alkaline ph & turn red in acidic
ph

Tyndalization sterilzation

Small inverted tube (Durham's tube) is put in

the test tube containing the media to test for
gas production

Identfy bacteria by their biochemical uses

activity(type of sugar fermented with acid only
or acid & gas
 Anaerobic cultivation

It is essential for some bacteria which can not grow

.in presence of oxygen as clostridia
:This method of cultivation can be achieved by
:A)GasPak system
:b)addition of reducing substances to culture media
:This to adsorb oxygen by
Robertson’s cooked meat(contain reducing-1
substances e.g.haematin & glutathione,the media
sterilized by autoclave)
Thioglycollate broth(contain sodium-2
thioglycollate as reducing substance and
stetilized by autoclave)
.Deep agar(by cultivation deeply) -3
What should be known in every
?media
Name of the)1
.medium,type,structure
.sterilization )2
.inoculated organism )3
Disease caused by this)4
.organism
Other media for isolation )5
.Name of the medium)1

.sterilization )2

.inoculated organism )3

.Disease caused by this organism)4

test for confirmation )5