Bacte ria l

Culture Media
basics
Dr.T.V.Rao MD
Majo r C on tri bution to C ul tu re
Medi a
Ag ar - Ag ar Fr au Hess e’s
contr ib uti on
 Ag ar – Ag ar
 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
 Aga r - Ag ar
 Complex polysaccharide
 Used as solidifying agent for culture media
in Petri plates, slants, and deeps
 Generally not metabolized by microbes
 Liquefies at 98°C
 Solidifies ~42°C

 Dr.T.V.Rao MD’s ‘e’ learning series

 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
 Cu lt ure med ia
 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
 Cu lt ure a nd M ediu m
 Cult ur e is the term given to
mi croo rga ni sms t ha t are cul ti va ted i n t he
lab for th e pu rpose of ident ifyi ng an d
stu dyin g th em.

 Medium is the term gi ve n t o t he

combi na ti on of i ngr edi ent s th at wi ll
supp ort t he g rowt h an d cult iva tion of
mi croo rga ni sms b y p rovi din g all t he
ess enti al nut ri ent s req uir ed f or the
grow th (t ha t i s, mult ipl ica ti on) in ord er
to cul ti va te these microorga nisms in
larg e numb ers to s tu dy them.
 Sp ecif ic 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 pyrimidines
 Need for Cu lture M ed ia
 It is usually essential to obtain a culture by
grwoing 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
 Bas ic req uire men ts of
cu lt ure me dia
 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
Pou rin g t he Cu lt ure P la tes
 Pet ri dis h w it h Med ia
 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
 Cl assi fica tio n o f C ulture
media
 Based on the consistency:

Li qui d -- Peptone water, Nutrient

broth
Semi sol id -- Nutrient agar stabs
Sol id -- Blood agar, Serum agar

 Based on Oxygen requirement:

-- Aerobic medium
-- Anaerobic media
 Aer ob ic M ed ia
 Si mpl e medi a
 Comp lex med ia
May b e Sy nt het ic o r Def in ed
Med ium
 - Enriched media
- Differential media
- Enrichment media
- Selective media
 Semisyntetic Medium
- Sugar media
- Transport media
 Aer ob ic med ia
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

 Liq uid M ed iu m
 Difficulat to identify
all types of organisms
 Suitable for isolation
of bacteria from Blood
culturing and water
analysis
Pep ton e
 Peptone contain partially
digested proteins
 Proteases
 Polypeptides
 Aminoacids
 Inorganic salts
Phosphates
Potassium and
Magnesium
Riboflavin
Meat exract called as Lab
lemco
Nu trien t A ga r
 Contain 2% agar
added to Nutrient
agar commonly used
 Concentration can be
increased to 6% to
prevent swarming
 Can be reduced to
0’5%
Pigm ent p ro du cing
Stap hyl oco cci
 Comp lex med ia
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
 Enric hed Med iu m
 To culture medium
Blood serum or egg
are added to medium
 eg Blood agar
 Chocolate agar
 Egg
Diff eren t typ es o f h emo lysi s
on Bl ood A ga r
 Other Enrichments – Chocolate Agar
 Several organic
materials are added
to the basic
constituents of the
Medium such as
Blood, yeast, yeast
extract etc
Ch oco late a gar
 Enric hmen t M ed iu m
 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
 Sele ctiv e med ia
 Serve the same purpose as Enrichment
media but are solid in consistency
- Wilson & Blair’s medium -
- Lowenstein Jensen’s medium -
 Use: To cultivate Salmonella, Shigella &
Mycobacteria
 Deoxy ch ola te cit rate Ag ar
 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
Indi ca tor Medi um Wi ls on -Bl ai r
medi um
 Indicate by change of
color Sulphite to
sulphide in Wilson-
Blair medium
 S.typhi reduces
sulphite to sulphide in
the presence of
Glucose
Differ enti al M edi um
M ac C on key' s ag ar
 Bringing out different
characters of bacteria
their atypical characters
 Mac Conkey’s medium

Contain peptone, Lactose

Agar, Neutral red and
taurocholate and show
grwoth of Lactose
fermenters as pink
colored colonies
 MacCon key a gar
 MacConkey agar is useful
medium for cultivation of
enterobacteria
 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
La cto se f erm enti ng an d No n
lacto se f ermenti ng
 Carb oh ydrate med ia
 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 Durham’s tubes to detect gas
production and steam for 30 min
 Use: To test the fermenting ability of an
organism
 Carb oh ydrate med ia
 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
 Su gar Med iu m
 Sugars are fermenting substances
 Monosaccharide – peptone, arabinose,xylose
and hexose's, dextrose and mannose
 Disaccharides Sucrose and Lactose
 Polysaccharides – Starch and Inulin
 Alcohols – Glycerol. Sorbital
 Sugar medium contain 1% sugar
 Durham’s tube indicates production of gas
 Hiss Serum sugars apart from sugar , serum is
added.
Su gar Med iu m
 Sugar medium
contain 1% sugar
 Durham’s tube
indicates production
of gas
 Hiss Serum sugars
apart from sugar ,
serum is added.
Ure ase Tes t
Loeff ler’ s s eru m s lop e
Lowen stein J en sen M ediu m
 Tran sport M ediu m
 Stuart’s medium
contain reducing
agents to prevent
oxidation.
 Charcoal to neutralize
certain bacterial
inhibitors to
Gonococci,
 Hiss Serum Sugars
Sugar Medium with Serum enrichment
Anaero bic M edi um

 Rob erts on’s

co oked me at
medi um
 Thiglyclolate liquid
medium
 Anaerob ic C ulture M et hods
Ana ero bi c j ar
 Anaerobic
jar

Figure 6.5
Sabo ur aud' s D extr ose a ga r
co mmo nly used Fu nga l
Is ol atio n M edi um
Sab ou ra ud's Dextros e A ga r
Dextrose - 4 gm%
Neopeptone - 1 gm%
Agar - 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
 Robe rts on s’co oked M ea t
Medium
 Place meat in 1 ounce
bottles to the depth
of 2.5 cms and cover
it with 15 ml of broth
 Autoclave at 1210 c
for 20 min
 After sterilization,
adjust the pH to 7.5

 Use: To cultivate the

anaerobic bacteria
Lowenstein Jensen Medium - cultivation of
Mycobacterium tuberculosis
Lowe nstei n- Jensen ’s m edi um
 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

Sterilizat ion o f c ultu re med ia
 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 –-- Lowenstein-Jensen’s
medium, Loeffler's serum slope by inspissation
 Discarded culture plates are to be sterilized by
autoclaving prior to washing
Colon ies of Ba ct eri a on
Cu lt ure pla tes
Sa lmon ella Sh igella a gar
TCBS med iu m
Blood cu lture – ‘ Liq uid
Medium ’
 Carb oh ydrate med ia
 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
Muller Hin to n Ag ar for
Antibiot ic Tes tin g
An tib iotic Te stin g o n
Blood A ga r M ediu m
 St ora ge of cu ltu re med ia
 Prepared m edia in
ind ivi dual s cr ew cap ped
bottl es can be stor ed for
we eks at room temp
 Pou re d plates deterior ate
qui ckl y and of ten
cont ami nated, hence col d
stor age i s nece ss ary
 For smal ler lab s do me stic
refr ig era tors & for larg er
labs ins ulated col d
room (4-5 o c)
 Deep freeze refrigerators
for preservation of sera,
antibiotics & amino acids
(-10 to - 400c)
Cr eated f or Dr.T.V.Rao MD’s ‘e’
Lea rn ing Pr ogr amme

Dr.T.V.Rao MD
Email
[email protected]