CULTURE MEDIA

Dr. Somya Sinha

Department of Microbiology
Dr. S.N. Medical college
CONTENTS
 A short History & brief introduction
 Basic CONSTITUENTS / COMMON INGRIDIENTS of
culture media
 Adjustment of pH & Sterilisation od prepared media
 Classification &/or varieties of culture media
 Storage of culture media
 Quality control
A short history
 The original media used
by Louis Pasteur were
liquids such as urine or
meat broth.
A short history
 The earliest solid medium-
cooked cut potato used by
Robert Koch
 Later on, introduction of
gelatin to solidify but
wasn’t satisfactory
 The use of agar – by Frau H
esse, the wife of one of the
investigators in Koch’s
lab.,who had seen her
mother using agar to make
jellies!
Basic requirements of culture
media
 Nutrients - Energy source - Carbon source -
Nitrogen source
 Mineral salts – Sulphates, 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
Basic CONSTITUENTS / COMMON
INGREDIENTS of culture media
 Water
 Electrolyte
 Peptone
 Meat Extract
 Blood Or Serum
 Agar
Temperature & Adjustment of pH
 Cultivation of most medically important bacteria
require temperature of 35-37 degree celcius.
 Enrichment by incubation at other temperatures. [ C.
jejuni at 42 degree celcius & Listeria monocytogenes at
temp. between 20-40 degree celcius ]
 Na, Mg phosphate buffers, citrate
 pH should be measured after cooling
Sterilisation of prepared media
 Media are sterilized in the autoclave at 121 0 c for
15’ under 15 lbs 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
Classification &/or varieties of
culture media
 Based on the consistency:
-Liquid -- Peptone water, Nutrient broth
- Semisolid -- Nutrient agar stabs
- Solid -- Blood agar.
 Based on Oxygen requirement:
- Aerobic media
- Anaerobic media- Robertson cooked meat medium &
thioglycollate broth
 Based on nutritional components
- Simple-peptone water
- Complex-blood agar
- Synthetic-Davis n Mingioli

 Based on functional use

- Basal-Nutrient broth, Peptone water
- Enriched-Blood agar, Chocolate agar
- Enrichment-Tetrathionate broth, Selenite F broth
- Selective-DCA, BSA, LJ media
- Transport-VR media, Stuart, Cary Blair
- Anaerobic-Robertson’s cooked meat media, TG,
- Differential-MacConkey, XLD,
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

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 organism

Sheep Blood agar plates
Chocolate agar
•Ingredients are essentially same as blood agar
•Melt the sterile nutrient agar by steaming and cool to about
750 c
•Add blood to the molten nutrient agar and allow to remain
at 750c after gently mixing till it is chocolate brown in
colour
•Pour in Petri dishes/ test tubes for slopes as desired

Use:
To culture H. influenzae & N. gonorrhoeae
Loeffler’s serum slope
 Loeffler’s serum slope
 N. broth 100ml+Serum300ml+1 gm% Glucose
•Dissolve glucose in NB & sterilize by autoclaving
•Add serum aseptically
•Distribute in small screw-capped bottles
•Sterilize by inspissation, at 82oc for 1 hr x 3 days

•Use: To cultivate Corynebacterium diphtheria

DIFFERENTIAL MEDIA- MACCONKEY’S MEDIUM
•Contains lactose as substrate and Neutral red as an
indicator
•Bacteria fermenting lactose produce acid and this
changes the colour of the indicator and the colonies
turn pink
Use: To differentiate lactose fermenters (E. coli,
Klebsiella) from non-lactose fermenters
(Salmonella,Shigella) & to cultivate Enterobacteria
 Enrichment media
 A liquid culture medium to which certain substances
are added which enhance the growth of the pathogenic
organisms and suppress the unwanted bacteria
 TETRATHIONATE BROTH,
Selenite-F broth – Salmonella typhi
Alkaline-peptone water Ph- 9.0 –V. cholerae
Use: To prevent the
non pathogenic
or commensal bacteria from overgrowing the pathogenic
bacteria
SELECTIVE MEDIA
•Serve the same purpose as Enrichment media but are
solid in consistency
 Four commonly used Selective media & their uses–
DCA ,BSA, Wilson &Blair’s medium, Lowenstein
Jensen’s medium
 Use: To cultivate Salmonella, Shigella & Mycobacteria
Lowenstein-Jensen’s medium
•Mineral salt soln- 600ml Malachite green soln - 20ml
(2gm% in D.water)Beaten egg - 1000ml (20-22 eggs)
•Mix & Distribute in Mc Cartney bottles
 Sterilize by Inspissation
 Use: To cultivate Mycobacteria
Deoxycholate Citrate Agar[DCA]
 Nutrient agar, sodium deoxycholate, sodium citrate,
lactose, neutral red.
 For culture of salmonella & shigella
Bile Salt Agar[ BSA]
 Nutrient agar,
Sodium taurocholate (0.5%), pH 8.2
- For culture of Vibrio cholerae
Robertson’s Cooked Meat medi
 Fresh bullock heart- 500 gms
 Distilled - 500 ml
 NaoH 1 mol/lit - 1.5ml
 Mince the heart, place in alkaline boiling water and
simmer for 20 min to neutralize the Lactic acid
 Drain off the liquid through a muslin filter, press the
minced meat in a cloth while hot and dry by spreading
it on a cloth
Transport media
•Are used in case of delicate organisms whenever there is
a delay in the transportation of the specimen to the lab
•To Maintain viability of them & to prevent the
multiplication of non-pathogenic bacteria
 Stuart’s medium- Gonococci
 Cary-Blair’s medium- V. cholerae
 V-R medium- V. cholerae
MULLER HINTON AGAR
- Originally used for isolation of pathogenic Neisseria
spp.
- Now used with high potency antibiotic disks for
detection of antibiotic sensitivity pattern by Kirby-
Bauer technique.
XLD MEDIUM
 Xylose Lysine Deoxycholate Agar
 Red colony with black centre : most strains of
salmonella ,Edwarsiella
 Red colony without black centre : shigella, providentia,
pseudomonas, H2S negative strains of salmonella
 Yellow colony : klebsiella, proteus & most strains of E.
coli.
Media preparation room
 Large autoclaves and a hot air oven, Two portable
autoclaves, Steam sterilizer, Inspissator, Distilled water
plant, Physical balance, Electronic balance
 Storage space at room temp for glassware and reagents
 Walk-in-cooler(40c to 80c)/refrigerators to store the
prepared media and heat liable reagents
 A small incubator for sterility check
 Seitz filter
 Stock of standard cultures to check performance of
prepared media
Media for preservation & Storage of cultures

 Cooked meat broth

 Nutrient agar slopes
 Semi solid nutrient agar stabs
 Blood agar or heated Blood agar slopes in small screw
capped bottles.
 Egg saline medium
Storage of culture media
 Prepared media in individual screw capped bottles can
be stored for weeks at room temp
 Poured plates deteriorate quickly and often
contaminated, hence cold storage is necessary
 For smaller labs domestic refrigerators & for larger labs
insulated cold room(4-5oc)
 Plates of agar media can be held for short periods not
exceeding 7-10 days
Quality control
 Surveillance procedures of equipments
 Purchase of quality reagents &ingredients from reputed
manufacturers
 Maintenance of ledger/register for - Issuing the reagents &
chemicals - Noting down the performance
 Ledger must be checked periodically
 Must be supervised by an experienced staff
 Dehydrated media & reagents must be labeled Label –
“Batch No” & “Date of expiry”
Quality Control (contd)
 Performance of plated media
 Samples of plates from each batch are selected for
performance testing & are inoculated with appropriate
standard stock cultures
 For each type of medium, at least 2 or 3 microorganisms
having growth characteristics with +ve & -ve results to be
used
 For biochemical tests, always test reagents with controls
 Media are released for use into the diagnostic laboratory
only if the results indicate satisfactory performance
THANK YOU