 Artificial Culture Medium constitutes the food

materials for the cultivation of

microorganisms, which simulates the In-
Vivo composition & conditions of tissues &
body fluids for the successful growth of
bacteria, fungi & parasites In-Vitro.

 Liquid medium – diffuse growth

 Solid medium – discrete colonies.
 Colony – macroscopically visible collection of

millions of bacteria originating from a single

bacterial cell.
 Diagnosis of infectious diseases
 Culturing bacteria - the initial step in

studying its morphology and its identification.

 Determination of Antibiotic sensitivity pattern
for optimal antibiotic selection.
 Growth for research ,vaccine preparation,
genetic studies, antigens for developing
serological assays etc.
 Culturing bacteria provides a reliable way
estimating their numbers (viable count).
 Culturing on solid media is another convenient
way of separating bacteria in mixtures.
 The original media
used by Louis Pasteur
were liquids such as
urine or meat broth.
 The earliest solid
medium was cooked
cut potato used by
Robert Koch.
 Later he introduced

gelatin, but it was not

satisfactory as gelatin
is liquified at 24ºC
and also by many
proteolytic bacteria.
Before the use of agar, attempts were made to use
gelatin as solidifying agent. Gelatin had some
inherent problems; it existed as liquid at normal
incubating temperatures (35-37oC) and was
digested by certain

Agar
 Used for preparing solid medium
 Obtained from seaweeds.
 No nutritive value, free from growth
promoting or growth retarding substances
 It is not hydrolysed by most bacteria
 However, it may be a source of calcium &
organic ions.
 Most commonly, it is used at concentration of
1-3% to make a solid agar medium.
 Not affected by the growth of the bacteria.
 Melts at 98oC & sets at 42oC
 The word "agar" comes from the Malay word agar agar (meaning
jelly). It is also known as kanten, China grass, or Japanese isinglass.
 Agar is chiefly used as an ingredient in desserts throughout Japan.
 It is an unbranched polysaccharide obtained from the cell
membranes of some species of red algae such as the genera Gelidium
and Gracilaria, or seaweed (Sphaerococcus euchema).
 Commercially it is derived primarily from Gelidium amansii.
 Agar is composed of two long-chain polysaccharides (70% agarose
and 30% agarapectin).
 New Zealand agar has more gelling capacity than the Japanese agar.
 Agar is available as fibres (shreds) or as powders.
 Must give a satisfactory growth from single
inoculum.
 Should give rapid growth.
 Should support growth of most pathogens easily.
 Should enable to demonstrate all characteristics
required.
 Satisfactory pH about 7.2
 Surveillance procedures of equipments
 Quality reagents & ingredients from reputed
manufacturers
 Maintenance of records for
- Issuing the reagents & chemicals
- Noting down the performance
 Must be supervised by an experienced staff
 Dehydrated media & reagents must be labeled
Label – “Batch No” & “Date of expiry”
 Media should be prepared as per S.O.P, poured in
laminar flow, under sterile conditions, flame is passed
over the media to avoid air bubbles & allowed to cool.


 Sterility check:
Prepared culture media plates must be kept in
incubator for 24-48 hrs.at 37oc. If contamined,
discard.
 pH check
 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
 Quality must be assessed as a routine in the lab
i.e., In-house media, dehydrated media & ready-
to-use media operate their own QC system

 Ensures prompt detection of poor performance

that may be attributable to
- Deterioration in the storage of Stock
- Lab. errors in weighing, measuring & pH
control
- Variation in the quality of the local water
- Lab’s sterilizing procedures
 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
1) Water
2) Agar
3) Peptone
4) Casein hydrolysate
5) Meat extract
6) Yeast extract
7) Malt extract
8) Blood
9) Serum
 Tap water – often suitable if mineral content is
low.
 Glass distilled or demineralised water
 Copper distilled water cannot be used
 Solid medium is made
by adding Agar
 Prepared from variety
of seaweeds -
Geledium, Pterocladia,
Eucheuma and others.
 The product is dried,
clarified and supplied
as powder or dried
strands.
 Peptone is a byproduct of
protein (plant or animal) digestion.
 Proteins are often obtained from heart muscle,
casein, fibrin or soya flour and is digested
using proteolytic enzymes such as pepsin, trypsin
or papain.
 The final product contains peptones, proteoses
and amino acids besides a variety of inorganic
salts including phosphates, potassium and
magnesium.
 Casein hydrolysate is obtained from hydrolysis of
milk protein casein using HCl or trypsin
Requirement of a good peptone include:
 Its ability to support growth of moderately

exacting bacteria from small inocula.

 Absence of fermentable carbohydrates.
 Low content of contaminating bacteria.
 Very low content of copper.

Peptones for special purposes

• Neopeptone
• Proteose peptone
• Mycological peptone
 As substitute for an infusion of fresh meat.
 The product of hot water extraction of lean
beef are concentrated by evaporation.
 A typical commercial preparation (Lab-
Lemco, Oxoid Unipath) contains a wide
variety of water soluble compounds –
 - Protein degradation products, eg. Gelatin ,
albumoses, peptones, proteoses &
aminoacids. Other nitrogen compounds –
creatine, creatinine, carnosine, anserine,
purines and glutathione
 Mineral salts
 Accessory growth factors.
 Some carbohydrates.
 Washed cells of brewer’s or baker’s yeast.
 Contains – amino acids, growth factors (vit B)

& inorganic salts

 Carbohydrate – glycogen, trehalose &

pentoses
 Substitute meat extract in culture media
 By extracting the soluble materials from sprouted
barley in water at about 55ºC, to yield a brown
viscous material.
 It consist of maltose, starch, dextrin and glucose,
protein and protein breakdown products.
 Wide range of mineral salts, growth factors
( thiamin, nicotinic acid, riboflavin, biotin,
pantothenic acid, pyridoxin, folic acid & inositol.
 For use in mycological media it must not contain
added sugar or cod liver oil.
 Blood may be collected from horse, rabbit,
sheep oxen or from man.
 Collect with aseptic precautions. Sterile blood
is immediately distributed in in 5 or 10 ml in
sterile screw capped bottles & stored in
refrigerators.
 Do not freeze.
 Non-coagulating by defibrination or by the
addition of citrate or oxalate.
 Defibrination is recommended because it
involves no additive that might alter the
nutritive properties of the medium.
 Difibrination – a bottle containing glass beads
is half filled with blood, stoppered at once
and shaken continously for 5 min.
 Oxalated blood – by bleeding the animals into

bottles containing 10 ml of a 10% solution of

neutral potassium oxalate/litre of blood.
 Citrated blood – blood is collected and gently

but thoroughly shaken in a bottle containing

sodium citrate 60mg/10 ml of blood
 Serum can be filter-sterilised
(membrane/Seitz filter) .
 Can also be prepared from unsterile

defibrinated or oxalated blood.

 May be stored in refrigerator at 3-5ºC until

use.
 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
 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)
 Deep freeze refrigerators for preservation of sera,
antibiotics & amino acids (-10 to - 400c)
 Classification
I. Based on their consistency
a) Liquid medium
b) Solid medium
c) semi solid medium
 Based on Oxygen requirement:
-- Aerobic medium
-- Anaerobic media
II. Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
Solid media – contains 2% agar
 Colony morphology, pigmentation, hemolysis can
be appreciated.
 Eg: Nutrient agar, Blood agar

Liquid media –no agar,Bacteria grow fast in liquid

media.
 For inoculum preparation, Blood culture, for the
isolation of pathogens from a mixture.
 For obtaining bacterial growth from blood or water
when large volumes have to be tested.
 For preparing bulk culture of antigens and
vaccines.
 Eg: Nutrient broth
Semi solid medium – 0.5% agar.
 Eg: Motility medium
 Simplemedia
 Complex media

- Enriched media
- Differential media
- Enrichment media
- Selective media
- Sugar media
- Transport media
 Also called basal media.
 Consist of meat extract, peptone, sodium
chloride and water.
Examples- nutrient broth
nutrient agar
peptone water
Nutrient broth
Three types of nutrient broths-
 meat infusion broth
 meat extract broth
 digest broth
Peptone water : is prepared by adding 1 gm
peptone , 0.5 gm sodium chloride to distilled
water.
 Used chiefly as the basis for carbohydrate

fermentation media.
 pH-7.4-7.5
 Autoclaved at 121ºC for15 mins.
1.Meat infusion broth - is aqueous extract of
meat to which peptone is added.

Composition -
 lean meat, ox heart or beef, water, peptone

and sodium chloride.

 Meat should be fresh.
 pH-7.5
 Method of sterlisation- autoclaving at 121ºC

for 15 mins.
2.Meat extract broth - is a mixture of meat
extract with commercial peptone.
Composition –
 peptone, meat extract, sodium chloride and

water
 pH-7.5
 Autoclave 121ºc for 15 minutes.
3.Digest broth - aqueous extract of lean meat
that has been digested with a proteolytic
enzyme so that additional peptone need not
be included.
- Hartleys digest broth
- Horse flesh digest broth.
 Nutrient agar-
- Nutrient broth
solidified by addition
of agar,
- Japanese agar-2%,
-NewZealand 1.2%
 Concentration can be

increased to 6% to
prevent swarming
 Thesemedia have added ingredients for
special purposes or for bringing out special
characteristics or providing special nutrients
required for the bacteria under study
 In this media
substances such as
blood, serum or egg
are added to the basal
medium.
 They are used to grow
bacteria which are
more exacting in their
nutritional needs.
EXAMPLES
Blood agar: widely
used in medical
bacteriology.
Blood agar Chocolate agar
Medium is prepared by adding
sterile blood to sterile nutrient
agar that has been melted and c
ooled to 50ºC.
Suitable for Haemophilus influenzae and other
fastidious organisims like Neisseriae and
Pneumococcus .
The heat ruptures the RBC’S
and liberates nutrients.
Nutrient agar is melted, cooled
in a water bath at 75ºC.
10% blood is mixed with agar by gentle agitation
from time to time till chocolate brown (10 mts.
It is then poured as plates or slopes.
Heating the plates of blood agar in hot air oven
at 55ºC for 1-2 hrs.
Lysed blood agar:
 Suitable for subculture of gonococci and in
sulphonamide sensitivity test.
 Blood is lysed by heating at 55 to 60º C for one
hour or by freezing or thawing or with saponin
treatment.
 Nutrient agar is melted and cooled to 50ºC and
lysed blood agar added and pour plated.
Serum agar and broth –
 generally used as slopes.
 10% sterile serum is added to sterile nutrient

agar
Example-
Loefflers serum slope - used as enriched
media for Corynebacterium diphtheriae.
Composition
Serum of ox, sheep,or horse
• Nutrient broth, glucose, NaCl.
• pH 7.6
Enrichment media
 Liquid media used to isolate

pathogens from a mixed culture.

 Media is incorporated with

inhibitory substances to suppress

the unwanted organism.
 Eg:

◦ Selenite F Broth – for the isolation of

Salmonella, Shigella
◦ Alkaline Peptone Water – for Vibrio
cholerae
Selective media
 The inhibitory substance is added to a solid

media.
Eg:
 Mac Conkey’s medium for gram negative

bacteria
 TCBS – for V.cholerae
 LJ medium – M.tuberculosis
 Wilson and Blair medium – S.typhi
 Potassium tellurite medium – Diphtheria bacilli
Mac Conkey’s medium TCBS
Indicator media
 These media contain an indicator which

changes its colour when a bacterium grows

in them.
 Eg:

◦ Blood agar
◦ Mac Conkey’s medium
◦ Christensen’s urease medium
Differential media
 A media which has substances incorporated in

it enabling it to distinguish between bacteria.

 Eg: Mac Conkey’s medium

◦ Peptone
◦ Lactose10% aqueous solution
◦ Agar
◦ Neutral red2% in 50% ethanol
◦ Taurocholate
 Distinguish between lactose fermenters & non
lactose fermenters.
 Lactose fermenters – Pink colonies
 Non lactose fermenters – colourless colonies

Mac Conkey agar

Transport media
 Media used for transporting the

samples.
 Delicate organisms may not

survive the time taken for

transporting the specimen without
a transport media.
 Eg:

◦ Stuart’s medium – non nutrient soft

agar gel containing a reducing agent
◦ Buffered glycerol saline – enteric
bacilli
 Transport medium should be non-nutritive, semi-
solid, and reductive and should be able to hamper
self-destructive enzymatic reactions within the
cells and in addition, must inhibit toxic oxidation
reactions
 Used in case of delicate organisms whenever there
is a delay in the transportation .
 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
 Used for transportation and preservation of
microbiological specimen.
 Composition:

Sodium chloride, Potassium chloride,

Calcium chloride, Magnesium chloride
Monopot. phosphate, Disod. phosphate
Sodium thioglycollate, Charcoal, Agar
 Reduced environment d/t sodium thioglycollate
 Charcoal - neutralize materials that are toxic to sensitive
pathogens like Neisseria gonorrhoeae .
 Ca, Mg, K and Na salts help the survival of gonococcal
cells and also control permeability of bacterial cells.
 Phosphates buffer the medium.
 Transport Medium Stuart is recommended for the
preservation and transportation of Neisseria
species and other fastidious bacteria.
 Composition

Sodium glycerophosphate
Sodium thioglycollate, Calcium chloride
Methylene blue, Agar
 Reduced environment d/t sodium thioglycollate
 Calcium chloride along with Na glycerophosphate act

as good buffering agent and also maintains osmotic

equilibrium in the medium..
Anaerobic media
 These media are used to grow anaerobic

organisms.
 Eg: Robertson’s cooked meat medium,

Thioglycolate medium.
 Prepared from pure chemical substances.
 Exact composition of the media is

documented.
 Suitable for studies such as metabolic

requirements and for research purposes.

 With this technique, colonies of specific
microorganisms can be recognized by their
colour at a glance.
 This technology is based on soluble colourless
molecules (called chromogens), composed of a
substrate (targeting a specific enzymatic activity)
and a chromophore.

When the target organism’s enzyme cleaves the
colourless chromogenic conjugate, the
chromophore is released.
 In its unconjugated form, the chromophore
exhibits its distinctive colour and, due to reduced
solubility, forms a precipitate.
 The result is very specific and distinctive.
(colour-based differentiation method)
 clearly distinguishable with the naked eye under normal
lighting conditions.

 It allows for easy differentiation of microorganisms without

the complex and costly traditional detection procedures
employed in traditional agar testing techniques (No
subcultures).
 By saving time and labor, it increases the efficiency of
laboratory testing.
 By shortening the timeframe for identifying pathogens, it
helps to prevent the spread of infections.