EXPERIMENT 7 PREPARATION OF MEDIA

Structure
7.0 Objectives
7.1 Introduction
7.2 Experiment
7.2.1 Principle
7.2.2 Requiretnents
7.2.3 Procedure
7.2.4 Observation
7.2.5 Result
7.3 Precautions

7.0 OBJECTIVES
After going through this experiment, you will be able to:
explain the role of nutritional requirement in the cultivation of microorganism in
laboratory;
acquire knowledge about various types of microbiological media used for diffeient
purpose; and
prepare the media for microbiological analysis.

7.1 INTRODUCTION
For any bacterium to be propagated for any purpose it is necessary to provide the
appropriate biochemical and biophysical environment. The biochemical (nutritional)
environment is made available as a culture medium. Media are the substances over
which micro-organisms such as bacteria, viruses, fungi etc are cultured or grown to
harvest in a larger amount. You all know that individual micro-organism is too small and
therefore they are required in larger amount for their evaluation. Depending upon the
special needs of particular bacteria (as well as particular investigators) a large variety
and types of culture media have been developed with different purposes and uses. Culture
media are employed in the isolation and maintenance of pure cultures of bacteria and are
also used for identification of bacteria according to their biochemical and physiological
properties.

7.2 EXPERIMENT
7.2.1 Principle
The ingredients in culture media range from pure chemical compounds to complex materials
such as extracts or digests of plant and animal tissues. If all the ingredients of a culture
medium are known, both qualitatively and quantitatively,the medium is called a chemically
defined medium. These media are of great value in studying the nutritional requirements
( * , -3?ic;oorganisrnsor in studying a great variety of their metahlic activities. In a complex

m ,exact chemical cornposition is not known, and such a medium is often

~ i ~ e d i t ~lire
prepared qrnm complex ~materials,e.g., body fluids, tissue extracts and infusions,
"pr ?" .,.
Commonly Used Constituents in Microbiological Media:

i) Carbohydrates: Carbohydrates and alcohols are added as carbon and energy

sources, which stimulate growth of microorganisms. Carbohydrates are also used
to aid in microorganism identification and differentiation.

ii) Agar: Agar is the major solidifying agent used in bacteriological media. It is an
impure polysaccharide gum obtained from certain marine algae. It is added as a
powder at a more or less standard concentration (1.5% for plates and slanted media,
0.5% or less for "semisolid media), usually after the other medium components
have been added and dissolved in the water. Agar dissolves at approximately 100°C,
and an agar-containing medium thus heated will not solidify until the temperature is
brought down to about 43°C. Once solidified, the medium will not melt until brought
back up to about 100°C.
iii) Body Fluids: Whole or defibrinated blood, plasma, serum or other body fluids are
frequently added to culture media for the isolation and cultivation of many pathogens.
Body fluids contribute many growth factors and/or substances which detoxify certain
inhibitors.

iv) Buffers: These compounds are incorporated to maintain the optimum pH range of
the organism. Substances like sodium and potassium phosphates and calcium
carbonate prevent marked changes in pH which otherwise would result from
microbial production of organic acids or bases.

v) Extracts (Beemeast): Eucaryotic tissues (yeast, beef muscle, liver, brain, heart,
etc.) are extracted by boiling and then concentrated to a paste or dried to a powder.
These extracts are frequently used as a source of amino acids, vitamins and
coenzymes, including many needed as growth factors by fastidious organisms. Trace
elements and other minerals and usually some sugar are also present.

vi) Peptones: These complex mixtures of organic and inorganic compounds are obtained
by digestion of protein-containing tissues of animals and plants such as meat scraps,
beef muscle, gelatin, milk protein (casein) and soybean meal. Peptones primarily
contain peptides and single amino acids. Being crude digests of complex materials,
they contain a great variety of other organic and inorganic materials. Examples are
Tryptone (a pancreatic digest of casein), Phytone (a papaic digest of soybean meal)
and simply Peptone (a digest of beef muscle).
vii) pH Indicators: An acid-base indicator is often added to differential media to detect
changes in hydrogen ion concentration during the growth of an organism such as in
Carbohydrate Fermentation Broth, Kligler Iron Agar, Simmons Citrate Agar,
MacConkey Agar and Glucose OIF Medium. Brom-cresol purple, brom-thymol
blue and phenol red are commonly used; for each of these, an acidic pH turns the
indicator a yellow colour.
viii) Reducing Agents: Certain chemicals may stimulate growth by reducing the
oxidation-reduction potential in the environment. Cystine and thioglycollate are
reducing agents often used for the cultivation of anaerobes.

ix) Selective Agents: Antimicrobial agents such as crystal violet, bile salts, brilliant
green, potassium tellurite, sodium azide and antibiotics can be employed in selective
media to suppress or inhibit the growth of certain groups of microorganisms while
allowing growth of desired organisms. These agents are usually bacteriostatic.
Classification of Culture Media
A classification of media based on their respective uses as follows. Note that these
categories can overlap.
Practical Manual - a) A minianal medium is one, which supplies only the minimal nutritional requirements
Fundamentals of Food of a particular organism. As an example, a typical, prototrophic strain of E. coli is
and Meat Science
able to synthesizeall of its cell components from a simple solution containing several
"mineral salts" plus glucose as the source of carbon and energy - such as the
medium given above. Minimal media vary in composition according to the minimal
nutritional requirements of the particular species under study.
b) An all-purpose medium is rich in a wide variety of nutrients (including many
growth factors) and will, therefore, support the growth of a wide range of bacteria.
All-purpose media include Nutrient Agar, APTAgar, Plate Count Agar, Heart Infusion
Agar, and Brain Heart Infusion Agar.
c) A selective medium supports the growth of desired organisms while inhibiting the
growth of many or most of the unwanted ones - either by purposely adding one or
more selective agents which "poison" certain types of organisms or by including or
deleting certain nutrients such that the desired organisms and few others are able to
grow. Examples on how these things may be accomplished are as follows:
i) MacConkey Agar: This is an example of a medium where selective agents
are added which directly suppress the growth of undesired organisms as much
as possible. The particular selective agents chosen for this medium -bile salts
and crystal violet - inhibit gram-positive bacteria, allowing the near-exclusive
growth of gram-negative bacteria.
ii) Nitrogen-Free Broth: Here the medium is made selective by the deletion of
a required element; no nitrogen compounds are present. Therefore, the only
organisms which can grow after inoculation into this medium are those, which
can utilize gaseous nitrogen (N2) which diffuses in from the atmosphere.These
are the nitrogen-fixing bacteria. While this medium does not utilize selective
agents, it is still restrictive to an extensive number of various organisms.
iii) Succinate Broth: In this example, a particular nutrient utilized by the desired
organism - and few others - is included as the only carbon source. This
medium is used for the enrichment of the purple non-sulfur photosynthetic
bacteria; most other organisms tend not to metabolize succinate under the
anaerobic conditions utilized. This is another example of a restrictive medium
not utilizing selective agents.

d) Differential Medium is one which allows two or more different organisms to

grow, but it containsdyes andlor other components upon which different organisms
act in various ways to produce a variety of end products or effects, often detected
by variations in colour. Examples:
i) MacConkey Agar: This medium is used in plates. Organisms which ferment
the lactose in the medium will lower the pH due to the production of acids.
The pH indicator (neutral red) will turn red, and the colonies will consequently
have a reddish appearance. Other colonies on the same plate which do not
contain lactose-fermenting cells should appear whitish. (As this medium also
appears in the above category, it is termed a selective-differentialmedium.)
ii) Carbohydrate Fermentation Broth: This medium is used in tubes, usually
with Durham tubes. Organisms which ferment the particular carbohydrate in
the medium (e.g., glucose, lactose, sucrose) will cause the pH indicator to
change colour. Also, if insoluble gas (H2) is produced during fermentation, a
bubble will be seen in the inverted Durham tube.
ifi) Other examples of differential media include Motility Medium (exploiting a
morphologicalcharacteristic-production of flagella), Nutrient Gelatin, Starch
Agar, Kligler Iron Agar and Blood Agar.
Some of the common media used in microbiological examination of food products are as Preparation of Media
follows:
Nutrient Broth and Agar
Nutrient broth is a liquid medium commonly used for the cultivation of microorganisms
which are non-demanding in their nutritional requirements e.g. water borne organisms,
air, soil and dust flora. Addition of agar to the broth gives a solid medium (Nutrient agar)
used for cultivation and determination of bacteria.
MacConkey's Broth and Agar
This is a selective medium used for detection, isolation and enumeration of colifoms and
intestinal pathogens. The medium is used for performing the presumptive coliform test.
Production of acid (indicated by the medium turning red or yellow) and gas in Durham
tube is considered as indication of presence of coliforms in the given sample.
Eosine Methylene Blue (EMB) Agar
It is a differentialplating medium, used for the isolation and differentiation of gram negative
enteric bacilli. Typical coliform organism (E. coli) form bluish black colonies with green
metallic sheen, while Enterobacter aerogenes forms mucid pink colonies. Salmonella
and Shigella organisms form translucent, amber coloured or colourless colonies.
Endo Agar
Endo agar is used for the confirmation of the presumptive test for coliforms. Tubes of
liquid media showing a positive presumptive reaction for colifoms are subcultured on to
Endo agar..Lactose fermenting colifoms give rise to red colonies with a metallic sheen
while non-coliforms form colourless colonies.
Tryptone Glucose Extract Agar
This medium is recommended as standard plate count agar for enumeration of bacteria
in water and thermophilic bacteria in products.

7.2.2 Requirements
Generally following equipments, glass wares and chemicals are required for
preparation of media.
i) Autoclave (Portable)
ii) Balance
iii) Heating mantle1 water bath
iv) pH meter
v) Laminar air flow
vi) Stirrer
vii) Pipettes

viii) Distilled water

ii) Media
x) Test tubes
xi) Beakers
Practical Manual - xii) Cotton plugs
Fundamentals of Food
and Meat Science xiii) pH paper
xiv) Measuring cylinder

Nutrient Broth and Agar

i) Beef extract
ii) Peptone
iii) Agar
iv) Sodium chloride

V) Distilled water

vi) Sterilized test tubes, bottles and flasks

vii) pH indicator
viii) Lovibond pH comparator
ix) Autoclave
MacConkey's Broth and Agar

i) Sodium glycol or taurocholate or bile salt

ii) Peptone
5) Lactose
iv) Sodiumchloride
v) Andrade's indicator

vi) Bromocresol purple solution

vii) Test tubes
viii) pH indicator
ix) Flask
x) Lovibond comparator
xi) Autoclave
EMB Agar
i) Peptone
ii) Lactose

iii) Sucrose
iv) Dipotassiun Phosphate
v) Agar
vi) EosinY
vii) Methylene blue
 Preparation of Media
viii) Test tubes or flasks

ix) Lovibond comparator

x) Autoclave
Endo Agar
i) Peptone
u) Lactose
iii) Dipotassium phosphate

iv) Sodium sulphate

v) Basic fuchsin
vi) Distilled water

vii) Test tubes or flasks

viii) pH indicator

ix) Lovibond comparator

x) Autoclave
Tryptone Glucose Extract Agar
i) Tryptone
ii) Yeast extract
iii) Glucose
iv) Agar
v) Test tubes or flasks

) pH indicator
vii) Lovibond comparator
viii) Autoclave

7.2.3 Procedure
General steps for preparation of microbiological media usually involve the following:

1) Carefully weigh the proper amount of the dehydrated base medium or the correct
proportion of constituent ingredients and dissolve in appropriate volume of distilled
water and heat.
.'.
2) Determine the pH of the medium, and adjust if necessary with dilute acid or alkali.

3) If a solid medium is desired, add agar (1.5-2%) and boil the medium to dissolve the
agar.

4) Distribute the medium into tubes or flasks. The amount of medium distributed per
container should be limited so that no point within the volume of the medium is more
than 2.5 cm from the top surface of the container.

5) Autoclave at 121°C for 15 minutes. Some media (or specific ingredients) that are
heat labile are sterilized by filtration.
 -
Practical Manual
Fundamentals of Food
and Meat Science
 Add Andrade's indicator to give clear amber colour or bromocresol purple to give a Preparation &Media
light purple colour.
Dispense 8 rnl into sterilized test tubes in which Durham tubes are placed inverted.
Sterilize at 15 lbs pressure for 20 minutes.
For preparing agar add agar to the broth medium, dispense into flasks or bottles and
then sterilize.
EMB Agar
The composition of medium is as follows:
- -

Ingredient Grams/ Litre

--

Peptone
Lactose
Sucrose
Dipotassium
Phosphate

Agar
Eosin Y
Methylene Blue

Final pH 7.2+ 0.2

Suspend 36g in 1OOO ml distilled water.

Boil to dissolve the medium completely.
Dispense and sterilize as mentioned earlier.
Cool to oxidize the Methylene blue (i.e to restore its blue colour) and to suspend the
flocculant precipitate which is an essential part of the medium. ,
Endo Agar
The composition of the medium is as follows:

Ingredient Grams/ Litre

Peptone 10.0
Lactose 10.0
Dipotassium phosphate 3.5
Sodium sulphite 2.5
Basic fuchsin 0.5

Agar 15.0

Final pH
Practical Manual - Suspend 41.5 g in 1000 ml distilled water.
Fundamentals of Food
and Meat Science Heat to boiling to dissolve the medium completely. ,
Distribute into the tubes or flasks and sterilize by autoclaving.
Shake the flask to evenly disperse the characteristic flocculant precipitate that forms
during heating.
Ttyptone Glucose Extract Agar
The composition of medium is as follows:

Ingredient Grams/ Litre

Tryptone 5 .O
Yeast extract 3.0
Glucose 1.O
Agar 15.0

Final pH
Weigh and dissolve the ingredients in 1000ml distilled water.
Heat to dissolve the medium completely.
Dispense into tubes or flasks and sterilize.
Composition of Diffemt Media Generally Used for Microbial Study
1. Standard Phte Count Agar (SPCA)
Tryptone . 5.Og
Yeast extract 2.5g
D-glucose - 5.0g

Agar - 15.0g

Distilled water - lOOOml

PH 7.0
2. Nutrient Agar for Bacterial count: It has already been discussed.
3. Potato Dextrose Agar for Fungal Count
Potato, peeled and diced - 200g

Boil 200g of peeled and diced potato for 1 hour in a litre of water. Filter and make up the
volume to llitre and add rest of the constituents.
 4. Vwlet Red Bile Agar (VRBA)for colifonn count Preparation of Media

Yeast Extract 3.0g

Peptone 7.0g
Sodium Chloride 5.0g
Bile Salts 1.5g
Lactose - 1O.Og
Neutral red 0.03g
Crystal violet 0.002g

Agar 13.0g
Distilled water 1OOOml

pH 7.4
7.2.4 Observations
The prepared media should be stored in a cool, dry place in the appropriate container.
Observe the pH using the pH meter or colour indicator solutions. At times, tubes and
flasks containing media may be incubated at 37°C for 48 hours before their use to detect
and weed out any possible contamination.

7.2.5 Result
After performing this experiment you will observe that suitable media for growth of
bacteria is nutrient agar (pH 6.8-7.0) or plate count agar (pH 7.0), whereas for yeasts
and moulds it is potato dextrose agar (pH 5.6) and malt agar (pH 5.4).

PRECAUTIONS
a Only the fresh packs should be used for media preparation and packs should not be
kept open for a longer as most of media powder are hygroscopic.
a Ensure before uFe that medium is not deteriorated physically and use before the
expiry date printed on the label of the container.
a Only the clean glasswares should be used for preparation of media.
a Weighing should be accurate and pH must be adjusted before putting for autoclave.
a If possible, prepare media just before use. Repeated storage and autoclaving must
be avoided.
a Avoid excessive heating or scorching of the medium. Agar media should not be
held at high temperature (40" to 45°C) for longer time as agar tends to clump.
a The media must be cooled under laminar flow to a temperature range of 42-45OC
before pouring over innoculum. Never cool under tap water or in septic area.
a If the medium is not used the same day it is prepared, store it properly in moisture
1 proof containers to prevent drying of the medium.

! a Prepare medium in such quantities that if stored, it will be used before loss of ,
moisture through evaporation that becomes evident.
i
Practical Manual - To prevent contamination and excess evaporation of water from a medium in flask
Fundamentals of Food and tubes during storage, optionally fit aluminium foil or plastic with loose rubber
and Meat Science
bands before autoclaving in order to allow air to escape and to prevent the container
from bursting.
Avoid over loading autoclaves so that the rate of air exhaust and heating is not
appreciably delayed.
Flask or test tubes should be plugged with cotton or capped with paper,
After sterilization gradually reduce the pressure within the autoclave (using no less
than 15min) since liquids may be at a temperature above their boiling point at
atmospheric pressure. If the pressure is lowered too rapidly, liquids may boil over
and come out from the container.