Basics of Culture Media preparation along with their types and uses. Useful for basic understanding for freshers in biotechnology.

2.  Media: A growth medium or culture medium is a solid, liquid or semi-solid
designed to support the growth of microorganisms or cells.
 It is either an organic or a synthetic substance that provides both the biophysical
and the biochemical factors necessary for the growth of bacteria.
WHY DO WE REQUIRE A MEDIA?
 Microorganisms need nutrients, a source of energy and certain environmental
conditions in order to grow and reproduce. In the environment, microbes have
adapted to the habitats most suitable for their needs, whereas in laboratory,
these requirements must be met by a culture medium.
 This is basically an aqueous solution to which all the necessary nutrients have
been added.

3. The general composition of a medium is as follows:
 H-donors and acceptors (approximately 1-15 g/L)
 C-source (approximately 1-20 g/L)
 N-source (approximately 0.2-2 g/L)
 Other inorganic nutrients e.g. S, P (50 mg/L)
 Trace elements (0.1-1 µg/L)
 Growth factors (amino acids, purines, pyrimidine's, occasionally 50 mg/L,
vitamins occasionally 0.1-1 mg/L)
 Solidifying agent (e.g. agar 10-20 g/L)
 Solvent (usually distilled water)
 Buffer chemicals

4.  Required amount of chemicals is dissolved in distilled water using
Erlenmeyer flasks or rimless clean test tubes.
 pH is adjusted by adding 1N of NaOH or N/10th of HCl
 15ml capacity of test tube  5ml of liquid medium is taken
 250ml flask  100ml of media
 These are plugged with non adsorbent cotton and are covered using brown
paper.
 This is kept for autoclaving – sterilization done at 15lbs/inch^2 at a
temperature of 121 degree Celsius for 15min
 Later cooled down and poured in petri plates or test tubes which solidifies to
form a medium.

5.  Nutrient Agar is a general purpose, nutrient medium used for the
cultivation of microbes
Composition of Nutrient Agar
o 0.5% Peptone - principal source of organic nitrogen for the growing bacteria.
o 0.3% beef extract- water-soluble substances which aid in bacterial growth, such
as vitamins, carbohydrates, organic nitrogen compounds and salts.
o 1.5% agar- solidifying agent.
o 0.5% NaCl- maintains a salt concentration in the medium that is similar to the
cytoplasm of the microorganisms.
o Distilled water-Water is essential for the growth of and reproduction of micro-
organisms and also provides the medium through which various nutrients can be
transported.
o pH is adjusted to neutral (7.4) at 25 °C.

6. Preparation of Nutrient Agar
1. Suspend 28 g of nutrient agar powder in 1 liter of distilled water.
2. Heat this mixture while stirring to fully dissolve all components.
3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
4. Once the nutrient agar has been autoclaved, allow it to cool but not
solidify.
5. Pour nutrient agar into each plate and leave plates on the sterile
surface until the agar has solidified.
6. Replace the lid of each Petri dish and store the plates in a refrigerator.

7. Uses of Nutrients Agar:
1. It is frequently used for isolation and purification of cultures.
2. It can also be used as a means for producing the bacterial lawns
needed for antibiotic sensitivity tests. In actuality, antibiotic sensitivity
testing is typically performed on media specially formulated for that
purpose.

8. Chemical
composition
Synthetic or chemically
defined medium
Semi synthetic or
undefined medium
 These media are prepared by mixing all
the pure chemicals of known
composition
 e.g. Czapek Dox medium.
 Such are those media, where exact
chemical composition is unknown
 e.g. potato dextrose agar or MacConkey
agar medium.

9. consistency
Solid or
synthetic
medium
Liquid medium
or broth
Semi-solid or
floppy agar
medium
 When 5-7% agar agar or
10-20% gelatin is added the
liquid broth becomes
solidified.
 used for making agar
slants or slopes and
agar stab.
 no agar is added or used
while preparing the
medium.
 After inoculation and
later incubation, the
growth of cells becomes
visible as small mass on
the top of the broth.
 prepared by adding half
quantity of agar i.e.
about 0.5% in the
medium.
 This type of medium
may be selective or
diffrential type of
medium

10. o Culture media
• Culture media contain all the elements that
most bacteria need for growth and are not
selective, so they are used for the general
cultivation and maintenance of bacteria kept in
laboratory culture collections.
o Minimal media
• Minimal media are those that contain the
minimum nutrients possible for colony growth,
generally without the presence of amino acids,
and are often used by microbiologists and
geneticists to grow "wild-type"
microorganisms.

11. o Selective media
 Selective media are used for the growth of only selected microorganisms. For
example, if a microorganism is resistant to a certain antibiotic, such
as ampicillin or tetracycline, then that antibiotic can be added to the medium to
prevent other cells, which do not possess the resistance, from growing.
 YM (yeast extract, malt extract agar) has a low pH, deterring bacterial growth.
 MacConkey agar is for Gram-negative bacteria.

12. o Differential media
• Differential or indicator media distinguish one microorganism type from
another growing on the same medium.[7] This type of media uses the
biochemical characteristics of a microorganism growing in the presence
of specific nutrients or indicators (such as neutral red, phenol red, eosin
y, or methylene blue) added to the medium to visibly indicate the
defining characteristics of a microorganism.
• Examples of differential media include:
• Blood agar (used in strep tests) contains bovine heart blood that
becomes transparent in the presence of β-hemolytic organisms such
as Streptococcus pyogenes and Staphylococcus aureus.
• MacConkey agar is differential for lactose fermentation.

13. o Transport media
• Transport media should fulfill these criteria:
• Temporary storage of specimens being transported to the laboratory for
cultivation
• Maintain the viability of all organisms in the specimen without altering their
concentration
• Contain only buffers and salt
• Lack of carbon, nitrogen, and organic growth factors so as to prevent microbial
multiplication
• Transport media used in the isolation of anaerobes must be free of molecular
oxygen
• Examples of transport media include:
• Thioglycolate broth is for strict anaerobes.
• Stuart transport medium is a non-nutrient soft agar gel containing a reducing
agent to prevent oxidation, and charcoal to neutralize.
• Certain bacterial inhibitors are used for gonococci, and buffered glycerol saline for
enteric bacilli.

14. o Enriched media
Enriched media contain the nutrients required to support the growth of
a wide variety of organisms, including some of the more fastidious ones.
They are commonly used to harvest as many different types of microbes
as are present in the specimen. Blood agar is an enriched medium in
which nutritionally rich whole blood supplements the basic
nutrients. Chocolate agar is enriched with heat-treated blood (40–
45 °C), which turns brown and gives the medium the color for which it
is named.