PREPARATION OF CULTURE MEDIA

POTATO DEXTROSE AGAR (PDA) FOR

FUNGI AND NUTRIENT AGAR (NA) FOR
BACTERIA

SUBMITTED TO: DR. SATISH SHARMA

SUBMITTED BY: JAYA MANDAL
SUBJECT: FUNDAMENTALS OF PLANT PATHOLOGY II
SUBJECT CODE: PATH 271
ENROLLMENT NO.: 2019MGU0020
 POTATO DEXTROSE AGAR (PDA)
• Potato Dextrose Agar (PDA) is used for the cultivation of fungi. Potato Dextrose Agar
(PDA) is a general purpose medium for yeasts and molds that can be
supplemented with acid or antibiotics to inhibit bacterial growth. It is recommended
for plate count methods for foods, dairy products and testing cosmetics. PDA can be
used for growing clinically significant yeast and molds. The nutritionally rich base
(potato infusion) encourages mold sporulation and pigment production in
some dermatophytes.
• PDA is recommended by APHA and FDA for plate counts of yeasts and molds in the
examination of foods and dairy products. It is a general purpose medium for yeasts
and molds that can be supplemented with acid or antibiotics like Chloramphenicol,
Tartaric Acid and Chlortetracycline can be added as selective agents and to inhibit
bacterial growth. PDA can be used for growing clinically significant yeast and molds.
PDA is also useful for maintaining stock cultures of certain dermatophytes.
 PREPARATION OF PDA
Ingredients:
• Potato infusion: 200 gm
• Dextrose: 20 gm
• Agar: 20 gm
• Distilled water: 1 liter
PROCEDURE:
To prepare potato infusion, boil 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30 min.
Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated
form).
Mix with Dextrose, Agar and Water and boil to dissolve.
Autoclave 15 min at 121°C.
Dispense 20-25 ml portions into sterile 15 × 100 mm petri dishes.
• Final pH, 5.6 ± 0.2.
PREPARING FROM COMMERCIAL MEDIUM
POWDER
Ingredients:
Commercial PDA Powder: (20 gm dextrose, 15 gm agar, and 4 gm potato starch)39 gm
• Distilled water: 1 lit.
PROCEDURE:
Add 39 gm of Commercial PDA Powder to 1 Litre of Distilled water.
Boil while mixing to dissolve.
Autoclave 15 min at 121°C
In addition, Potato Dextrose Agar with Chlortetracycline contains:
Chlortetracycline: 40.0 mg
In addition, Potato Dextrose Agar with Chloramphenicol contains:
Chloramphenicol
25.0 mg (Final pH of 5.6 +/- 0.2 at 25 degrees C)
In addition, Potato Dextrose Agar with TA contains:
Tartaric Acid: 1.4 gm (Final pH of 3.5 +/- 0.3 at 25 degrees C.)
APPEARANCE AND STORAGE

• Appearance: Light amber colored clear to slightly opaque gel

• STORAGE
• Media is both light and temperature sensitive. Store plates away from direct light at
2-8ºC. Plates may be used for one week when stored in a clean sterile area. Media
should not be used if any signs of deterioration, contamination, and/or expiration
date has passed.

STERILIZE ALL BIOHAZARD WASTE BEFORE DISPOSAL.

 NUTRIENT AGAR (NA)
Nutrient Agar is a general purpose, nutrient medium used for the cultivation of microbes supporting
growth of a wide range of non-fastidious organisms. Nutrient agar is popular because it can grow a
variety of types of bacteria and fungi, and contains many nutrients needed for the bacterial growth.

What Grows on Nutrient Agar?

• Microorganisms need food, water and a suitable environment in order to survive
and grow. Nutrient agar provides these resources for many types of microbes,
from fungi like yeast and mold to common bacteria such as Streptococcus and
Staphylococcus.
• The microbes that can be grown on complex media such as nutrient agar can be described as
nonfastidious organisms. Nonfastidious organisms are microbes that are able to grow and thrive
without special nutritional or environmental conditions.
• Some bacteria cannot be grown with nutrient agar medium. Fastidious organisms (picky bacteria)
may need a very specific food source not provided in nutrient agar.
INGREDIENTS
0.5% Peptone: It is an enzymatic digest of animal protein. Peptone is the principal
source of organic nitrogen for the growing bacteria.
0.3% beef extract/yeast extract: It is the water-soluble substances which aid in
bacterial growth, such as vitamins, carbohydrates, organic nitrogen compounds and
salts.
1.5% Agar: It is the solidifying agent.
0.5% NaCl: The presence of sodium chloride in nutrient agar maintains a salt
concentration in the medium that is similar to the cytoplasm of the microorganisms.
• 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.
pH is adjusted to neutral (7.4) at 25 °C.
 PROCEDURE
Suspend 28 g of nutrient agar powder in 1 litre of distilled water.
Heat this mixture while stirring to fully dissolve all components.
1. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
2. Once the nutrient agar has been autoclaved, allow it to cool but not solidify.
3. Pour nutrient agar into each plate and leave plates on the sterile surface until the
agar has solidified.
4. Replace the lid of each Petri dish and store the plates in a refrigerator.
PRINCIPLE OF NUTRIENT AGAR
Nutrient agar is made with various nutrients which allow the growth of a wide variety of microorganisms that do not
usually require specific nutrients or supplements.
The primary constituents of the media are peptone, beef extract, and agar. In addition to these nutrients, some
vitamins and some trace ingredients necessary for the growth of bacteria are also added.
The peptone is the source of nitrogen or protein that acts as a source of amino acids for the bacteria.
The beef extract is the primary source of carbon which is essential for the formation of carbohydrates in the
bacteria. It also contains other components like some vitamins, different trace minerals, organic compounds, and
salts, which further enhance the growth of different organisms.
Besides, sodium chloride is added to the medium in order to maintain the osmotic equilibrium of the medium and
prevent the change in pH of the medium during growth.
The distilled water provides a medium to dissolve the nutrients so that it is easier for the bacteria to absorb them.
Agar is the solidifying agent that provides a stable surface for the organism to grow on, which allows for the
observation of colony morphology and enumeration of the organism.
• The medium is made up of basic nutrient that is essential for a wide variety of organism, which makes it a general
media that can be used for various purposes.
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.