Dept. of Chem. Engg. Biochem. Eng. Lab. N.I.T.

Srinagar

EXPERIMENT No. 2
Preparation of Basic Solid Media

Table of Contents
 Objectives
 Introduction
 List of Reagents and Instruments
 Procedures
 Discussions
 Questions
 Comments

Objectives
To prepare basic solid media as agar slants and agar plates

Introduction
Liquid solid media containing nutrient (broths) are usually solidified by the addition of agar.
Agar-agar (often called simply agar) is a complex polysaccharide (carbohydrate) consisting of 3,
6-anhydro-L galactose and D galacto-pyranose, free of nitrogen, produced from various red algae
belonging to Geladium, Gracilaria and other genera. It liquefies on heating to 96 0C and hardens
into a jelly on cooling to 40-45 0C. The solidified medium kept in a Petri dish provides an
artificial environment suitable for rapid growth of microorganisms. While in the liquefied state,
solid media can be added in test tubes, which are either allowed to cool and harden in a slanted
position producing agar slants or allowed to harden in a upright position producing agar deep
tubes.

List of Reagents and Instruments

A. Equipment

 Erlenmeyer flasks
 Culture tubes
 Petri dishes, 100*15mm
 Bunsen burner
 Autoclave
 Balance
 Magnetic Stirrer or stirring rod
 Incubator, 37ºC
 Refrigerator

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Dept. of Chem. Engg. Biochem. Eng. Lab. N.I.T. Srinagar

B. Reagents

 Nutrient media:
o Yeast extract
o Peptone
o Agar
o Glucose
 HCl, 0.1 N solution
 KOH, 0.1 N solution

Procedures

1. Petri dish and agar slant preparation

Mix the following nutrient ingredients in proportion to the amount required. Note that a Petri
dish pour requires about 12 ml and a slant needs 6 ml. Adjust the pH to the desired value
(pH=7.0) with 0.1N HCl or 0.1N KOH. If a large number (more than 5) of plates are to be
poured, mix the nutrient in 1-2 liter flasks. It is difficult to handle flasks larger than 2 liters with
one hand. If only a few (less than 5) are needed, the nutrient can be divided and poured into test
tubes, each holding enough media for one Petri dish. The advantage of using test tubes is that
they can be autoclaved separately and may later be heated to melt the agar in a beaker of boiling
water. The liquefied agar may then be poured into a Petri dish directly from the test tube.
However, the use of test tubes is not practical when making large quantities of agar plates.

YPG (east extract- Peptone- Glucose) Agar

o Yeast extract 5g
o Peptone 10g
o Glucose 5g
o Agar 15g
o Add water to make 1 liter

Cover the flask with a beaker or a piece of aluminum foil. Autoclave the media for 20 minutes at
121 0C temperature and 15 psi pressure. The heat of sterilization will dissolve the agar. Set the
media on a bench top and cool until the flask can be handled with your bare hands. Wrap paper
towel around the neck of the flask when pouring agar into Petri dishes just in case the media
reheats the glassware as the hot liquid passes through the neck in the process of pouring. Of
course, one cannot pour out a block of solidified agar if the flask is cooled excessively. The agar
will solidify at approximately 42ºC; thus, the temperature window for handling the liquefied agar
is quite narrow. If the agar starts to solidify at the bottom as a result of not pouring fast enough,
the flask can be gently heated with a flame to raise the temperature and melt the agar.

Stack sterile Petri dishes in a row, three or four per stack, on a disinfected lab bench. The number
of dishes per stack can be adjusted according to the size of the student's hand. With the right
hand holding the neck of the flask, open the cover of the lowest Petri dish with the left hand just
wide enough to pour the media. (The top cover is the larger dish of the two; the actual dish is the

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Dept. of Chem. Engg. Biochem. Eng. Lab. N.I.T. Srinagar

smaller one.) Pour about 12 ml into each Petri dish. If too little agar is poured, there may not be
enough to cover the dish or the agar plate will dry up easily. If too much is poured, the cover dish
will come in contact with the nutrient agar, leaving no room for microbial growth. The plates are
rendered useless either way. After pouring the bottom dish, pour the second one from the bottom,
and so on until the entire stack is poured. Proceed to the next stack until all dishes are poured.

Leave the plates undisturbed until the agar solidifies. The plates may now be streaked.

Un-inoculated agar plates may be stacked and stored upside down in a refrigerator for about 3
months if not used immediately. The inverted position ensures that water does not condense on
the bottom of the cover plate and retards the loss of moisture. An inoculated agar plate is also
incubated and stored upside down to retain moisture. In addition, the water condensate can act as
a medium for transport of cells from one location on an agar plate to another. The slants may be
stored in a refrigerator for over a year if culture tubes with screw caps are used.

Incubation of the inoculated plates should last for about 48 hours. They are stored in a
refrigerator, and the culture can stay viable for over one month in agar plates or for one year in
sealed slants. Prolonged storage of the plates in the incubator dries up the agar and kills the
culture. The severe loss of moisture can be visually detected by the appearance of macroscopic
cracks that develop in the agar plate.

The used agar plates and other biological wastes should be autoclaved in a pan to destroy all
biological activities before being disposed.

Questions

1. Why is the agar in test tubes allowed to solidify in a slanted position?

2. During the preparation of a streak plate, why flame the loop between different sets of
strokes?

Observe and report

Task

1. Cough and thumb some of the agar plates and leave others untouched. Incubate them at
37 0C. Comment your observation after 2 – 3 days.

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