CLB10603 Lab Manual / Appendix A

SECTION OF BIO-ENGINEERING
TECHNOLOGY, UniKL MICET.

Media preparation

Objectives :

After completing this laboratory, student should be able to prepare

microbiological media on their own.
Student will be exposed to media preparation and aseptic technique
involve during media preparation.

Introduction

There is a wide variety of media which are used in microbiology, but the procedures used
in their preparation are generally the same. They include weighing out the dehydrated
media, dissolving it in dH2O, sterilizing it (usually by autoclaving), pouring the plates,
preincubation to check for contamination and to dry out the plates, and storage.

Agar is particularly suited as a solidifying agent because it will not melt until the medium
is heated to near boiling(95°C-100°C), but will remain melted until cooled to around
42°C. It is not degraded by the vast majority of bacteria, and therefore maintains its
structure during bacterial growth, and because it is not a nutrient for bacteria, it also
allows strict control of growth factors.

Because medium boils up during decompression from autoclaving, we will autoclave

only 600 mL of medium in a 1 liter bottle. You will need to calculate the amount of
powder needed per 600 mL of medium since the directions on reagent bottles are for 1
full liter. Teams of 4 students will prepare a media assigned to them.

Materials and Methods

Equipments Materials
Spatula Dehydrated media (Nutrient Agar

ndh/dbt/July 2010 1
CLB10603 Lab Manual / Appendix A

Balance Distilled water

250 ml Beaker
250 ml Erlenmeyer flask
Stirring hot plate
Stirrer
100 ml graduated cylinder
10 ml graduated cylinder
test tubes
Sterile disposable Petri dishes.
Tube rack
Specific agar or broth powder

Example of an Agar Preparation Method for Mac Conkey Agar.

If you want to prepare agar/broth other than Mac Conkey, determine how many agar or
broth powder that you need by referring to the information given at the label of that
precise media (Nutrient agar powder, nutrient broth powder, potato dextrose agar powder
etc).

A. Determining the amount

1. Consult the label on the bottle to determine what amount of nutrient agar powder
is used per given volume of water.

2. Use proportionate method to calculate the amount desired.

3. The following procedure will show how to prepare nutrient agar with 100 ml of
water.

4. Since the label instructs to dissolve 23 grams in 1 liter of water (for Mac Conkey
agar), the following calculation algebraically solves for the grams of powder to be
mixed with 100 ml of water.

x 23g

100ml 1000ml

23g  100ml
x
1000ml

ndh/dbt/July 2010 2
CLB10603 Lab Manual / Appendix A

2300g
x  2.3g
1000ml

5. Repeat the same procedure for macconkey agar powder.

B. Weighing the dry media

1. Place a piece of weighing paper or weighing dish on a balance and tare it so the
balance reads 0.00 grams.

2. With a spatula, transfer small amounts of nutrient agar/ mac conkey agar powder
to the weighing paper until the balance reads 2.30 g.

3. Carefully transfer this powder into a 250 ml beaker.

4. With a 100 ml graduated cylinder, measure out 100 ml of distilled water. Pour this
water into the beaker containing the 2.3 g of nutrient agar powder.

B. Dissolving the media

1. Put small stirrer into the beaker and place the beaker over a stirring hot plate.

2. Continuously stirring the mixture will prevent the agar from charring to the
bottom of the beaker.

3. Initially the mixture will be very cloudy. Keep stirring until the medium clears
and has an amber color.

4. Make sure not to let the suspension over boiled. (Note: at 100 °C, the low surface
tension of agar gives it tendency to rapidly froth and boiled out of the beaker. To
prevent this, immediately turn off the stirring hot plate or remove the beaker as
the solution begins to show signs of foaming).

C. Preparing slant tubes and Erlenmeyer flask before sterilization.

1. Place 3 test tubes into a tube rack.

2. To prepare slant tubes, measure out 7 ml of the hot nutrient agar in a 10 ml

graduated cylinder. (Note: initially the beaker of nutrient agar might be very
hot. Use autoclave gloves or some other protection when handling this
container).

ndh/dbt/July 2010 3
CLB10603 Lab Manual / Appendix A

3. Pour into the first tube and use this tube as a visual guide to fill 2 more tubes.
If précised volume is desired, use a graduated pipette to transfer the agar.

4. After filling the tubes, pour the remaining nutrient agar into a 250 ml
Erlenmeyer flask. This agar will be used after sterilization to prepare the
plates.
a. (Note: be sure u cover the mouth of the tubes and pour the remaining nutrient
agar into Erlenmeyer flask before it cools and solidifies, around 42 °C. To
ensure thorough heating during sterilization, the medium should still be in a
melted state when it is placed in the autoclave).

5. Using a small piece of aluminum foil, cover the mouth of the tubes and the
Erlenmeyer flask.

6. Pour into a 1000 mL bottle with a funnel. Cap loosely, label bottle with name
of medium and your group's name, place in autoclave.

C. Autoclaving the media

(After all student groups have placed their tubes and flasks in autoclave)

1. Set for slow exhaust, autoclave the medium at 15 lbs. pressure for 15 minutes (for
most media). This takes at least 45 minutes. Usually the whole process
(autoclaving and cooling) will take as long as 3 hours. Therefore plan your time
wisely.

2. For the proper and safe operation of the particular autoclave being used, always
refer to procedure manual that came with the autoclave. For instance, if larger
loads are used, the time of sterilization needs to be increased to insure thorough
sterilization of all items within the chamber.

3. Remove from autoclave (CAUTION: HOT STEAM), allow to cool to 50-60 °C

(feels hot, but possible to hold).

4. For protection from steam and hot surfaces, use autoclave gloves while removing
the tubes from the autoclave.

D. Preparing slant agar after sterilization.

1. Convert each of the 7 ml agar tubes to slants by laying them on their side.

2. Use a slanting device or a piece of tubing to adjust the liquid level so the agar fills
the bottom curvature of the tubes and extends ½ to ¾ of the distance to the tubes
opening.

ndh/dbt/July 2010 4
CLB10603 Lab Manual / Appendix A

3. Leave the tubes undisturbed until the agar has solidified and cooled to room
temperature. (Note: As agar cools, it begins to solidify around 42 °C. the medium
visibly turns from clear to translucent).

E. Pouring the plates after sterilization.

1. Place unopened, sterile Petri dishes on a benchtop. (Note: To minimize the risk of
contamination from the air during pouring and cooling, disinfect the benchtop in
the work area and shut off any fans and close doors to minimize air currents in the
room).

2. Using aseptic techniques, hold the flask at an angle, remove the aluminum foil
and briefly flame the mouth of the flask with a Bunsen burner.

3. Lift the Petri dish cover and use it to shield the dish while pouring the sterile agar
into the plate.

4. Gently fill the bottom until it is completely covered to a depth of a round 5 mm.
this will take about 15 – 20 ml of agar.

5. To minimize condensation during cooling, replace the cover slightly ajar to allow
water vapor to escape. (Note: To avoid excessive condensation inside the plates,
first allow the nutrient agar to cool to 50 °C before pouring. If you cannot pour
the plate immediately, the flask can be placed in a 50 °C water bath to maintain
this temperature).

6. Repeat this procedure on the remaining Petri dishes.

7. Leave the plates undisturbed until the medium solidifies, then close the plate by
fully replacing the lids.

ndh/dbt/July 2010 5