Name: ____________________________________ Date of activity: ______________

Sec: _______ Group: _______ Laboratory instructor signature: _______________

Subject Pharm 204 – Pharmaceutical Microbiology with Parasitology

Title Preparation and Sterilization of Microbiological Culture Media
Activity No No. 4
Introduction The survival and growth of microorganisms depend on the available nutrients and
favourable growth environment. In the laboratory, the nutrient preparations that
are used for culturing microorganisms are called media. There are three physical
forms of media- liquid or broth media, semisolid media, and solid media. The major
difference among the media is the presence of a solidifying agent (agar). Liquid
media such as nutrient broth can be used to propagate large number of
microorganisms in fermentation studies. Semisolid media are used in determining
bacterial motility and promoting anaerobic growth in order to observe colony
appearance and for pure culture isolation.

A liquefied media is usually poured into a test tube. A semisolid media, while in
liquefied state, can be poured either in a test tube or plate (dish). If the medium in
the test tube is allowed to harden in a slated position, the tube is designated as agar
slant but if the medium is a tube is allowed to harden in an upright position, it is
called as agar deep tube or butt and if the agar is poured into a Petri plate, the plate
ids designated as agar plate.

Sterilization is a process of rendering a medium or material free of all forms of life.

The three basic ways of sterilizing media and supplies are autoclaving, dry heat
sterilization, and Ultraviolet radiation. The most useful approach is autoclaving in
which items are sterilized by exposure to steam at 121.5 degrees Celsius and 15 lbs
of pressure for 15 minutes or longer depending on the item. Under this condition
even endospores will not survive longer than about 12 to 13 minutes. This method
is rapid and dependable.

Objective(s) At the end of the activity, the student should be able to:
1. Identify the different types of culture media and the ingredients, their
preparation and uses; and
2. Prepare the microbiological culture media used for bacterial growth.
Materials and Nutrient agar (5 g) Analytical balance (1)
Equipment Distilled water (225 ml) Sterile Petri Plates (4 pairs)
Autoclave Erlenmeyer flask (1)
(*use pressure cooker and electric Spatula (1)
stove / water bath as improvised
autoclave) Graduated cylinder (1)
Thermometer (1pc) Beaker (1)
Timer
Magnetic stirrer with * Others: Alcohol lamp,
magnetic bar denatured alcohol and
Aluminum foil match sticks
Stirring rod (1) * Pen, paper for labelling,
Name: ____________________________________ Date of activity: ______________

Sec: _______ Group: _______ Laboratory instructor signature: _______________

 tape

Name: ____________________________________ Date of activity: ______________

Sec: _______ Group: _______ Laboratory instructor signature: _______________

Preparation
1. Clean and disinfect the working area before the experiment.
2. Wear mask, head cap, lab gown and latex gloves.
3. Sterilize all the materials needed in the experiment.
4. Prepare all the materials needed.

Procedure/ Instruction
PREPARATION OF CULTURE MEDIUM
Nutrient Agar Plate
1. Dissolve 5 grams of Nutrient agar powder in 225 ml of distilled water in an Erlenmeyer flask.
(Follow the direction written on the container of the medium for the media preparation.) Mix
and heat over the magnetic stirrer until the medium becomes clear or transparent.
2. Sterilize the culture media in the autoclave at 15 psi, 121.5 degrees Celsius for 15 mins.

DISPENSING MEDIUM
Nutrient Agar Plate
1. Lay out four (4) sterile Petri dishes on the table with the cover partially open and dispense the
medium inside the inoculating hood.
2. Dispense the nutrient agar medium 10 to 15 ml and allow 5 to 10 minutes to elapse before
completely covering the dish to avoid contamination.
3. When the medium is solidified, label the nutrient agar plate. Wrap and label the plate.

Note: if the agar is not to be inoculated yet, refrigerate the agar plate in an upside down position.
If the medium to be inoculated is taken from the refrigerator, allow at least 10-20 minutes to
elapse for the medium at room temperature for inoculation.

Data and Draw and label the preparation and dispensing of the culture medium.
Observation

Discussion

Conclusion

Post Lab

1. What are different measures taken to prevent contamination of the

culture media?
2. Discuss the classification of culture media according to physical state, composition, and uses

3. After culture medium is sterilized and dispensed, how would you test if the medium is really free from
contamination?