LAB 3 : Preparation and Sterilization of Culture Media

Introduction :

            Culture media are available commercially as powders; the require only addition of water. Nutrient medium is a general preparation for culturing microorganisms which are not nutritionally fastidious.The broth contains:

1.50 g/L "Lab-lemco" powder (a beef extract)
1.50 g/L yeast extract
5.00 g/L peptone （a nitrogen source)
5.00 g/L sodium chloride
15.00 g/L agar powder

The agar has the same composition, except that it contains 15g/L agar. The final pH of both media is 7.4.

     Autoclaving is a process that use moist heat and pressure so that all parts of the material to be sterilized reach 121^oC for 15 minutes. An autoclave is, in essence, a large pressure cooker; a chamber which may be sealed off against surrounding air. Materials for sterilization are placed in the chamber, the door is sealed, and pressurized steam is forced into the chamber. The incoming steam displaces cooler air through an exhaust valve; this valve closes when the cell cooler air has been vented.

     Steam is continually forced into the chamber until the pressure reaches 103kPa above atmospheric pressure; at sea level, this pushes the temperature in the chamber to 121^oC. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121^oC throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilized objects can then be removed.

Objective :

To prepare sterile nutrient agar for culturing microorganisms.

Materials and reagents :

Commercial nutrient agar

Balance

Distilled water

Scott bottles

Measuring cylinder

Beaker

Glass rod

Procedures :

1. The amount of broth ( with agar ) is weighed appropriately in the Scott bottles and is  dissolved with distilled water. The mixtureis then mixed well.
2. The bottles is loosely recap and set aside for sterilization.
3. All media is sterilized at 121^oC for 15 minutes.
4.  After autoclaving, the media is removed. The broth preparation is allowed to cool then the cap of each bottle is tighten.

Discussions :

1. Precautions 

a)     The pan and the balance must be cleaned before weight the culture medium powders. This is to ensure that more accurate amount of culture medium powders is weighed.

b)     The door of the balance machine should also be closed before weighing the substances and pressed the ‘tare’ button after the pan or beaker is put onto the balance to avoid zero error.

c)     Before use the apparatus, rinsed all the apparatus with distilled water to avoid contamination.

d)     The Scott bottles` cap have been heated in autoclave should be recapped loosely before putting into the autoclave machine. This is to prevent the Scott bottles from breaking during autoclaving process.

e)     Use the rod to stir before pour the media into the Scott bottles to ensure that all powders from cultural medium were dissolved in the distilled water.

2. Working of Autoclave 

     Most autoclaves contain a sterilizing chamber into which articles are place and a steam jacket where steam is maintained. As steam flows from the steam jacket into the sterilizing chamber, cool air is forced out and a special valve increases the pressure to 15 pounds/square inch above normal atmospheric pressure. The temperature rises to 121.5 ^oC  and the superheated water molecules rapidly conduct heat into microorganisms. The time for destruction of the most resistant bacterial spore is now reduced to about 15 minutes. For denser objects, up to 30 minutes of exposure may be required. The conditions must be carefully controlled or serious problems may occur.

3. Effectiveness of Autoclave or Optimum Conditions

    Sterilization in an autoclave is most effective when the organisms are either contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under these conditions, steam at a pressure about 15 psi; attaining temperature (121 ^oC) will kill all organisms and their endospores in about 15 minutes.

Conclusion :
The process that we go through when preparing culture media are important before autoclaving. We are able to prepare culture medium with correct quantities of ingredients. Autoclaving is the effective way use in sterilization.



References :  
http://microbiologyon-line.blogspot.com/2009/08/autoclaving-real-sterilization.html
http://en.wikipedia.org/wiki/Autoclave
http://www.cabri.org/guidelines/micro-organisms/M203Ap1.html