Lab 3 report written by Tang Su Xian

Name: Tang Su Xian
Matric card no.: 111431

Lab 3: Preparation and sterilization of culture media

Introduction:

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

3.0g/L “Lab – lemco” power ( a beef extract )

2.0g/L yeast extract

2.0g/L peptone( a nitrogen source)

5.0g/L sodium chloride

15.0g/L agar powder

  

  The ready-made nutrient agar contains 15 g/L agar and the same contents as the manually-made nutrient medium. The final pH of both media has to be 7.4.

    

  Autoclaving is a process that use moist heat and pressure so thst all parts of the material to be sterilized reach 121 ̊C 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 pressurixed 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 103 kPa above atmospheric pressure; at sea level, this pushes the temperature in the chamber to 121 ̊C. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121 ̊C throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilixed objects can then be removed.

Objective:

To prepare sterile nutrient agar for culturing microorganisms

Discussions:

1.)    There are several precaution steps to be taken in this experiment, which include:

a.)    The pan and the balance must be cleaned before weight the culture medium powders. This is to ensure that the culture medium powders are not containminated by other things and can get more accurate amount of culture medium powders.

b.)    Besides, distilled water should be used to mix with the culture medium powders instead of using tap water. This is because the tap water might contain high amount of minerals.

c.)     The culture medium should be stirred during the heating process of culture medium until the medium boils and the colour of medium changes from murky to clear. This is to ensure that all the agar powder has dissolved in the culture medium.

d.)    The caps of two Scott bottles that filled with distilled water and culture medium that have been heated should be recaped loosely before putting into the autoclave machine. This is to prevent the Scott bottles from broke during autoclaving process.

2.)    Sterilization of culture medium

a.)    Although sterilization of culture media is best carried out in a steam autoclave at temperatures between 121-134°C, it has to be recognised that damage is caused to the medium by the heating process.

b.)    It is important, therefore, to optimise the heating process so that a medium is sterile after heating but minimal damage is caused to the ingredients of the medium. As a general rule it is accepted that short-duration, high-temperature processes are more lethal to organisms and less chemically damaging than are longer, lower temperature processes e.g. 3 minutes at 134°C is preferable to 20 minutes at 115°C..  

3.)    When removed the Scott bottles from the autoclave machine,they should be allowed to cool down in a laminar air flow cabinet. Alternatively Scott bottles may be sterilized in a jar which is covered by a piece of felt which effectively protects the Scott bottles from infection by air- borne microorganisms. Caps of Scott bottles are screwed down tightly after the culture medium have been cooled to ambient temperature.

4.)    Laminar air flow is based on the flow of air current to create uniform velocity, along parallel lines, which helps in transforming microbial culture in aseptic conditions. When fresh air is passed in the laminar air flow it replaces the comtamintaed air inside and keeps it contamination free. 

Conclusion:
I have learned the way to prepare and sterilize the culture medium. During the preparation of culture medium, I found that there are a lot of precaution steps are needed to be taken in order to prepare a sterilized culture medium successfully.

References:

http://www.bc-technology.info/laminarflow-working-benches.html

http://www.secondaryteachersstore.net/all_about_agar.pdf
http://en.wikipedia.org/wiki/Laminar_flow_cabinet

http://en.wikipedia.org/wiki/Autoclave