LaB 3 : NuRaIn BiNtI aRzMi~111400

LAB 3: PREPARATION AND STERILIZATION OF CULTURE MEDIA


Introduction


Culture media are available commercially as powders; they only require the addition of water. Nutrient medium is general purpose preparation for culturing microorganism not nutritionally fastidious. The broth contains:
3.0 g/L “Lab-lemco” powder (a beef extract)
2.0 g/L yeast extract
5.0 g/L peptone (a nitrogen source)
5.0 g/L sodium chloride
15.0 g/L agar powder
The agar has the same composition, except that it contains 15 g/L agar. The final pH of both media is 7.4.
Process that use moist heat and pressure so that all parts of the material to be sterilized reach 121’C for 15 minutes is called autoclaving. This process is in essence, a large pressure cooker; a chamber which may be sealed off against surrounding air. For sterilization, the materials 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.
Then 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. Large volume required more time.


Objective


To prepare sterile nutrient agar for culturing microorganisms


Discussions


      Plant tissue culture media are generally sterilized by autoclaving at 121 °C and 1.05 kg / cm2(15-20 psi). The time required for sterilization depends upon the volume of medium in the vessel. It is advisable to dispense medium in small aliquots whenever possible as many media components are broken down on prolonged exposure to heat. There is evidence that medium exposed to temperatures in excess of 121 °C may not properly gel or may result in poor cell growth.
      Several medium components are considered thermolabile and should not be autoclaved. Stock solutions of the heat labile components are prepared and filter sterilized through a 0.22 µm filter into a sterile container. The filtered solution is aseptically added to the culture medium, which has been autoclaved and allowed to cool to approximately 35-45 °C. The medium is then dispensed under sterile conditions. Experimentation with your system is recommended.
      Liquid media which are sterilized in their final containers should be cooled down to room temperature as rapidly as possible. Screw caps should then be tightened.
      Containers of agar media which have been sterilized should be placed in a 50°C water bath and the medium dispensed as soon as it reaches this temperature, or within a maximum of 3 hours in the bath. The medium should be mixed thoroughly, without bubble formation and aseptically dispensed into sterile containers. Do not expose dishes of agar media to sunlight; it causes excessive condensation on the lids and may cause the formation of inhibitory substances by photo-oxidation.


References


• http://www.sigmaaldrich.com/life-science/molecular-biology/plant-biotechnology/tissue-culture-protocols/media-sterilization.html
• http://www.cabri.org/guidelines/micro-organisms/M203Ap1.html