Matric No : 111427
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:
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 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 degree 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 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 degree C. The high pressure prevent solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121 degree C 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.
Discussions
1) We need to weigh and mix all the ingredients into the beaker to prepare culture medium. After mixing the ingredients, we need to add 200 mL distilled water into the beaker which has been measured using measuring cylinder.
2) After preparing culture medium solution, we heat up culture medium until it boils. While heating, we use rod to stir the media so that the all powder form culture medium dissolved in water. Next, we pour whole culture medium into Scott bottle and loosely recap the bottle.
3) Culture medium in Scott bottle need to be autoclaved. Autoclaving is most effective and most efficient means of heat sterilization.Culture medium should be sterilized reached 121 degree C for 15 minutes to ensure there are no microorganisms contamination.
4) The autoclave operates by using steam under high pressure as sterilizing agent. High pressure enable steam to reach high temperature which can increase heat content and killing power.
5) After autoclaving, Scott bottles were removed from autoclave machine. Sterile media is then put into vertical laminar flow cabinet ( biological safety cabinet) for the following process.
6) Vertical laminar flow cabinet provide a clean, safe environment for sterile media. It use a high-efficiency particulate air (HEPA) filter and blowers that generate a non-mixing stream of air.
Conclusion
We are able to prepare culture medium with correct quantities of ingredients. Preparing culture media need to be go through some process such as weighing the ingredients, mixing ingredients with distilled water, heating media until it boils and autoclaving. Finally, we have prepared culture medium successfully.
References
http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/cell-culture-equipment/laminar-flow-hood.html
http://www.aircleansystems.com/process_vertical.htm
http://www.cabri.org/guidelines/micro-organisms/M203Ap1.html
http://www.cib.csic.es/en/servicio.php?iddepartamento=23
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