LAB 3: PREPARATION AND STERILIZATION OF CULTURE MEDIA

Introduction
          Naturally, microorganism exists in all places and surrounding environment. So, it is difficult to find the original habitat of the microorganism. Factitious condition in laboratory is created to cultivate this microorganism. A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants. There are different types of media for growing different types of cells. The most common culture media for microorganisms are nutrient broths and agar plates, specialized media are sometimes required for microorganism and cell culture growth. While, 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” poeder (a beef extract)
2.0g/L yeast extract
5.0g/L peptone (a nitrogen source)
5.0g/L sodium chloride
2.0g/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.

Media preparation guidelines:
- Always read the label on the container of the medium before use. Verify and record the medium, the manufacturer ,and the expiry date.
- Accurately weigh or measure all ingredients and add them according to the instructions for the procedure being used.
-Use distilled or deionized water.
-If the medium contains agar, stir continuously and heat to boiling before dispensing into bottles. Agar melts (as evident by medium clearing in boiling aqueous solution. Agar is generally used at 15g/L, although this may vary depending on the medium and use.

          An autoclave is an instrument used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C or more, typically for 15–20 minutes depending on the size of the load and the contents. The various media constituents and other reagents used in cell cultures must be carefully sterilized either by autoclaving or by filtration. 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.

Objective
Refer to the lab manual.

Material and reagents
Ready-made commercial nutrient agar
Balance
Distilled water
Scott bottles
Measuring cylinder
Beaker
Forcep
Universal bottles

Procedure
Refer to the lab manual.

Discussion
          These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar. Bacteria had grown in liquid cultures often form colloidal suspensions.
          There are many steps to be taken in the process of preparing culture medium. We cleaned the pan and inside of the balance with a brush before we weighted the culture medium powder so that it is free from anything on or around its surface that can affect our readings. The “tare” button can only press after the container is put onto the balance to get the accurate reading. In addition, the distilled water was measured using measuring cylinder. The water should not pour all into beaker, because there should be some distilled water reserved for washing of leftover powder from the weighing container into beaker. The correct amount of distilled water is added to make sure we produced the correct composition of culture media. After the distilled water is added to the culture medium powder, we using rod to stir the media so that the all powder form culture medium dissolved in water. In the final step when the entire medium has been poured into the Scott bottles, we labelled each of the bottles and loosen the cap of the bottle before putting them into autoclave machine. This is because the autoclave works under high steam pressure. Therefore we loosen the cap to allow the expansion of the bottle so that the bottle will not break. After autoclaving, the Scott bottles were removed from the autoclave machine and the cap of the bottle is tightened. The bottles should be turned over again for a few times so that no agar will solidify at the bottom of the bottle. This is to make sure that the culture agar can be used for the pour-plate in the next laboratory work.

Conclusion
          Bacteriological media have a wide range of types. Nutrient Agar is a complex medium because it contains ingredients with contain unknown amounts or types of nutrients. Nutrient Agar contains Beef Extract (0.3%), Peptone (0.5%) and Agar (1.5%) in water. Beef extract is the commercially prepared dehydrated form of autolysed beef and is supplied in the form of a paste. Peptone is casein (milk protein) that has been digested with the enzyme pepsin. Peptone is dehydrated and supplied as a powder. Peptone and Beef Extract contain a mixture of amino acids and peptides. Beef Extract also contains water soluble digest products of all other macromolecules (nucleic acids, fats, polysaccharides) as well as vitamins and trace minerals. Although we know and can define Beef Extract in these terms, each batch cannot be chemically defined.
          There are many media ingredients which are complex: yeast extract, tryptone, and others. The advantage of complex media is that they support the growth of a wide range of microbes. Agar is purified from red algae in which it is an accessory polysaccharide (polygalacturonic acid) of their cell walls. Agar is added to microbiological media only as a solidification agent. Agar for most purposes has no nutrient value. Agar is an excellent solidification agent because it dissolves at near boiling but solidifies at 45°C. Thus, one can prepare molten (liquid) agar at 45°C, mix cells with it, and then allow it to solidify thereby trapping living cells. Below 45°C agar is a solid and remains so as the temperature is raised melting only when >95°C is obtained.
          The ready make nutrient agar is autoclaved at 121°C for 15 minutes. Autoclaving is a process that use moist heat and pressure so that all parts of the material to be sterilized. An autoclave is, in essence, a large pressure cooker. A chamber must be sealed off against surrounding air. Materials for sterilization are placed in the chamber. The door is sealed, and pressurized steam is forced into chamber. The incoming steam displaces cooler air through an exhaust valve. This valve closes when the cell cooler air has 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°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 sterilized objects can then be removed.

Reference
http://en.wikipedia.org/wiki/Growth_medium
http://en.wikipedia.org/wiki/Agar_plate
http://en.wikipedia.org/wiki/Autoclave
http://www.studentsguide.in/animal-biotechnology/animal-cell-and-tissue-culture/preparation-and-sterilization-of-medium.html
http://www.bd.com/ds/technicalCenter/inserts/Nutrient_Agar.pdf
http://www2.fiu.edu/~makemson/MCB2000Lab/Exp3MediaPrep.pdf
http://en.wikipedia.org/wiki/Agar
http://www.cabri.org/guidelines/micro-organisms/M203Ap1.html