Lab 4271

INTRODUCTION

A culture media is special medium used in microbiological laboratories to grow different kinds of microorganisms. A growth or a culture medium is composed of different nutrients that are essential for a microbial growth.

Since there are many types of microorganisms, each having unique properties and requiring specific nutrients for growth, the culture media are of many types based on what nutrients they contain and what function they play in the growth of microorganisms.

A culture may be solid or liquid. The solid culture media is composed of a brown jelly like substance known as agar. Different nutrients and chemicals are added in it to allow the growth of different microorganisms.

culture media

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.When microbiological media has been made, it still has to be sterilized because of microbial contamination from air, glassware, and hands. Within a few hours there will be thousands of bacteria reproducing in the media so it has to be sterilized quickly before the microbes start using the nutrients up. The sterilization process is a 100% kill, and guarantees that the medium will stay sterile unless exposed to contaminants by less that adequate aseptic technique to exposure to air.

Media sterilization is carried out with the autoclave, basically a huge steam cooker. Steam enters into a jacket surrounding the chamber. When the pressure from the steam is at a certain point in the jacket, a valve allows the steam to enter the chamber. The pressure will go up over 15 pounds per square inch (psi): at this point the timer begins to count down---usually for 15 minutes, depending on the type of media. The high pressure in a closed container allows the temperature to go above the highest temperature one could get by just boiling, around 121 degrees C. Therefore, the parameters for sterilization with an autoclave are 121 C at >15 psi for 15 minutes. Fifteen minutes is the thermal death time for most organisms (except some really hardy sporeformers).

An autoclave

OBJECTIVE

To prepare sterile nutrient agar for culturing microorganisms.

DISCUSSION

The culture media are prepared by weighing as below ;

0.6 g/L “Lab-lemco”powder (a beef extract)

0.4 g/L yeast extract

1.0 g/L peptone (nitrogen source)

1.0 g/L sodium chloride

3.0 g/L agar powder

Beef extract

Yeast extract

Peptone

Sodium chloride

Agar powder

Here 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 weigh 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 be pressed 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 produce the correct composition of culture media. After the distilled water is added to the culture medium powder, we used rod to stir the media so that all powder from culture medium dissolved in water. Then, the entire medium has been poured into Sccott bottle and loosen the cap of the bottle before putting into the autoclave machine. 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 bottle was removed from the autoclave machine and the cap of the bottle is tightened. The bottle 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.