Title: Media Preparation,sterilization and Aseptic Technique

Title: Media preparation,sterilization and aseptic technique

Objectives:

1. To describe different types of culture media and their composition
2. To demonstrate skills in the preparation of agar, agar deep and agar slant
3. Describe the importance of aseptic technique in microbiology

Introduction:

The cultivation of microorganisms can only be carried out when certain factors are fulfilled such as proper nutrients. There are more than 500 types of media used to identify the microorganisms as they varies in nutrient content and consistency. Media can be classified into three; liquid or broth, semisolid and solid.

Liquid media is a water based solution hence it tends to flow freely in the container. It has nutrient broth, citrate broth, and even litmus milk which is an opaque liquid. Liquid media is solidified by agents namely agar or silica gel. Whilst semisolid medium has a clot-like consistency, it has solidifying agent of low percentage to ensure that it will not turn firm. This jellylike media is used to localize a reaction at a specific site and the location of the growth is used to specify the non-motility. Contrary to liquid, solid media is useful in isolation and cultivation of bacteria. The common agent used for solid media is agar because it can reverse the solid media to liquid at boiling temperature which is 100℃. Synthetic media is composed of known composition and concentration. Non synthetic media which is complex must have one chemically undefinable or more.

Sterilization is needed in culturing media because it is important to clear the medium from other microorganisms. Sterilization can be done by autoclaving and membrane filtration. Autoclaving is carried out for 15 minutes in 15 pound pressure with 121℃ autoclave machine. Autoclaves should be checked of interval period of time to ensure it functions efficiently. A autoclave tape is plastered on the bottle and after 15 minutes, black lines can be seen on the tape that indicates the process is completed. Next, the aseptic technique is used to prevent unwanted contaminants from the environment entering the culture as contaminants move freely in dust particles. The light of Bunsen burner should be adjusted to blue flame as it is more visible. This burning process will conduct the air convection to take place.

[pic 1]

Methodology:

Materials and Apparatus:

Nutrient agar

Distilled water

Laboratory glass bottle

Petri dish

Screw cap test tube

Bunsen burner

Incubator

Measuring cylinder

Weighing boat

Spatula

Procedures:

Media preparation and sterilization

1. 2g of nutrient agar was weighed and placed on weighing boat using a spatula.
2. 100 ml of distilled water was measured using a measuring cylinder and poured          into a laboratory glass bottle along with the nutrient agar. The bottle was         shaken to mix the ingredients.
3. The mixture was autoclaved at 121℃ and 1.5lb pressure for 15 minutes.
4. A bottle of prepared nutrient agar was used to prepare agar, agar deep and agar         slant. The bench was cleaned with 70% ethanol and Bunsen burner was lighted         up with a lighter. The flame was adjusted until blue flame showed.
5. Screw cap test tube A was lifted until its position was almost the same level as the blue flame and 3/4 of the prepared nutrient agar was poured to make agar deep and repeated with test tube B with 1/2 nutrient agar for agar slant.
6. Screw cap test tube B was placed on the rack and tilted to get the correct degree of slant. Both test tubes were left to solidify before disposing.

Aseptic technique

1. Petri dish A,B and C each labelled open, closed and dirty&clean(divided) respectively were poured with the prepared nutrient agar. Petri dish A was left open and B was closed with its lid while C was touched with unwashed hand for dirty part and touched with washed hand for clean part. All 3 petri dishes were covered with their lid and placed invertedly in the incubator of 37℃.
2. After at least 24 hours, the petri dishes were collected and results were observed.

Results:

 [pic 2]       [pic 3]

1. Left in open air                                                B) Left in closed air

[pic 4]

C) A; clean, B; dirty

Discussion:

This experiment was conducted mainly to prepare culture media for bacteria cultivation purpose. In preparing the media, there are several technique that are needed to use in order to prevent from contamination. There are different types of culture media with different composition. The media prepared were incubated for 24 hours and the bacteria growth was observed.

After observing, petri dish A was found out to have only one small spot in the medium culture. While in theory, Louis Pasteur found that medium exposed to open air will have microbial growth because microorganisms in the dust particles has entered the contaminated container. Hence, supposedly petri dish A should show big spot that indicates the growth in the medium but the observation is different because it was left in open air of laboratory. Laboratories must maintain their clean air and suitable pH to reduce contamination levels and the surface is often sanitized with proper sanitizer. This can be done by air filtration system in the laboratories.

Petri dish B was left closed with its lid and the observation showed that there was no growth at all. This is because the microorganism cannot enter the dish in short amount of time, given that it was only left for 15 minutes before incubated for 24 hours. Meanwhile, petri dish C that were divided into two parts have shown quite a result. A area was touched with clean hand that was washed for 5 minutes approximately using Dettol handwashing liquid and this rinsed most of the microorganisms. Hence, it showed small colony of bacteria. B division was touched with unwashed hand so the result is different from A division as it has bigger colony of bacteria.