Bacteriological Media and Sterilization

Purpose:

To understand the basic principles of Media preparation, and dispensing of media
To learn and the practice sterilization of media like Nutrient Broth Media and Nutrient Agar Media
To prepare solid media like nutrient agar plates to use following experiments.
To prepare deep agar and agar slant.
To be familiar with calculation of content of any kind of media.
To become familiar with sterilization techniques.

Procedure:

PREPARING AND DISPENSING MEDIA & STERILIZATION OF MEDIA AND EQUIPMENT
(Exercise1& Exercise2)

Combined & Simplified protocol:
Dissolve 1,2g Nutrient Broth and 2,25g agar in 250 ml of flask by using microwave oven up to boiling point for 2 or 3 minutes
Label each two tubes with group number, section number, date,
And then label them as deep and slant.
Pour 5 ml to deep labeled tube and 8 ml to slant labeled tube by using pipette.
Sterilize two tubes and 250ml of flask at 121 °C for 15 minutes by using autoclave. (It will take around 45 min.)

After autoclaving all media, pour media into Petri dishes under the flow, which has a sterile, aseptic environment. (Around 50 ml for each Petri dish). Leave them for a while to dry, and then keep them in the refrigerator for future studies.
Then, to obtain slant agar, keep tubes nearly horizontal to obtain a large surface area.

Discussion:

In this experiment, we have learned basic molecular biology techniques and equipments such as media preparation and autoclave respectively. After careful weighting of components of media and correct addition of accurate amount of water, we dissolved by using oven. We used oven because it gives boiling temperature (100 °C), so that agar dissolves easily at this temperature. Moreover, we performed dispensing technique under laminar flow, so that we saved our liquid media from any possible contamination from air flow based contaminations.

In our media, Nutrient broth was the main source of energy, mineral, carbon, nitrogen, in other words, all essential things for heterotrophic organisms. We used agar as a solidifying agent to prepare solid media. Our media was one of the complex media that can be used to grow variety of organisms on it, even auxotroph microorganisms.

We used heat sterilization, in particular, steam sterilization. Steam sterilization is the best sterilization technique for non-labile equipment and though, stable media. When we compare it with other methods like UV or Filtration sterilization, Steam sterilization is the best one because no organism or spore or viruses can escape from heat inactivation.

Boiling temperature (100 °C) of water is enough to kill all kind of living bacteria but to kill other living things such spores and viruses further increasing of temperature is necessary. Therefore we use a special equipment lie autoclave to increase temperature by increasing pressure (15psi) inside of the machine so that water boils at 121 °C instead of 100 °C. As a result we can sure about sterilization of media.
We prepared “deep agar” tube (standing straight) and “slant” tube standing in a slant position as the name suggests. Actually keeping the tube in this position aims to increase the surface area. In the “deep agar” tube bacteria are grown at the bottom, whereas in the “slant” tube bacteria are grown on the surface.

Media preparation, sterilization and Petri plate techniques are important because they allows researchers to study unique colonies selected, and store them in an controlled environment.

In conclusion, these exercises were successful and they were a good start to study and learn microbiology, because they are essential of microbiology studies. New procedures were practiced, and further understanding of media preparations, sterilization methods, and dispensing methods were gained.

REFERENCE:
No specific reference…
Lab course notes….
And lab manual of microbiology (bio355)…

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