Activity No.

2 Report Sheet

MEDIA PREPARATION
Name: Buenaflor, Andrea Mae P.
Enorio, Isabelle D.
Reyes, Shylene Kaye A.
Rivera, NJ Aleah F.
Yap, Fhardina D.

I.Introduction
Several kinds of microbiological media are used to cultivate bacteria and fungi. Depending
on the microorganism that is being isolated or identified, a specific culture medium must be
utilized. The medium may be given additional nutrients to increase its protein or sugar content.
Several pH indicators are frequently used to differentiate bacteria depending on their metabolic
activities; the indicators may change color when mildly basic or acidic. The medium may also be
supplemented with growth stimulants, NaCl, and pH buffers to prevent it from deviating too far
from neutral as the microorganisms metabolize.
The traditional process for creating media involved adding each ingredient gradually, like
in a recipe. Only when creating a customized medium to grow a particular picky organism where
certain growth agents, nutrients, vitamins, and so forth have to be given in specific amounts.
Chemically defined media is the name given to this medium (synthetic). And the most common
bacteria that we want to cultivate can do nicely with the media that we regularly use in the lab.
While some of our media is purchased.

Nowadays, creating complicated media is actually very easy:

• rehydrate the medium's powdered form
• dissolve the agar powder, stir and boil the agar medium (if making an agar medium rather
than a broth medium)
• dispense the substance into tubes.
• sterilize the tube medium with an autoclave.
• pour the autoclaved agar medium for making plates into sterile petri dishes.

Even after making microbiological media, it must be sterilized due to microbial

contamination from air, glassware, hands, and other sources. Thousands of bacteria will reproduce
in the medium within a few hours, so it must be sterilized quickly before the microbes start
depleting the nutrients. The sterilization process ensures that the medium will remain sterile unless
exposed to contaminants via a less-than-adequate aseptic technique (air exposure).

Media should always be stored in a cold, moist environment to prevent evaporation,

preferably in screw-capped tubes or bottles. Therefore, it is not advised to keep sterile media for
an extended period of time unless stability has been proven. Media tubes should be reheated right
before use if they have been stored for any period of time. To release dissolved gasses, liquid
media should be heated for a short time in a boiling water bath or in flowing steam and then
immediately cooled in cold water without agitation soon before inoculation. All bacteria prefer a
damp surface, thus agar tubes should be melted and then allowed to solidify.

II. Objectives
• To learn about the environmental and nutritional requirements for cultivating organisms
cultured in a lab.
• To comprehend the use of an autoclave in the decontamination or sterilization process.
• To become familiar with the methods for creating the media required for cultivating
microorganisms.

III. Requirements:
A. Materials: Erlenmeyer Flask, 10mL Graduated Cylinder, Petri Plate, Electronic Balance,
Hot Plate, Stirring Rod, Test Tubes, Test Tube Racks, Beaker, Autoclave
B. Chemicals: Distilled Water, Media Base Powder

III. Procedure
1. Start by adding 250 ml of distilled water to a 500ml or 1L flask to make the TSB (broth).
Turn on the hot plate and insert the stir bar to gently disrupt the surface. 3.25 grams of the
TSB powder should be added to the flask, and it should dissolve (will happen quickly). At
this point, there is no need to use heat.
2. Pipette out 5 ml of the green cap after the powder has been dissolved.
3. With the remaining solution (about 100ml) still stirring, add 2 grams of agar powder.
4. The next step will require you to apply heat to the mixture. The agar has a strong tendency
to boil over when it reaches 100⁰C. Watch the flask at all times once you see steam coming
off of it.
5. Remove the mixture from the heated plate as soon as it begins to boil (paper towels around
the flask neck). DON'T just turn off the heat and leave the flask alone. Turning off the heat
won't stop the flask from boiling over since the metal plate retains a lot of heat.
6. The agar dissolves as it is heated up and turns a clearer, darker tan. The remainder of the
agar medium in the flask will be poured into one large flask to save it for the preparation
of petri plates after it has been taken off the heat and pipetted into 5 tubes for slants.
7. Put the remaining melted agar and all of the tubes that you have pipetted out in the plastic
autoclave racks.

IV. Conclusion
We came to the conclusion that there are essential things that should and shouldn't be done
when preparing the cultured medium, the agar. The agar must be dissolved before heating the
mixture in the Erlenmeyer flask with the distilled water because if not, the agar will burn. Then,
the mixtures were heated in the microwave to completely dissolve the agar rather than on a hot
plate since we did not have enough time. But because the Erlenmeyer flask that our group used
was too large to fit in the microwave, we were unable to complete this step. This phase of the
procedure was noted by our group, as we presumed that the absence of this could contribute to the
results. The mixture was then autoclaved for sterilization.
In preparing the broth, we dissolved the TSB powder in a 1 L flask containing 250 ml of
distilled water. Hence, this distilled water is important as a medium through which nutrients can
be transferred. Realizations have been made that, in doing the procedures, microbial contamination
could easily happen. With that, ensuring proper protective apparel is important, as well as in
quickly sterilizing the medium. Many sources and factors from the surroundings could affect the
results of the laboratory activity.
Documentation
V. References
• Microbial Culture Media Preparation. (2023). Merck. Retrieved March 9, 2023,
from https://www.sigmaaldrich.com/PH/en/applications/microbiological-
testing/microbial-culture-media-preparation
• Mettler-Toledo International Inc. all rights reserved. (2022, November 16).
Culture Media Preparation. Mettler-Toledo International Inc. All Rights
Reserved.
https://www.mt.com/ph/en/home/applications/Laboratory_weighing/Culture-
Media-Preparation.html
• Al-Tameemi, H. (2018, July 12). Medical Microbiology Laboratory (culture
media preparation). https://www.slideshare.net/HusseinAltameemi2/medical-
microbiology-laboratory-culture-media-preparation
• Aryal, S. (2022, August 10). Nutrient Agar: Composition, Preparation and Uses.
Microbiology Info.com. https://microbiologyinfo.com/nutrient-agar-composition-
preparation-and-
uses/#:~:text=Water%20is%20essential%20for%20the,various%20nutrients%20c
an%20be%20transported.