LAB TOPIC : CULTURE MEDIA AND STERILIZATION

1.0 INTRODUCTION

A culture medium or growth medium is a liquid or gel designed to support the growth of
microorganisms. There are different types of media suitable for growing different types of cells.
These are the most common growth media, although specialized media are sometimes required for
microorganism and cell culture growth. Some organisms, termed fastidious organisms, need
specialized environments due to complex nutritional requirements. Bacteria, for example, are
obligate intracellular parasites and require a growth medium containing living cells. Many human
microbial pathogens also require the use of human cells or cell lysates to grow on a media. Culture
media contains the nutrients needed to sustain a microbe. Culture media can vary in many
ingredients allowing the media to select for or against microbes. Glucose or glycerol are often used
as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources in culture media.

2.0 OBJECTIVE

This experiment’s objective is to prepare culture media from basic ingredients and ready-
made dehydrated media. Students will learn about the right ways to prepare the media without
contamination. The media prepared will be used in the following experiment. Additionally, after
using the equipment and material, students will learn about the procedures for sterilizing it and the
medium to make sure no living microbes remain on it. Microbes on the equipment have the
potential to contaminate the lab space or have an impact on the results of the experiment. Finally,
by participating in this experiment, students will gain practical experience as well as knowledge
of appropriate lab procedures.
3.0 METHODS

BROTH GRANULE
AGAR GRANULE
4.0 RESULTS

Calculations :
1. Broth Agar from Nutrient Agar
60
𝑥 20𝑔 = 1.2𝑔
1000
7
2. Granule Agar from MacConkey Agar
60
𝑥 50𝑔 = 3𝑔
1000

3. Broth from Nutrient Broth

30
𝑥 8𝑔 = 0.24𝑔
1000

4. Broth from Nutrient Agar

30
𝑥 20𝑔 = 0.6𝑔
1000
5.0 DISCUSSION

Bacteria require oxygen to live. The culture medium allows bacteria to grow in two
different environments which are aerobic and oxygen-rich surface. Nutrient agar contains nutrients
that are suitable to subculture a wide range of microorganisms. Besides, Nutrient broth is the
nutrient agar that lack of the solidifying agent, agar powder. They remain in liquid form at room
temperature. Therefore, they are used to grow fastidious organisms. We can enrich the nutrient
broth with blood, serum and sugars. can grow on the surface of the broth agar. Bacterial growth in
broths is indicated by the development of a cloudy appearance. Thus, growing non-fastidious
microorganisms with no nutritional requirements is the aim of nutrient broth.

The components in these media is agar. Agar functions as a solidifying agent, similar to
gelatin. Agar gives media a gel-like consistency that creates a firm surface for bacterial growth. In
the absence of agar, discrete colonies would not develop and the medium would instead resemble
a liquid soup. Next component is peptone. Peptone is the result of breaking down proteins, mainly
animal proteins, by enzymes. To make their own proteins, bacteria require a source of nitrogen
and amino acids, which peptone provides.

A culture medium is simply water and nutrients that support microbial growth. Selective
media are used to select for the growth of a particular selected microorganism. The general purpose
medium that will support the growth of a wide variety of bacteria include nutrient agar and tryptic
soy agar. A medium may be enriched, by the addition of blood or serum.

Sterilization procedures eliminate all viable microorganisms from a specified region. All
the components such as culture dishes, test tubes, flasks, pipettes, transfer loops, and media must
be free from any microorganisms before starting to establish a pure culture of microorganisms.
The containers, liquids, and instruments must be exposed to high-temperature levels to prevent
contamination. This experiment must be held in an aseptic condition to prevent microbes from
unintentionally being released into the environment and contaminating lab users in the laboratory.
In addition, this technique is to prevent the transfer of bacteria from the surrounding environment
into a culture medium and to grow and maintain the bacteria in the culture.

Bacteria live in every corner of our environment. We come in contact with bacteria on a
daily basis. Therefore, we need to take precautions to prevent bacteria from spreading into the air
or damaging the results of the experiment. Firstly, we must always sterilize all the equipment and
materials that we use during investigation by autoclaving. Secondly, we should always disinfect
work areas before and after using 75% alcohol and always use disinfectant soap to kill all the
bacteria that may attach to our hands. In addition, do not eat or drink in the lab, nor store food in
areas where microorganisms are stored and store the medium in a contamination-free environment
for later use.

6.0 QUESTIONS

1. Why are pH buffers added to the growth media?

Buffers are significant in maintaining the proper environment within microorganisms and
within other cells. In the microbial lab, several solutions and growth media are buffered to
prevent sudden and adverse changes in the acidity or alkalinity of the environment
surrounding the microorganisms.

2. Why do you have to boil agar first before dispensing it into tubes?
We need to boil the agar first in order to make sure the agar powder is completely dissolved
and the agar is distributed evenly throughout the media. This is important because undissolved
agar can cause clumping or unevenness in the agar when it solidifies. In addition, to reduce the
possibility of post-sterilization contamination, which could happen if the dry powder stays above
the water's surface.

3. Why do we use 70% alcohol instead of 95% alcohol during disinfectant?

 We use 70% alcohol because this alcohol contains between 60% and 90% alcohol with 40%
purifies water that can kill bacteria, fungi, and viruses quickly. 70% alcohol penetrates the cell
wall more completely which permeates the entire cell, coagulates all proteins, and therefore
the microorganism dies. Thus, 70% alcohol solution in water is more effective than 95%
alcohol and has more disinfectant capacity. Extra water content slows evaporation, therefore
increasing surface contact time and enhancing effectiveness. In addition, this 70% alcohol is
safer to use and store such as in sanitizer.

4. What are the two sterilization methods?

The two sterilization methods are Dry heat and Autoclave. By burning and heating the coil
until the ember, the coil wire can be made sterile. Burning is an effective way to get rid of
germ- and other organism-infected material. Therefore, by oxidizing the bacteria's
protoplasm, hot air will kill the bacteria. Due to the weak conductivity of air, complete
sterilization requires high temperatures and prolonged exposure. We use autoclaves because
they provide a physical process of disinfection and sterilization. They combine stem,
pressure, and time to operate.

7.0 CONCLUSION

This experiment aims to prepare culture media from basic ingredients and ready-made
dehydrated media. The media we prepared are important to see the growth of microorganisms and
this media can also be used in other experiments. From these experiments, we are able to prepare
the media the right way without contamination. We also have learned the steps to ensure there are
no living microorganisms on the apparatus and material after using it. As we know, contamination
from the microorganism can affect the results of experiments. Therefore, precautions are essential
in this experiment especially to get good media.
8.0 REFERENCES

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