MLT 415 – Fundamentals of Microbiology

Objectives:

1. To prepare nutrient agar and Mueller Hinton agar media.

2. To weight the media using the balance.
3. To melt the agar media following instructions on the bottle.
4. To sterilize by autoclaving for 15 min at 121oC.
5. To melt the nutrient agar and Mueller Hinton agar media.
6. To aseptically prepare nutrient agar and Mueller Hinton agar.
7. To test for sterility the media prepared.

Principles:

Once the microscopic morphology and staining characteristics of a

microorganism present in a clinical specimen are known, the
microbiologist can make appropriate decisions as to how it should be
cultivated and what biological properties must be demonstrated to
identify it fully. First a suitable culture medium must be provided and it
must contain the nutrients essentials for the growth of the
microorganisms to be studied. Most media designed for the initial growth
and isolation of microorganisms are rich in protein components derived
from animal meats.

A growth medium or culture medium is a liquid or agar designed to

support the growth of microorganisms or cells. A culture medium is a
gelatinous substance that contain essential nutrient to cultivate the target
microorganisms for further purpose. Today, there are many types of
culture media available commercially. Some media can be made of cooked
blood, meat, milk & various other products. Culture media are available
commercially as powders; they require only the addition of water.

There are three state of culture medium which is solid, semi solid
and liquid. The major culture medium used widely today is agar and broth.
The type and state of culture media used is depending on the type of
organisms to be cultured. Some of it is chemically defined media which

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mean the media that the exact chemical composition is known. It is used
to grow fastidious organisms. Meanwhile some of the culture medium is a
complex media which mean that the exact chemical composition is not
known. Most of the bacteria and fungi are grown with this

The reason why agar was chosen to be the medium for bacteria
culture is gelatine was difficult to prepare and difficult to use at room
temperature, let alone at the higher temperature of an incubator, and
many bacteria digest the protein. Meanwhile agar has characteristics of
melting only when boiled, rarely being digested by bacteria, and providing
a substance in which other nutrients could be dissolved, proved to be a
suitable material on which to grow bacteria.

Nutrient broth is in liquid form while an agar plate is in a solid form.

The nutrient broth added with agar powder so that when the suspension
cools down, it will coagulate as a semi-solid medium. An agar plate is a
sterile petri dish that contains agar and nutrient. Selective growth
compounds may also be added to the media, such as antibiotics. The
microorganisms will grow on the surface of the agar and this makes
examination work so much easier as the colonies of microorganisms
remain stationery and clearly visible.

The Nutrient agar preparation is similar to the preparation of a

Mueller Hinton (MH) agar. The composition of both types of growth media
is as listed below:

1. Nutrient agar (pH 7.4 at 25OC)

i. ‘Lab-Lemco’ powder : 1.0 g/L

ii. Yeast extract : 2.0 g/L
iii. Peptone : 5.0 g/L
iv. Sodium Chloride : 5.0 g/L
v. Agar : 15.0 g/L

2. Mueller Hinton (MH) agar (pH 7.3 at 25OC)

i. Beef dehydrated : 300.0 g/L

infusion

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ii. Casein hydrolysate : 17.5 g/L

iii. Starch : 1.5 g/L
iv. Agar : 17.0 g/L

A culture medium must be sterilised before use so that no unwanted

microorganisms grow which may contaminate the growing sample. This is
because the nutrient broth and the agar-nutrient broth contained essential
nutrients for the growth of the microorganisms.

An autoclave is, in essence, a large pressure cooker; a chamber

which may be sealed off against surrounding air. Autoclaving, sometimes
called steam sterilization, is the use of pressurized steam to kill infectious
agents and denature proteins. Steam is continually forced into the
chamber and cause the chamber has high pressure. The purpose of the
high pressure inside the chamber is to prevent solutions from boiling over
at this temperature. This kind of "wet heat" is considered the most
dependable method of sterilizing laboratory equipment and
decontaminating biohazardous waste. Usually it utilises moist heat and
pressure that reach 121°C in 15 minutes to disinfect medical supplies and
laboratory equipment, extending their useful lifespan.

Materials/Equipment:

1. Sterilize petri dish

2. Schott bottle
3. Incubator
4. Bunsen burner
5. Tripod stand
6. Wire Gauze
7. Spatula
8. Glass stirrer
9. Weighing boat
10. Analytical Balance
11. Distilled Water
12. 500ml Beaker
13. Measuring Cylinder
14. Mueller Hinton (MH) agar powder
15. Autoclave

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16. Autoclave tape

Procedures:

1. The 500 ml of MH agar was prepared

2. The label on a bottle of dehydrated MH agar was read. It
specifies the amount of dehydrated powder required to make
1 liter of medium.
3. It was specifies that 38g of dehydrated power is needed to
make 1 liter of medium. The amount of powder needed for
500 ml was calculated and the weighted using analytical
balance. The calculation showed that to make 500ml of
medium, 19g of dehydrated MH agar powder was needed.
4. 500 ml of distilled water was measured using measuring
cylinder and poured in the 1000 ml beaker.
5. The weighed, dehydrated agar powder was added into the
distilled water while stirring with a spatula to prevent lumping.
6. The tripod stand and the Bunsen burner were set up and the
beaker was place on the top of the tripod stand over the wire
gauze.
7. The dehydrated agar solution was boiled slowly while stir it
gently.
8. When the agar powder mixture is completely dissolved, the
beaker was removed from the flame.
9. The agar solution was poured into Schott bottle and autoclave
at 121°C for 15 mins. The agar solution was kept warm inside
the autoclave overnight.
10. When the Schott bottle of sterilize agar is returned to
you, the cap of the bottle was removed with your right hand.
11. The melted sterile agar was poured quickly into a series
of sterilize petri dishes. The petri dish was filled to about one-
third capacity with melted agar.
12. The petri dish was inverted when the agar seem to be
solidified to prevent condensing moisture from accumulating
on the agar surfaces.
13. One inverted agar plates were placed inside the
incubator overnight for quality check purposes which to

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determine and ensure that the agar plates are sterile and not
contaminated.
14. The rest of agar plates were placed in the chiller for
further purposes.

Results:

1. The agar plat store inside the incubator (35oC overnight).

Observati There is no bacteria growth on the culture

on: medium. The agar is appearing to be yellowish in
colour and there is no white spot appears on the
surface of the agar.

Discussion:

In this study, we focused on preparation of agar plate culture media.

In the process of preparing culture medium, there are many precautions
steps to be taken. This is because to ensure the accuracy composition of
nutrient so the bacteria could grow perfectly inside the culture medium.

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Before the culture agar powders could be weighed, the analytical

balance must be cleaned with a brush or tissue so that it is free from any
dust or unwanted residue.. The door of the balance machine should also
be closed before weighing any substances and ‘tare’ button can only be
pressed after the container is put onto the balance. Also, the balance
should be allowed to stabilize for 1 minute after it has been on. These
steps are important in order to obtain an accurate reading.

Moreover, the volume of distilled water must be measure accurately

using measuring cylinder. This is because the label on the agar powder
bottle clearly state the specific volume of distilled water needed for
specific amount of agar powder. Furthermore, when measuring the volume
of distilled water, the measuring cylinder must be put on flat stable
surface. The level of eyes must be same as the level of the curved surface
of liquid (meniscus) to get the accurate reading. The distilled water in the
measuring cylinder should not be poured all out into the beaker, because
there should be some distilled water reserved for the washing of leftover
powders from the weighing boat into the beaker.

The culture medium should also be heated by Bunsen burner. The

medium is stirred properly and observed from time to time and only can
be removed when the medium has turned to slightly clear yellowish
colour. This step is to ensure that all the agar powder has dissolved
completely in the culture medium. However make sure that the culture
medium is not overcooked. This can be seen through the changes of
medium colour during the heating process. The overcooked culture
medium usually appears in dark yellowish colour.

In the final step the culture medium will undergo the sterilizing
process. Sterilization is defined as the complete destruction of all forms of
microbial life, including bacterial spores. Steam sterilization generally
refers to heating in an autoclave employing saturated steam. High
pressures enable steam to reach high temperatures, thus increasing its
heat content and killing power.

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In this step, when all of the medium have been poured into the Scott
bottles, we have to label the bottle properly for identification and loosen
the cap of the bottles before putting them into the autoclave machine.
This is because the autoclave machine operates under high pressure.
Loosening the the cap will allow expansion of the bottle so that the bottles
will not be broken or worst explode. The autoclave tape also attached on
the bottle as a determination sign that the bottle has been undergoing a
proper autoclave process. The autoclave will be run for 15 minute at
temperature 121oC.

However, after the autoclaving process has ended, the Scott bottles
will be remaining in the autoclave overnight in warm condition around
50oC. The culture medium can be removed from the machine and the cap
of the bottles should be tightened. The bottles should also be turned over
again for a few times to allow a balance distribution of agar in the bottle
so that no agar will solidify at the bottom of the bottle. This step is to
make sure that the culture agar can be pour inside the sterilize petri dish.

The sterilize culture medium in the Scott bottle then poured into the
sterilize petri dish. The melted culture agar is filled about one third of the
petri dish. The aseptic technique need to be adhering during this process
to avoid any cross contamination on the culture agar. Heat the mouth of
the Scott bottle when pour the culture medium into the sterilize petri dish.
The cooled MH agar then keeps inside the chiller for future purpose.

However to indicate that the agar store is free from any

contamination, one petri dish filled with the culture agar is store inside the
incubator overnight. This step was taken by observe if there is any
organisms growth on the surface of the culture agar. If there is a sign of
organisms growth on the surface of culture agar, it mean that the culture
agar already being contaminate but if there is no sign of organisms growth
on the surface of culture agar, it safe to assume that the culture agar is
not contaminate.

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Based on the observation, there are no sign of a colony of bacteria

appear on the surface of the prepared culture agar. Therefore it is indicate
that the agar is not contaminate with any bacteria and can be used to
culture the bacteria.

Conclusion:

From these experiments, we have learned the correct techniques in

preparing the culture medium. Culture media must be stored at the
specified temperature, under specified conditions such as pH and
humidity. Avoid exposing the culture medium to the sunlight all the time.
To prevent humidity of laboratory, all plastic containers must be sealed.
There is specific temperature for sterilization of culture media.

The culture media need to be sterilized to make sure all pathogen

was damaged. Besides that we managed to know the sterilization method
and also know how to operate the autoclave. Preparation and sterilization
of culture media are very important to prevent unwanted microorganisms
to growth on the culture agar. Any of the precaution steps should be
carried out carefully to ensure unwanted errors to occur. For example if
there is a present of bubble in the culture agar when pouring into the
sterilize petri dish, remove it by using the sterilize pipette. Other
precautions are we must read the label and instruction on the container
before use.

In the progress of experiment, use distilled water to clean all the

apparatus. Then, measuring cylinder is used to measure the volume of
distilled water required accurately. Therefore using a suitable measuring
cylinder is very important. Since there variety of measuring cylinder with
different volume capacity, it is essential to use the correct one based on
desire volume. The culture medium need to be heated stir the mixture

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continuously to ensure that the nutrient powder dissolves completely. Last

but not least, autoclaving is a good sterilization process

Questions:

1. Define a culture medium.

Culture medium is any liquid or solid prepared specifically for the
growth, storage, or transport of microorganisms or cells. The variety
of media that exist allow for the culturing of specific microorganisms
and cell types, such as differential media, selective media, test
media, and defined media. Solid media consist of liquid media that
have been solidified with an agent such as agar or gelatine. It
contain all of the nutrients and physical growth parameters
necessary for microbial growth

2. Discuss some of the physical and chemical factors involved in the

composition and in the preparation of a culture medium.
a) Nutrient ingredients
There are two type of energy source used by the bacteria
which is the radiant energy (light) for phototrophs
microorganisms and the oxidation and reduction of chemical
compounds for chemotrophic microorganisms. Glucose is the
main carbon source. For carbon source the autotrophs is a
group of microorganisms that require only carbon dioxide as a
carbon source. Meanwhile the heterotroph is a group of
microorganisms that require organic forms of carbon since
they cannot synthesize organic molecules from inorganic
nutrients. Nitrogen is needed for the synthesis of molecules
such as amino acids, DNA, RNA and ATP. Depending on the
organism, nitrogen, nitrates, ammonia, or organic nitrogen
compounds may be used as a nitrogen source. Therefore
peptone are widely used as the nitrogen source in the agar.

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There are varieties of mineral inside if culture medium such as

sulphur which needs to synthesize sulphur-containing amino
acids and certain vitamins. The phosphorus is needed to
synthesize phospholipids, DNA, RNA, and ATP while the
potassium, magnesium, and calcium is required for certain
enzymes to function.

b) pH and buffering
Microorganisms can be placed based on their optimum pH
requirements. The suitable pH is needed for the
microorganisms to grow. There are three classes of
microorganisms based on their optimum pH requirement. The
first class are neutrophils. It is a group of microorganisms that
grow best at a pH range of 5-8. Second class is acidophilic, a
microorganisms that grow best at a pH below 5.5 and the last
class is alkaliphiles, a bacteria which grow best at a pH above
8.5.

c) Heat (to reconstitute)

The culture agar is heated using Bunsen burner to dissolve the
agar powder completely before autoclave. However make sure
that the agar is not overcook. Then the culture agar is pouring
into Schott bottle for autoclaving at 121oC.

d) Heat (to sterilize)

The sterilization of culture agar is best carried out in a
autoclave at temperatures 121°C. However it has been
recognised to cause a damage to the culture medium. Heat-
treatment of complex culture media results in nutrient
destruction. It can be cause either by direct thermal
degradation or by reaction between the medium components.
It is important to optimise the heating process so that a
medium is sterile with a minimal damage to the agar
ingredients.
e) Other

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Each organism has their own requirements of molecular

oxygen (O2) as well as other atmospheric gasses, such as
carbon dioxide (CO2). There are 5 categories of organism
oxygen requirements which are the obligate aerobes,
microaerophilic, the obligate anaerobes, the aero-tolerant
anaerobes and the facultative anaerobes. For the first
categories, the obligate aerobes is a group of organisms that
grow only when oxygen is present while the microaerophilic
organisms only require the low level oxygen for growth (2-
10%). Obligate anaerobes organisms grow only in the absence
of oxygen and most of them will die in presence of oxygen. For
aero-tolerant anaerobes, the organisms cannot use oxygen to
transform energy but can grow in its presence while the
facultative anaerobes organisms can grow with or without
oxygen. This is because they obtain their energy through
aerobic respiration if oxygen is present, but use fermentation
or anaerobic respiration if the oxygen is absent.

3. At what temperature does agar solidify? And what temperature does

agar melt?
In the room temperature (25oC-28OC), the state of agar is in solid
state remaining firm at temperature as high as 65°C. However when
the temperature reach 80oC-85oC, the agar will begin to melt.

4. What would happen to plates poured with agar that is too hot?
Could they be used?
The condensation will occur in the culture plate. There is also
potential that many of the microorganisms will be killed.

5. What would happen to plates poured with agar that is too cool?
Could they be used?
The lumps will occur in the plate or the agar will not pour. If agar is
cooled more than 45oC although not be poured into the plate, the
agar will begin solidify and get into a gel form and if it still in the
condition that it can be poured then it will make the loan uneven
into which streaking will be difficult and result will be not clear.

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References:

1. Jeffrey C. Pommerville (2017), Fundamentals of Microbiology, 11th

Edition, Jones & Bartlett Learning, LCC , Ascend Learning Company.
2. Mark Slingo. (2017). Aseptic Technique and It’s Important in
Microbiology, 24–28. https://healthyliving.azcentral.com/Aseptic-
technique-and-it-is-important-in- microbiology-12580895.html
3. Jackie Reynolds, (2012), the Microbiology Laboratory- Lab Procedure
Manual, Chapter 18, 19, 21. 
4. Laboratory Practical (2018), Laboratory Exercise, MLT 415, HS241,
Title: Preparation of Culture Media, Week 7, pg 1-2.
5. Azlin Sham Rambley (2018), Lecture Note, MLT 415, HS241, Chap. 9,
Title: Media, pg 1-38
6. Azlin Sham Rambley (2018), Lecture Note, MLT 415, HS241, Chap 6,
Title: Bacterial Physiology and Metabolisms, pg 1-25.
7. Divakaran A/L Chandrahasan (2012), Media Preparation, Isolation of
Pure Culture and Bacterial Growth, 1-6.
https://www.academia.edu/12455926/SY_10401_
MEDIA_PREPARATION_ISOLATION_OF_PURE_CULTURE
8. Sajetra, Shuhada, Alif and Ezzreen (2016), Preparation and
Sterilization of Culture Media,
https://group1ibg102.wordpress.com/2016/10/28/60/
9. http://www.studentsguide.in/animal-biotechnology/animal-cell-and-
tissue- culture/preparation-and-sterilization-of-medium.html
10. http://www.ehow.com/info_8131230_types-agar-plates.html
11. http://www.cabri.org/guidelines/micro-organisms/M203Ap1.html

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