BIO1080 Microbial diversity

Practical 1
Tutor: Dr Gabrielle Zammit, Demonstrator: Dr Michelle Ellul

Part A: Preparation of culture media

Objectives:
• Media preparation
• Sterile techniques

Preparation of Media
Introduction:
The survival and continued growth of microorganisms depend on an adequate supply of
nutrients and a favourable growth environment. As nutrients, most microbes must use
soluble low‐molecular‐weight substances that are frequently derived from the enzymatic
degradation of complex nutrients. A solution containing these nutrients is a nutrient
medium.
Culture media can be liquid, semisolid or solid. A liquid medium lacks a solidifying agent
and is called a broth.
A broth medium supplemented with a solidifying agent called agar will result in a solid or
a semisolid medium.
While in the liquefied state, solid media can be poured into Petri dishes, allowed to cool
and harden to produce agar plates, which provide a large surface area for the isolation
and study of microorganisms. If poured into test tubes and allowed to solidify while these
are placed in a slanted position, these are known as agar slants and are useful for
maintaining pure cultures over an extended period.

Preparation of TSA
• Prepare TSA by adding 12g of TSA powder to 300ml of distilled water. Add 4.5g of
sodium chloride to the medium. Carefully heat on the Bunsen burner stirring
(continuously) until clear.

Preparation of Agar slants (to be used in Practical 2):

• Distribute 100ml of the liquid TSA equally in the bijoux bottles provided
(approximately 10 bijoux bottles will be required).
• Do not tighten the bijoux cap. Place a small piece of Bowie Dick tape (Autoclave
tape) on the side to cover part of the neck and the cap. Label with your initials and
date, and place in autoclave at 121°C for 15 minutes.
• Once the autoclave cycle is complete and the temperature has fallen to about 75°C,
slowly open the autoclave lid and take out the bottles. Note the colour of the autoclave
tape and record results.
• Allow the bijoux bottles to cool and the agar to harden in a slanted position.

Last updated G. Zammit, February 2020 . Page 1 of 5

 BIO1080 Microbial diversity
Practical 1
Tutor: Dr Gabrielle Zammit, Demonstrator: Dr Michelle Ellul

Preparation of Agar plates (to be used in Practical 2):

• Allow the TSA to cool slightly and dispense the solution into a media bottle. Do not
tighten the bottle cap. Place a small piece of Bowie Dick tape (Autoclave tape) on
the side to cover part of the neck and the cap. Label with your initials and date
prepared, and place in autoclave.
• Once the autoclave cycle is ready and the temperature has fallen to about 75°C,
slowly open the autoclave lid and take out the media bottles. Note the colour of
the autoclave tape and record results.
• Once the bottles are cool, pour media into the Petri dishes provided. Label plates
with initials, date of preparation and medium used.
• Leave to solidify in an undisturbed area for later use in Practical 2.

Part B: Isolation of discrete colonies and Preparation of

an environmental mixed microbial culture
Objectives:
• Isolation of discrete colonies from a mixed culture using the streak‐plate method.
• Preparation of an environmental mixed culture, followed by isolation of discrete
colonies using the spread‐plate method.

Isolation of Discrete Colonies

Introduction:
Pure microbial cultures contain only one type of microorganism and are suitable for the
study of morphological, ultrastructural and biochemical properties. These pure cultures may
be obtained from a mixed microbial culture by using techniques designed to produce discrete
colonies. Bacterial colonies are individual, macroscopic masses of microbes that grow on the
surface of an agarised medium, each representing the mass of cells that arises from the
multiplication of a single microorganism.

Once discrete colonies have been obtained, they may be aseptically transferred onto a nutrient
agar plate for the isolation of pure cultures.

The techniques involved are the streak‐plate method and the spread‐plate method.

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 BIO1080 Microbial diversity
Practical 1
Tutor: Dr Gabrielle Zammit, Demonstrator: Dr Michelle Ellul

The Streak‐Plate Method:

(Using TSA plates that are provided).

• Clean your working area with ethanol. Immerse a sterile wire loop in one of the mixed
bacterial cultures growing in TSB provided and spread lightly over the agar surface as
in Area 1 shown in Figure 2.1 below. Do not press the loop into agar surface whilst streaking.

• Flame the loop, and cool it (you may cool the loop by touching it against an unused
part of the agar surface close to the periphery of the plate). Rotate the Petri dish 90°,
then touch the loop to a corner of the culture in Area 1 and drag it several times across
the agar in Area 2. The loop should never come in contact with Area 1 after this.

• Reflame and cool the loop and again rotate the dish 90°. Streak Area 3 in the same
manner as you have done for Area 2 (Fig 2.1).

• Without reflaming the loop, rotate the dish 90° and spread the culture from the corner
of Area 3 across Area 4 (Fig 2.1), using a wider streak. Don’t allow the loop to come
into contact with any of the previously streaked areas.

Please note: Whenever possible, if working close to a flame, Petri dish covers should never be completely
removed. The cover should be raised and held at the smallest angle sufficient for the introduction of the
inoculating wire, and this should be done only for as long as it takes to inoculate each designated area of
the plate. If you find this difficult to do, please prepare the streak plate under the biosafety cabinet for
training purposes, until you get used to the technique. You may remove the Petri dish cover and place it
facing upwards on the surface of the biosafety cabinet while streaking.

• Make sure all the Petri dishes are properly labelled on the underside.
• Incubate all plates in an inverted position for 48‐72 hours at 30°C.

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 BIO1080 Microbial diversity
Practical 1
Tutor: Dr Gabrielle Zammit, Demonstrator: Dr Michelle Ellul

Isolation of Bacterial Colonies from an Environmental

Mixed Microbial Culture

Introduction
The techniques commonly used for the isolation of discrete colonies initially require that the
number of organisms in the inoculum be reduced. The resulting diminution of the population
size ensures that, following inoculation, individual cells will be sufficiently far apart on the
surface of the agar medium to ensure separate growth of the different species present.

Procedure:
Preparation of environmental mixed culture:
• Dampen a sterile cotton swab with sterile saline water (4ml of saline in a tube is
enough). Wring out the excess water by pressing the wet swab against the walls of the
tube.
• With the moistened cotton swab, obtain your microbial specimen from an
environmental source (this may include a table top, door knob, bathroom sink, etc.).
Do this by swabbing a surface traversing approximately a 9 cm2 area, a total of four
times in two directions.
• Place the contaminated swab back into the tube of sterile saline. Mix gently and allow
it to stand for 5 minutes.
• Perform the spread‐plate method explained below.

The Spread‐Plate Method:

(Using plates that have been provided).

• Make sure the bent L‐shaped rod is sterile.
• Pipette 0.5ml of the environmental mixed culture prepared above onto a labelled
nutrient agar plate.
• While rotating the Petri dish, lightly touch the sterile bent rod to the surface of the agar
and spread the culture throughout.
• Make sure to immediately replace the Petri dish cover.
• Make sure all plates are properly labelled on the underside of the Petri dish.
• Incubate all plates in an inverted position for 48‐72 hours at 30°C.
The mixed microbial cultures prepared using both the streak‐plate and the spread‐
plate method will be used next week for Practical 2.
You are also advised to share results from this section on the appropriate sheet that
will be available on the VLE.

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 BIO1080 Microbial diversity
Practical 1
Tutor: Dr Gabrielle Zammit, Demonstrator: Dr Michelle Ellul

Practical Report:

The following sections should be included: Date, Title, Introduction (including Aim),
Precautions, Results, Sources of Error, Discussion and References. In your report,
also refer to the additional hand-out provided (‘attachment to Practical 1’ on the
VLE)
N.B. All observations and results should be included in the ‘Results’ section, while a
discussion of results and answers to the review questions in the ‘Discussion’.
Include an account of the aseptic techniques used as precautions. Along with any other
Results you may wish to add, include the colour of the autoclave tape after autoclaving and
use a graphical representation to compare the bacterial counts obtained from different
environmental samples. Discuss differences in the Results obtained and try to infer why
the microbial diversity in some environmental samples might have been higher than others.

Review Questions:

1. Write about agar as a solidifying agent. How is agar used in the preparation of a
solid and a semisolid medium?
2. Write about different nutrient media and how standard general purpose, selective
and differential media are used to grow different microorganisms.
3. Discuss the use of the streak‐plate and spread‐plate methods.
4. What is the pour‐plate method?
5. Why does flaming of the loop occur during the streak‐plate method, besides for
sterilisation purposes?
6. Ideally, Petri dish covers should never be completely removed, but the cover should
be raised and held at the smallest angle possible. Why?
7. Agar plate cultures are always incubated in an inverted position. Why?
8. Can a pure culture be prepared from a mixed broth or a mixed‐agar slant culture?
Explain.
9. Observation of a streak‐plate culture shows more growth in Area 4 than in Area 3.
Account for this observation.

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